IMMUNOLOGY

SYLLABUS

2015 University of Texas Medical School at Houston

“The Immune System is a Vital Organ System, Necessary for Life.”

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IMMUNOLOGY SYLLABUS 2015 - TABLE OF CONTENTS Course Schedule iii

Course Description iv-vi

Essay Assignment + Exam/Essay Review Policy vii-ix

Clinical Correlation: Reading List x

Team Based Learning Exercise - Integrative Exercises xi-xii

Lectures & Clinical Correlations (Begins at Page Number 1)

OVERVIEW AND ELEMENTS OF THE IMMUNE SYSTEM Syllabus Page Medical Importance of the Immune System / How the Immune System Works 1 Cells and Organs of the Immune System 13 Innate Immunity/Inflammation 22

ANTIGENS, ANTIBODIES AND T CELL RECEPTORS - STRUCTURE AND ACTIVITIES Immunogens & Antigens 36 Antibody Structure and Function I+II 45 COMPLEMENT Complement 69 ANTIBODY, T CELL RECEPTORS, AND MHC – STRUCTURE AND ACTIVITIES Genetic Basis of Ab Structure 88 Role of MHC in the Immune Response 97 The T Cell Receptor: Structure and Genetic Basis 110 Adaptive Immune Response: I+II 120

CELLULAR ACTIVITIES AND IMMUNE MEDIATION Antigen-Antibody Interactions - ImmunoAssays 142 Antibody-Mediated Reactions 158 Cell-Mediated Reactions 169 IMMUNE SYSTEM AND INFECTIOUS DISEASE Immunology of HIV Infection 178 Infection and Immunity 190

MEDICAL APPLICATIONS OF IMMUNOLOGY (Immunopathology) Immune Regulation & Tolerance 202 Autoimmunity 206 Clinical Scenarios 213 Disorders of the Immune Response 214 Immunoprophylaxis (Vaccines) 224 Immunology of Cancer 235 Team Based Learning Exercise 241 Transplantation 242 Modern Immuno Therapy 249

Timeline of Immunology (located at end of syllabus) Glossary (located at end of syllabus) APPENDIX: Resource Information (located at end of syllabus)

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Cover Description: Principles of Modern Immunobiology. B.H. Park and R.A. Good. 1974. Lea & Febiger, Henry Kimpton Publishers, Philadelphia. p54.

The purpose of the Immunology course is to provide a basic knowledge of the immune response and its involvement in health and disease. A series of lectures cover course components; additional materials are presented through clinical correlations that focus on clinically applied immunological concepts. An effort has been made to increase clinical relevance and problem-solving skills through an essay assignment and through a team-learning exercise.

SCHEDULE - IMMUNOLOGY 2015Click on Lecture for Additional Immunology Information Medic Web Site

Links for Lectures

Session Date Time Instructor Topic

OVERVIEW AND ELEMENTS OF THE IMMUNE SYSTEM1 1/6/2015 10:00-10:50 Jeffrey Actor Medical Importance of the Immune System

2 1/6/2015 11:00-11:50 Jeffrey Actor Cells and Organs of the Immune Sytstem

3 1/9/2015 8:00-8:50 Jeffrey Actor Innate Immunity/Inflammation

ANTIGENS AND ANTIBODIES4 1/13/2015 10:00-10:50 Sudhir Paul Immunogens & Antigens

5 1/13/2015 11:00-11:50 Keri Smith Antibody Structure and Function I

6 1/16/2015 8:00-8:50 Keri Smith Antibody Structure and Function II

COMPLEMENT7 1/16/2015 9:00-9:50 Rick Wetsel Complement

ANTIBODIES, T CELL RECEPTORS, AND MHC - STRUCTURE AND ACTIVITIES8 1/20/2015 10:00-10:50 Steven Norris Genetic Basis of Ab Structure

9 1/20/2015 11:00-11:50 Jeffrey Actor Role of MHC in the Immune Response

10 1/22/2015 9:00-9:50 Jeffrey Actor The T Cell Receptor: Structure and Genetic Basis

11 1/22/2015 10:00-10:50 Jeffrey Actor Adaptive Immune Response 1

12 1/22/2015 11:00-11:50 Jeffrey Actor Adaptive Immune Response 2

13 1/23/2015 8:00-8:50 Keri Smith Antigen-Antibody Interactions

1/29/2015 1:00-3:00 1:00-3:00 Midterm Exam

CELLULAR ACTIVITIES AND IMMUNE MEDIATION14 2/3/2015 9:00-9:50 Steven Norris Antibody-Mediated Reactions

15 2/5/2015 10:00-10:50 Steven Norris Cell-Mediated Reactions

IMMUNE SYSTEM AND INFECTIOUS DISEASE16 2/6/2015 10:00-10:50 Steven Norris Immunology of HIV Infection

17 2/6/2015 11:00-11:50 Jeffrey Actor Infection and Immunity

MEDICAL APPLICATIONS OF IMMUNOLOGY (Immunopathology)18 2/10/2015 11:00-11:50 Dat Tran Immune Regulation & Tolerance

19 2/12/2015 10:00-10:50 Sandeep Agarwal Autoimmunity

20 2/12/2015 11:00-11:50 Sandeep Agarwal and Jeffrey Actor Immunology: Clinical Scenarios 

21 2/17/2015 11:00-11:50 William Shearer Disorders of the Immune Response

22 2/19/2015 11:00-11:50 Jeffrey Actor Immunoprophylaxis (Vaccines)

23 2/23/2015 10:00-10:50 Jeffrey Actor Cancer Immunology

24 2/26/2015 8:00-9:50 Jeffrey Actor Team Based Learning

25 2/26/2015 10:00-10:50 Keri Smith Transplantation

26 2/27/2015 11:00-11:50 TBA ImmunoTherapy

3/5/2015 1:00-4:00 1:00-4:00 Final Exam

3/18/2015 Essay Assignment Due Essay must be submitted prior to 5:00pm

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MEDICAL SCHOOL IMMUNOLOGY - 2015 COURSE DESCRIPTION Course Director: Jeffrey K. Actor, Ph.D. Department of Pathology, MSB 2.214 Office Tel: 713-500-5344 LECTURERS OFFICE TELEPHONE email

Jeffrey K. Actor, Ph.D. MSB 2.214 713-500-5344 Jeffrey.K.Actor@uth.tmc.edu Sandeep K. Agarwal, M.D., Ph.D. BCM 713-798-3390 skagarwa@bcm.edu Steven J. Norris, Ph.D. MSB 2.278 713-500-5338 Steven.J.Norris@uth.tmc.edu Sudhir Paul, Ph.D. MSB 2.230A 713-500-5347 Sudhir.Paul@uth.tmc.edu William T. Shearer, M.D., Ph.D. Texas Children's 832-824-1274 WTSheare@texaschildrens.org Keri C. Smith, Ph.D. MSB 2.248 713-500-2250 Keri.C.Smith@uth.tmc.edu Dat Q. Tran, M.D. MSE R428 713-500-5422 Dat.Q.Tran@uth.tmc.edu Rick A. Wetsel, M.D., Ph.D. SRB 430A 713-500-2412 Rick.A.Wetsel@uth.tmc.edu IMMUNOLOGY WEB PAGE: Medic Immunology Web Page 1) COURSE ORGANIZATION

The purpose of the Immunology course is to provide a basic knowledge of the immune response and its involvement in health and disease. All lectures will be presented in MSB 2.006. An effort has been made to increase clinical relevance and problem-solving skills through an essay assignment and faculty-presented clinical correlations, and a team based learning exercise. Any questions on the lecture material should be addressed to Dr. Actor or directly to that lecturer. If you have general problems or comments regarding the course, your grades, or the faculty, please contact the course director. If the problem is not resolved, you should make an appointment to see Dr. Robert L. Hunter (Chairman of Pathology) at MSB 2.136 (500-5301) or, finally, Dr. Patricia Butler (Assoc. Dean for Educational Programs) or Dr. Margaret McNeese (Assoc. Dean for Student Affairs). 2) COURSE MATERIALS

a) Lectures. The student is responsible for all material covered in lectures and faculty presented clinical correlations, as well as for any additional handouts or assignments (whether provided in this syllabus or at a later time). Immunology is a rapidly advancing area, so the lectures may contain new information not covered in the textbooks. Therefore you should make every effort to attend lecture and take complete and accurate notes. Streaming video is available on-line through the UT Med School student web pages. Additional information may also be available from the Conference Operations Office (LRC), and can be used to verify your notes.

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b) Reading. Two textbooks are required for the course. Chapter assignments are listed directly in the syllabus chapter. Required Case Studies are listed separately in this syllabus. R. Coico and Sunshine, G. Immunology: A Short Course. (6th Ed) John Wiley & Sons, Inc., 2009. (The 7th edition is set for release in March, 2015.) R. S. Geha and Notarangelo, L. Case Studies in Immunology: A Clinical Companion. (6th Ed)

Garland Publishing, New York, 2012.

The Coico et al. text was selected because it is well-organized, clearly and concisely written, and contains chapter summaries, study questions, and case studies. The lecture schedule is loosely organized to match the Coico et al. book chapters. Knowledge of the assigned reading is required, even if the material is not covered in the lectures. Modifications of the study questions may be used in the exams. The Geha and Rosen text provides examples of the role of immunology in health and disease, and is used extensively in the Clinical Correlations and as ‘Clinical Vignettes’ in the lectures. Cases from Geha and Notarangelo presented (in part or in full) during lecture are considered required reading. Please see the list of required associated cases assigned for each lecture, located under “Clinical Correlation Required Readings”. c) MEDIC IMMUNOLOGY Web Page. You are encouraged to make use of the MEDIC Immunology web site at: https://med.uth.edu/pathology/courses/immunology/. Materials are also on Blackboard. The website is actively updated during the course to include links for lecture materials and information that will assist in understanding of course materials. Alternative recommended texts available in the bookstore (not required, but potentially helpful):

Actor, J.K. Elsevier’s Integrated Immunology and Microbiology (2nd Ed.), Mosby/Elsevier, Philadelphia, 2012.

Actor, J.K. Introductory Immunology: Basic Concepts for Interdisciplinary Applications (1st Ed.), Academic Press/Elsevier, 2014.

Some other good textbooks (not required, but potentially helpful) may also be available in the bookstore:

Parham, P. The Immune System. 3rd Edition. Garland Publishing, New York, 2009. o Note: a 4th edition was due for release at the end of 2014.

Abbas, A. K. and Lichtman, A. H. Basic Immunology – Functions and Disorders of the Immune System, 4th Edition. Saunders-Elsevier. Philadelphia, PA. 2012.

Owen, J. Punt, J., and Strandford, S. Kuby Immunology (7th Ed.), W.H. Freeman and Company, New York, 2013. Murphy, K. Janeway’s Immunobiology (8th Ed.). Garland Publishing, New York, 2012 (updated 2014).

Each of these texts may be found at the LRC or the HAM Library, or available on-line for purchase and/or digital download.

d) Essay Assignment. Students must turn in one essay assignment worth 10 points. Students must attend one of the City-Wide Infectious Disease Rounds and provide a written review of one of the cases. The assignment and due date are described in detail elsewhere in the syllabus, as well as on Blackboard.

e) Team Based Learning. There will be one team based learning exercise as a portion of the course. The TBL is detailed later in the syllabus. The TBL is worth a maximum of 10 points.

f) Clinical Scenarios. In addition to the regular lectures, we may have a Clinical Scenario sessions during the semester. Past experience has shown that immunology (or any other medical topic) is easier to learn and remember if it is presented as clinical cases involving 'real' patients. In each clinical correlation, cases relevant to immunology will be discussed by faculty. The correlate scenarios are related to those presented in the Geha and Notarangelo text, but may vary to accommodate additional learning materials.

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g) Self Study Questions. Additional study questions are provided at the end of some lecture outlines. The purpose of these questions is to test your knowledge and extend your learning beyond rote memorization toward more 'cognitive' learning. The study questions will not be graded, but questions related to these assignments overlap with examination materials. Answers are typically (but not always) posted on the Immunology web site or on Blackborad.

h) Streaming video. Lectures will be made available for viewing via streaming video over the internet.

i) Office hours and other assistance. Students are encouraged to approach the lecturers if they need assistance in understanding the course material. Dr. Actor is also available at his office (MSB 2.214) by individual appointment or by phone or email. 3) GRADING

a) Examinations. There will be two major exams consisting of multiple choice, matching, and national board format questions. The midterm exam will contain 60 questions (worth 40% of your grade). A cumulative final exam will have 80 questions (worth 40% of your grade). Exam answers will be posted according to accepted policies of the University. Policies for review after each exam are set by the University (see posted document on University Policy on Exam Grading and Review sessions).*

b) Essay Assignment. The essay assignment is required and will be worth a possible 10 points. Grading is based on adherence to the format described in this syllabus, thoroughness, and application of your budding medical knowledge and logic; you are not expected to 'know it all' at this point. The due date is listed in the Essay instruction page in the syllabus, and posted on Blackboard. Essays may be turned in early. Assignments turned in late will only receive a maximum of half credit (no exceptions).

c) Team Based Learning. Questions answered for the TBL session are worth a maximum of 10 points.

d) Overall grade. The total possible points and grade assignments are given below. The total value of points for the course is 100 points. Midterm 40 points (60 questions) Final 40 points (80 questions) Essay Assignment 10 points Team Based Learning 10 points e) Final grade assignment. The final grade is based on percentage of points earned (max of 100 points) as related to total possible points (max of 100). Honors 90-100 % High pass 85.5-89.99 % Pass 69.5-85.49 % Below Pass 65.5-69.49 % Fail 65.49 % or below

*Immunology Exam Question Review Policy: Review of exams may be done on an individual basis. Upon written request, students may view questions missed. Any requests to review exams must be submitted immediately following release of scores; exam questions may only be reviewed within the two week period following release of scores. The Course Director has the right to limit question viewing. The Course Director (course policy) limits question viewing to a two week period after release of scores.

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IMMUNOLOGY ESSAY ASSIGNMENT The purpose of this assignment is to encourage you to explore an important form of information in medicine: grand rounds presentations. You are not expected to master the analysis of the information presented, but you should demonstrate that you have made an honest attempt to understand and interpret it. The assignment is worth a possible 10 points (maximum). The assignment is due any time on, or before, 5:00pm on March 18, 2015. Assignments turned in late will receive a maximum of only half credit. Completed assignments are turned in to the Health Education Office (MSB 2.120), or directly to Dr. Actor. Assignments are not accepted via e-mail.

YOU MUST TYPE YOUR ESSAY. THE ASSIGNMENT SHOULD BE APPROXIMATELY TWO-THREE PAGES IN LENGTH. THIS IS NOT A SHARED ASSIGNMENT; DUPLICATE ESSAYS (TURNED IN BY MORE THAN ONE STUDENT) WILL NOT BE ACCEPTED.

Examples of the essay assignment can be found posted on Blackboard. ______________________________________________________________________________ ASSIGNMENT: Grand Rounds Review. Attend one of the City-Wide Infectious Disease Conferences held every Wednesday at Noon in the auditorium behind the elevators on the ground floor of the DeBakey Building, Baylor College of Medicine (BCM Rm M112). (This building is the white building next to the new Baylor Graduate School Building and across the street from the Jones Library). Usually three cases are presented as unknowns, a differential diagnosis is made, and the outcome and ramifications of the case are discussed. The presenters often provide handouts for the case, but you may wish to take notes of the conference to help you glean out the information. Note that the infectious disease and microbiology aspects are generally covered in detail, whereas the immunology is often not discussed. Your job is to investigate the immunologic aspects of the disease and incorporate them into your interpretation and case description. This is an Immunology assignment; limit your discussion of Microbiology to pertinent information only. It is not permitted to record or capture pictures/slides of presented materials. No picture taking is allowed (due to HIPAA regulations, patient confidentiality, and proprietary information). Choose only ONE of the cases presented. The case must have an immunologic implication. (e.g. The infection was cleared by immune mechanisms, or the patient was immunodeficient and thus developed an unusual infection). a) Briefly describe the case, concentrating on the clinical manifestations (patient's symptoms + findings from examination and tests) that are most relevant. Use medical terms where you can, but define them in a few words. Include the diagnosis, treatment, and outcome (if presented). USE YOUR OWN WORDS. Include a copy of the handout for the case, if one was provided. There is no need to include copies of the presenter’s PowerPoint slides.

b) Using your microbiology and immunology texts, describe the organism(s) which caused the infection in this case. What is the normal course of disease, and how did they differ in this case? What treatments are generally effective, and were they effective in this patient?

c) The major portion of the essay should be devoted towards discussion of the immunologic implications and principles of the case. Describe in as much detail as possible the normal

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immune mechanisms to combat this infectious agent and how they affect the course of infection (e.g. Macrophages phagocytose and process the antigen and present antigen fragments in association with MHC Class II proteins to antigen-specific CD4+ helper T cells, role of complement, cell phenotypes involved, etc.). Be specific and included details! How was the immune response of this patient different than normal (if this is applicable)? Did the patient have an underlying condition that contributed to the development of this infection? Did the patient have cancer, AIDS, hereditary immunodeficiency or some other condition affecting the immune response? How did the immune response (or lack thereof) affect the outcome of this case? Did the immune response contribute to the pathogenesis of disease (i.e. is immunopathology involved)? Describe immunization or other immunologic procedures (such as passive transfer of antibodies) used in the prevention or treatment of this disease.

d) Cite references used in your analysis of the case. You will need to refer to published journal articles to obtain specific background information or methods needed to comprehend the case. Include as many references as needed to support ideas. Points will be subtracted if relevant citations are absent. You must include at least 2 primary publications (meaning: journal articles) published within the past 3 years. Web pages are not considered as primary references. You may also include syllabus chapters as references, but must also include additional references that demonstrate you have expanded your discussion to materials outside the course lecture presented materials. Syllabus chapters are NOT primary references. Up to 1 point is subtracted if the references are missing, incomplete, or inadequate to support your discussion. Recommended: use PubMed to find related articles for the report. e) Include a copy of the handout from the Grand Rounds session if one was available. Do not include copies of the PowerPoint presentation. f) The length of the essay should not exceed 3 pages (including references). 1 point is subtracted for going over the set page limit. In summary, make sure you: Describe clinical manifestations of the case. Discuss immunological aspects of case. Give full citations to cite your ideas, including use of current references from

journal articles. Attach a copy of the Grand Rounds handout for the case, if available. Do not

include a copy of the presenters PowerPoint slides. Turn in your assignment on time.

Essays may be submitted anytime prior to the stated deadline. Late submitted essays will automatically receive a 5 point deduction. ESSAY GRADES: Essays will be returned to students as quickly as grading allows. Inquiries regarding essay assignment grades received must be submitted within one week after receipt of returned assignments. Requests for review of essays past the one week period will be denied.

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Policy on Exam Grading (MS1 and MS2 courses) and Exam Review Sessions

Recommended by the Educational Policy Subcommittee: August 12, 2010 Approved by the Curriculum Committee: August 25, 2010

Revised by Educational Programs: August 31, 2010 Approved by the Curriculum Committee: September 15, 2010

Revised and Revisions Approved by the Curriculum Committee: May 18, 2011

The following policy delineates procedures related to exam grading and review/protest sessions to be followed by all first- and second-year courses.

1. Course directors will score examinations through LXR and post results on MS-Gradebook as soon as possible.

2. Course directors will use item statistics generated by LXR to identify problematic

questions. Upon review, if a course director determines that a question was written incorrectly (e.g. had more than one or no correct answer), then the director will give all students credit for that particular question.

3. Large group post-exam review sessions may be held to provide feedback on difficult

examination topics, in a manner deemed appropriate by the course director. Copies of the examinations will not be returned to the students during these sessions.

a. Course directors may meet with individual students to review examinations. The

format of these sessions, which may involve reviewing specific examination questions, will be determined by the course director on a case by case basis.

Immunology Exam Question Review Policy: Review of exams may be done on an individual basis. Upon request, students may view questions missed. Any requests to review exams must be submitted immediately following release of scores; exam questions may only be reviewed within the two week period following release of scores. The Course Director has the right to limit question viewing. Immunology Essay Grades Review Policy: Discussion of score received for the essay assignment may be done on an individual basis. Inquiries regarding essay assignment score received must be submitted within one week after receipt of returned assignments. Requests for review of essays past that one week period will be denied.

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CLINICAL CORRELATIONS 2015 Required Readings

2015 Immunology Clinical Correlation Required Readings: R. S. Geha and Notarangelo, L. Case Studies in Immunology: A Clinical Companion. (6th Ed) Garland Publishing, New York, 2012.

Reading for Lecture: Required Readings Cells and Organs 30. Congenital Asplenia Innate Immunity 15. Chediak-Higashi Syndrome

25. Neutropenia 26. Chronic Granulomatous Disease 27. Leukocyte Adhesion Deficiency

Antibody Structure and Function Multiple Myeloma (Blackboard file) 46. Hemolytic Disease of the Newborn

Complement 32. Factor I Deficiency 33. Deficiency of C8

MHC 8. MHC Class II Deficiency 12. MHC Class I Deficiency

T cell receptor 7. Omenn Syndrome T-Cell Lymphoma (Blackboard file)

Adaptive Immune Response 47. Toxic Shock Syndrome Antibody-Mediated Reactions 41. Autoimmune Hemolytic Anemia

50. Allergic Asthma 52. Drug-Induced Serum Sickness

T cell Mediated Reactions

45. Acute Infectious Mononucleosis 53. Contact Hypersensitivity to Poison Ivy

Immunology of HIV Infections 10. Acquired Immune Deficiency Syndrome (AIDS) Infection and Immunity 28. Recurrent Herpes Simplex Encephalitis

48. Lepromatous Leprosy Autoimmunity 36. Rheumatoid Arthritis

35. Systemic-Onset JID 40. Multiple Sclerosis 42. Myasthenia Gravis

Transplantation Kidney Graft Complications (Blackboard file) 11. Graft-Versus-Host Disease

Disorders of the Immune Response 1. X-linked Agammaglobulinemia 4. CVID 9. DiGeorge Syndrome 16. Wiskott-Aldrich Syndrome

Clin. Corr. Date Time Case Readings Class 2/12 11:00-11:50 AM 36. Rheumatoid Arthritis

37. Systemic Lupus Erythematosus TBL 2/26 8:00-9:50 AM Distributed Reading: Inflammatory Bowel Diseases (Crohn’s

Disease, Ulcerative Colitis, and Celiac) 39. Crohn’s Disease 44. Celiac Disease

Required readings complement lectures and presented materials. It is highly encouraged to view these clinical cases. Case materials may not be covered in full during lectures, however, all required case study readings contain material that

may be tested on exams. Assigned readings may be discussed in multiple lectures, in addition to the “assigned” lectures.

Spring Semester, 2015

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Team Based Learning Exercise

The Immunology course will have one Team Based Learning exercise where students will be required to address a clinically based scenario and provide answers to related questions. Students will be assigned specific reading prior to the session, which will assist in mastering of the material so as to allow participation in the group activities. Materials will include new material in Immunology, as well as materials already mastered in other courses. The format will be similar to the Clinical Applications course. The Team Based Learning Exercise is mandatory. The Team Based Learning Exercise encompasses a graded set of exercises related to multiple integrated aspects of a clinical scenario. The exercise is worth a maximum of 10 points towards your overall Immunology grade. The session will utilize a clinical scenario to present a problem. Students are divided into teams; utilizing the groups already in place for the Clinical Applications course. Approximately 5 problem questions arising from the clinical scenario are crafted to foster discussion within the teams; each team is required to come to a consensus as to the solution to the problem. Written justification may be required for the team solution, to be prepared and handed in for grading at the end of the session. Team Based Learning Exercise: Immunology

February 26th

8:00-9:50 a.m.

Persons missing the session must provide written notice explaining circumstances for not attending. Written approval must be obtained from the Office of Educational/Student Affairs prior to consideration for any makeup session or alternate assignment.

Spring Semester, 2015

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Clinical Applications: Integrative Exercises There will be a series of Clinical Applications (Integrative Exercises) throughout the first year, up to three of which are scheduled for the Spring 2015 semester. These exercises are designed to integrate content from the basic science courses and the ICM course and to help students develop reasoning skills they will utilize in their clinical years. The administration of these Exercises is held separate from the Immunology course, but material from the exercises will be subject to assessment in all of the first year courses. Attendance is required at these sessions and will be monitored. The dates of the Clinical Applications Integrative Exercises during the Spring semester are posted; see Blackboard to confirm times and room assignments: Students will be assigned to work in small groups of four to six students. These groups will remain together for all seven of the Integrative Exercises throughout the year. During the Integrative Exercises, each group will discuss a clinical problem that integrates material from the current basic science courses and will develop a team answer to a question regarding that clinical problem. The teams will then prepare a written justification for their answer for one of these problems. These justifications will be handed in for grading. Pre-reading and pretests may be posted to Blackboard as necessary for each exercise. You will receive email notifications regarding any pre-reading or pretest assignments. The graded responses from all of the sessions will contribute to the final grade in the Integrative Exercise course. Each of the group members will receive the same score. Students who have unexcused absences will receive a score of 0 for all responses for that Integrative Exercise session.

Information presented within any Clinical Application Exercise throughout the year is a potential source of testable material for exams

in any MSI class.

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MEDICAL IMPORTANCE OF THE IMMUNE SYSTEM [HOW THE IMMUNE SYSTEM WORKS]

Jeffrey K. Actor, Ph.D.

MSB 2.214, 713-500-5344 (Special thanks to Gailen D. Marshall, Jr., MD, PhD, and Steven J. Norris, PhD)

Objectives

1. To appreciate the components of the human immune response that work together to

protect the host 2. To understand the concept of immune-based diseases as either a deficiency of

components or excess activity as hypersensitivity

3. To introduce the 7 main concepts of the course

KEYWORDS immunodeficiency, hypersensitivity Required Knowledge: Hypersensitivity Chart Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York, NY. 6th edition, 2009. Chapter 1. Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/medical-importance-of-the-immune-system/ The chief function of immunity is to discriminate between self and non-self. The immune cells and organs of the body comprise the primary defense system against invasion by microorganisms and foreign pathogens. A functional immune system confers a state of health through effective elimination of infectious agents (bacteria, viruses, fungi, and parasites) and through control of malignancies by protective immune surveillance. In essence, the process is based in functional discernment between self and non-self, a process which begins in utero and continues through adult life. Immune responses are designed to interact with the environment to protect the host against pathogenic invaders. The goal of these chapters is to appreciate the components of the human immune response that work together to protect the host. Furthermore, we will strive to present a working clinical understanding of the concept of immune-based diseases resulting from either immune system component deficiencies or excess activity. Immunological memory as the basis for vaccine efficacy Continued discrimination for health depends upon immunological memory where the adaptive immune system can more efficiently respond to previously encountered antigen. This results in resistance to repeated infection with pathogens and the resulting clinical syndrome. This principle accounts for the clinical utility of vaccines which have done more to improve mortality rates worldwide than any other medical discovery in recorded history.

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The clinical immunologist is a physician who has specialized in the diagnosis and treatment of disorders of the immune system. Many other clinical specialties (such as oncology, hematology, infectious diseases, transplant surgery, etc.) also deal with immunologically-based diseases in their area of specialization. Much of the work of modern clinical immunologists revolves around refining diagnostic techniques for greater clinical utility and evaluating new therapeutic modalities such as recombinant cytokines and cytokine modulators. Protection against foreign pathogens Normal physiologic functions of the immune system include the ability to discern self from non-self, and recognition of foreign pathogens. This represents recognition of environmental challenges in an attempt to preserve homeostasis while responding to pathogenic agents. The goal is to respond with specificity, allowing sufficient intensity and duration to protect the host without causing damage to self. The Immune system protects against foreign pathogens, of which four major classes can be defined. These include (1) Extracellular bacteria, parasites and fungi; (2) Intracellular bacteria and parasites; (3) Viruses (intracellular); and (4) Parasitic worms (extracellular). Immunodeficiency and dysfunction as the basis of disease Immunological diseases can be grouped into two large categories – deficiency and dysfunction. Immunodeficiency diseases occur as the result of the absence (congenital or acquired) of one or more elements of the immune system. Immune dysfunction occurs when a particular immune response occurs that is detrimental to the host. This response may be against a foreign antigen or self antigen. It may also be an inappropriate regulation of an effector response resulting in the absence of a protective response. Notwithstanding, the host is adversely affected. A healthy immune system occurs as a result of balance between innate and adaptive immunity, cellular and humoral immunity, inflammatory and regulatory networks and small biochemical mediators (cytokines). Because specific mechanism affects prognosis as well as therapeutic approaches, Gel and Coombs classified these dysfunctional immune responses into hypersensitivity diseases. Hypersensitivities will be discussed throughout the course, and in much greater detail in a later chapter. There is considerable overlap in underlying mechanisms that contribute to the hypersensitive responses. The major mechanisms are outlined on the following page.

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Type I (also called immediate hypersensitivity) is due to aberrant production and activity of IgE against normally nonpathogenic antigens (commonly called allergens). The IgE binds to mast cells via high affinity IgE receptors. Subsequent antigen exposure results in crosslinking of mast cell bound IgE with activation of mast cells that release preformed mediators (e.g. histamine, leukotrienes, etc.) and synthesize new mediators (i.e. chemotaxins, cytokines). These mediators are responsible for the signs and symptoms of allergic diseases. [A = Allergic] Type II is due to antibody directed against cell membrane-associated antigen that results in cytolysis. The mechanism may involve complement (cytotoxic antibody) or effector lymphocytes that bind to target cell-associated antibody and effect cytolysis via a complement independent pathway (Antibody dependent cellular cytotoxicity, ADCC). Cytotoxic antibodies mediate many immunologically-based hemolytic anemias while ADCC may be involved in the pathophysiology of certain virus-induced immunological diseases. [C = Cytotoxic] Type III results from soluble antigen-antibody immune complexes that activate complement. The antigens may be self or foreign (i.e. microbial). Such complexes are deposited on membrane surfaces of various organs (i.e. kidney, lung, synovium, etc). The byproducts of complement activation (C3a, C5a) are chemotaxins for acute inflammatory cells. These result in the inflammatory injury seen in diseases such as rheumatoid arthritis, systemic lupus erythematosus, postinfectious arthritis, etc). [I = Immune Complexes] Type IV (also called Delayed Type Hypersensitivity, DTH) involves macrophage-T cell-antigen interactions that cause activation, cytokine secretion and potential granuloma formation. Diseases such as tuberculosis, leprosy and sarcoidosis as well as contact dermatitis are all clinical examples where the tissue injury is primarily due to the vigorous immune response rather than the inciting pathogen itself. [D = DTH] EXPANDED FIGURES OF HYPERSENSITIVITIES INCLUDED IN APPENDIX.

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Clinical suspicion for immunodeficiency may be made when patients present with chronic infection or chronic inflammatory status, poor wound healing, constant fatigue and malaise, or when unresponsive to vaccine administration. Certain infections with organisms may be suggestive of deficiency in an immune related component. Alternatively, disruptions in homeostasis may lead to immunodeficiency, such as those induced inadvertently by a physician through medical treatment (iatrogenic). The mechanisms for clinical immunodeficiency are varied, and will be examined (in part) throughout the remainder of the course. Therapeutic intervention for immune based diseases Therapy for these diseases has historically been nonspecific, centering on repair of the damaged tissues and inhibition of the aberrant immune responses with immunosuppressive drugs. Recent work using such cutting edge techniques as recombinant DNA technology, gene therapy, and stem-cell research have opened up an entire new avenue to address these diseases by providing diagnostic and therapeutic modalities not previously available. For immunodeficiency states, we have developed the g ability to replace elements through marrow transplants, recombinant immune molecule administration and, soon, gene therapy.

Introduction to 7 Main Concepts towards Understanding Medical Immunology

1. The chief function of the immune system is to distinguish between self and non-self.

Health – effective elimination or control of health-threatening agents Infectious agents – bacteria, viruses, fungi, parasites

Tumors – the immune system also plays an important role in the control of malignant cells through a mechanism called immune surveillance

Hyporeactivity – inability to recognize and control health-threatening agents (immunodeficiency)

Congenital immunodeficiency – immune defects due to genetic defects Acquired immunodeficiency – caused by multiple agents, including

Human Immunodeficiency Virus and tumors Malnutrition – severe malnutrition compromises the immune system Young/Old Age – increased susceptibility to infection Hyperreactivity – aberrant immune responses

Systemic autoimmunity – e.g. systemic lupus erythematosus Organ-Specific autoimmunity – e.g. thyroiditis

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Allergies and Asthma – aberrant immune response to environmental allergens or chemicals Immunopathology – general term for damage to normal tissue due to the immune reaction to infectious agents or other antigens (e.g. rheumatic fever, leprosy)

Figure: Immune based diseases can be caused by lack of specific functions (immune deficiency) or excessive activity (hypersensitivity).

2. The immune system consists of two overlapping compartments: the innate immune system and the adaptive immune system.

Innate immune system Most primitive type of immune system; found in virtually all multicellular

animals (arguably also in plants!) Always present and active, constitutively expressed Nonspecific; not specifically directed against any particular infectious agent or

tumor Same every time; no ‘memory’ as found in the adaptive immune system First line of defense against infection Includes:

o Physical barriers (skin, mucus lining of gastrointestinal, respiratory and genitourinary tracts)

o Phagocytic cells – neutrophils, macrophages o Protective chemicals – acid pH of stomach, lipids on skin surface o Enzymes – lysozyme in saliva, intestinal secretions; digests cell walls of

bacteria o Alternate complement pathway – cascade of serum proteins that are

activated by bacterial cell wall components

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Adaptive or acquired immune system Found only in vertebrates (fish, amphibians, birds and mammals) Must be induced to be active against infections or tumors Antigen-specific – adaptive immune responses recognize antigens, which can

be proteins, carbohydrates, lipids and nucleic acids. Memory – response against a given antigen is much stronger after the first

(primary) response. This heightened reactivity is called secondary responses, and is due to increased numbers of memory B and T cells to that antigen

Regulation – discriminates between self and non-self, prevents autoimmune reactions in most individuals

Cells of the Adaptive Immune Response include: o B lymphocytes – differentiate into plasma cells that produce antibodies o T lymphocytes – subdivided into CD4+ and CD8+ populations

Helper activity – help other lymphocytes respond to antigen (mostly CD4+ T cells, subdivided into phenotypic responders)

Delayed type hypersensitivity – activate macrophages to phagocytose, kill pathogens (mostly CD4+ T cells)

T cell-mediated cytotoxicity – cytotoxic T cells (mostly CD8+ T cells) bind to and kill target cells (e.g. virus-infected cells and tumor cells)

Suppressor T cells/Treg cells – down-regulate the responses of other lymphocytes

Table: Elements of Innate and Acquired Immune Responses Innate Adaptive Rapid response (minutes to hours) Slow Response (days to weeks) PMNs and Phagocytes B cells and T cells Preformed effectors with limited variability Pattern Recognition Molecules recognizing

structural motifs

B cell and T cell receptors with highly selective specificities

Soluble activators Proinflammatory mediators

Antibodies (humoral) Cytokines (cellular)

Non-specific Specific No memory, no increase in response upon

secondary exposure Memory, maturation of secondary response

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3. The antigenic specificity of the adaptive immune system is due to antigen- specific receptors.

Immunoglobulins (also called antibodies) – produced by B cell lineage

o IgM, IgD, IgG, IgA, and IgE subtypes o Surface immunoglobulin (Ig) – antigen-specific receptor of B

lymphocytes o Secreted immunoglobulin (Ig) – Ig molecules secreted by plasma cells

T cell receptor (TCR) – antigen-specific receptor of T lymphocytes

o and TCR subtypes

Coico and Sunshine, 2009. Fig. 1.3.

The basic reaction in immunology is the binding of antigen to an antigen-specific receptor. The affinity of this interaction is similar to that of an enzyme binding to its substrate.

Ag + Ab AgAb Typically, each antibody or T cell receptor molecule recognizes a single epitope, a

small region (e.g. 6-10 amino acids) of an antigen. In a given B- or T-cell, the antigen-specific receptors of all are identical.

o Exception – IgM and IgD can be coexpressed on certain B cells Each B cell and T cell has its own antigenic specificity, determined by the amino acid

sequence of its surface Ig or TCR. The region of the Ig or TCR that binds to the antigen is called the paratope.

In each person, there are ~106 to 108 different Igs and TCRs, giving rise to an almost endless supply of antigenic specificities. This is called diversity.

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4. The generation of antigen-binding diversity occurs prior to antigen exposure through a DNA rearrangement process called VDJ joining.

The “business end” of an antibody or TCR is the variable region. This region contains

the antigen-binding site that binds to the epitope (meaning: the conformational shape recognized).

Variable Region

Coico and Sunshine, 2009. Fig. 1.2.

The variable region is formed during B and T cell development. This process occurs prior to exposure to a given antigen.

The DNA encoding the variable region is subdivided into V, D, and J gene segments. There are multiple V, D, and J gene segments in the Ig and TCR genetic loci.

In most cells, these gene segments are spread out, so that all the V segments are together, all the D segments are together, and all the J segments are together. This is called the germline configuration, because it is the arrangement seen in sperm and ova.

The V, D, and J gene segments are brought together to form a contiguous exon encoding the variable region. The V, D, and J segments are selected randomly in each cell, giving rise to combinatorial diversity. This is similar to the “Pick 5” game in Texas lotto, in which a large number of different number combinations exist.

The light chain gene locus (and some TCR genes) has only V and J regions. There are several other mechanisms for generating diversity, as will be discussed in a

later lecture.

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5. To generate an active immune response against a certain antigen, a small number of B and T cell clones that bind to the antigen with high affinity undergo activation, proliferation, and differentiation into plasma cells (for B cells) or activated T cells. This process is called ‘clonal selection’.

B and T cells are resting cells that lack functional activity until they undergo

activation, proliferation, and differentiation into plasma cells or activated T cells. This process takes several days, which explains the lag between being exposed to an infectious agent and eventually getting better when the immune response ‘kicks in’.

Of the millions of different specificities of B and T cells produced, only a few will have surface Ig or TCRs that bind the antigen with high affinity. However, we produce B and T cells that will react with virtually any antigen, including those that are man-made and are not found in nature (e.g. di-nitrophenol).

In nearly all cases, activation of a B or T cell requires two signals: binding of the antigen-specific receptor to the antigen, and exposure to proteins called cytokines expressed by helper T cells.

The blast cells resulting from activation undergo proliferation, resulting in a ~100-fold expansion of the number of cells reactive to the antigen.

Some of these cells become effector cells (plasma cells and activated T cells that express activities that help to eliminate the pathogen. Others become memory cells that can give rise to secondary responses as described below.

Coico and Sunshine, 2009. Fig. 1.1.

…(106-108 clones)

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6. The adaptive immune system has memory, meaning that the response against an antigen is much greater after the first exposure.

The first response to an antigen is called the primary response, and responses

thereafter are called secondary responses. The different properties of secondary responses are due to memory cells generated

during the primary responses. Secondary responses have

o Higher antibody levels o Increased proportion of IgG and other immunoglobulin isotypes o Shorter lag period o Higher affinity for antigen

Vaccination is effective because it primes the immune system to provide secondary responses when the individual is exposed to an infectious agent.

Each exposure to an antigen tends to increase the secondary response. This is why booster immunizations are often used in vaccinations.

Coico and Sunshine, 2009. Fig. 4.12.

Figure: Primary and secondary antibody responses. The adaptive immune system has memory, allowing for maturation of a rapid secondary immune response with higher specificity and magnitude directed against foreign substances.

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7. The immune system is tightly regulated. Self-reactive B and T cell clones are generated as a natural part of the random VDJ

recombination process. The adaptive immune system has developed several mechanisms to eliminate or inhibit

self-reactive B and T cells. o Elimination of self-reactive cells during their development through apoptosis. o Permanent inactivation of self-reactive cells through a process called clonal

anergy. o Inhibition of self-reactive cells by suppressor cells, inhibitory cytokines, and

other factors Each immune response requires a combination of multiple factors, thereby limiting the

number of spurious responses.

SUMMARY – MEDICAL IMPORTANCE OF THE IMMUNE SYSTEM AND HOW THE IMMUNE SYSTEM WORKS

Thus it can be said that the healthy immune system occurs as a result of balance – between innate and adaptive immunity, cellular and humoral immunity, inflammatory and regulatory networks and even cytokine modulators. Disease occurs when the balance is altered either by deficiency or dysfunction. Current and future research efforts center about defining exact hypersensitivity and/or immunodeficiency mechanisms in specific diseases, developing diagnostic assays that have individual patient relevance and finding more specific agents that can regulate or eliminate aberrant immune responses while leaving the rest of the system intact. Research opportunities abound in the broadening area of clinical immunology. SUMMARY

The immune response is designed to interact with the environment to protect the host against pathogenic invaders.

Immune-based diseases are either because of a lack of specific elements (immune deficiency) or excess activity (hypersensitivity).

Hypersensitivity Chart: Know the differences between types of

hypersensitivity.

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1. The chief function of the immune system is to distinguish between self and non-self.

2. The immune system consists of two overlapping compartments: the innate immune system and the adaptive immune system.

3. The antigenic specificity of the adaptive immune system is due to antigen-specific receptors.

4. The generation of antigen-binding diversity occurs prior to antigen exposure through a DNA rearrangement process called VDJ joining.

5. To generate an active immune response against a certain antigen, a small number of B and T cell clones that bind to the antigen with high affinity undergo activation, proliferation, and differentiation into plasma cells (for B cells) or activated T cells. This process is called ‘clonal selection’.

6. The adaptive immune system has memory, meaning that the response against an antigen is much greater after the first exposure.

7. The immune system is tightly regulated.

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CELLS AND ORGANS OF THE IMMUNE SYSTEM Jeffrey K. Actor, Ph.D.

713-500-5344 Objectives: (1) Identify cell types involved in specific and non-specific immune responses. (2) Present the developmental pathway of immune system cells. (3) Understand structure and function of primary and secondary lymphoid organs. Keywords: Reticuloendothelial System, Leukocytes, Myeloid Cells, Lymphocytes, Antigen Presenting Cells (APC), GALT, MALT, BALT, Cluster of Differentiation (CD). Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York, NY. 6th edition, 2009. Chapter 2; Geha and Notarangelo. Case Studies in Immunology. Garland Publishing, New York, NY. 6th edition, 2012. Case 30: Congenital Asplenia. Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/cells-and-organs-of-the-immune-sytstem/ Immune system cells are derived from pluripotent hematopoietic stem cells in the bone marrow. These cells can be functionally divided into groups that are involved in two major categories of immune responses: innate (natural) and acquired. Innate immunity is present from birth and consists of non-specific components. Acquired immunity by definition requires recognition specificity to foreign (non-self) substances. The major properties of the acquired immune response are specificity, memory, adaptiveness, and discrimination between self and non-self.

The acquired immune response is subdivided into humoral and cellular immunity, based on participation of two major cell types. In Humoral Immunity, B lymphocytes synthesize and secrete antibodies. Cellular Immunity (CMI) involves effector T lymphocytes which secrete immunoregulatory factors following interaction with antigen presenting cells (APCs). Figure. The developmental pathway of pluripotent bone marrow stem cells. Coico and Sunshine, 2009. Fig. 2.1.

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Figure. The interrelationship between innate and acquired immunity. An intricate communication system allows components of innate and acquired immunity to work in concert to combat infectious disease. Coico and Sunshine, 2009. Fig. 2.12.

Cluster of Differentiation (CD): Cell surface molecules are identifiable by monoclonal antibodies. In humans, these molecules have been given number designations. The acronym CD describes the cluster of antigens with which the antibody reacts; the number describes the order in which it was discovered. As of 2010, the list of determinants officially identified 350 individual and unique markers (link to Human CD Molecules). Surface expression of a particular CD molecule may not be specific for just one cell or even a cell lineage. However, many are useful for characterization of cells. CD-specific monoclonal antibodies have been useful for 1) determining the functions of CD proteins; 2) identifying the distribution of CD proteins in different cell populations in normal individuals; 3) measuring changes in the proportion of cells carrying these markers in patients with disease (e.g. decrease in CD4+ T cells is a hallmark of HIV infection); 4) developing therapeutic measures for increasing or decreasing the numbers or activities of certain cell populations.

Figure. Nomenclature of Inflammatory Cells.

Reticuloendothelial System Cells of the RES provide natural immunity against microorganisms by 1) a coupled process of phagocytosis and intracellular killing, 2) recruiting other inflammatory cells through the production of cytokines, and 3) presenting peptide antigens to lymphocytes for the production of antigen-specific immunity. The RES consists of 1) circulating monocytes; 2) resident macrophages in the liver, spleen, lymph nodes, thymus, submucosal tissues of the respiratory and

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alimentary tracts, bone marrow, and connective tissues; and 3) macrophage-like cells including dendritic cells in lymph nodes, Langerhans cells in skin, and glial cells in the central nervous system. Leukocytes Leukocytes provide either innate or specific adaptive immunity. These cells are derived from myeloid or lymphoid lineage. Myeloid cells include highly phagocytic, motile neutrophils, monocytes, and macrophages that provide a first line of defense against most pathogens. The other myeloid cells, including eosinophils, basophils, and their tissue counterparts, mast cells, are involved in defense against parasites and in the genesis of allergic reactions. Cells from the lymphoid lineage are responsible for humoral or cell mediated immunity. Myeloid Cells Neutrophils: Neutrophils are the most highly adherent, motile, phagocytic leukocytes and are the first cells recruited to acute inflammatory sites. They ingest, kill, and digest pathogens, with their functions dependent upon special proteins, such as adherence molecules, or via biochemical pathways (respiratory burst). Eosinophils: Eosinophils defend against parasites and participate in hypersensitivity reactions via cytotoxicity. Their cytotoxicity is mediated by large cytoplasmic granules, which contain eosinophilic basic and cationic proteins. Basophils/Mast cells: Basophils, and their tissue counterpart mast cells, produce cytokines that help defend against parasites, and also cause allergic inflammation. These cells display high affinity surface membrane receptors for IgE antibodies, and have many cytoplasmic granules containing heparin and histamine. The cells degranulate when cell-bound IgE antibodies are cross-linked by antigens, and produce low-molecular weight vasoactive mediators (e.g. histamine). Monocytes/Macrophages: Monocytes and macrophages are involved in phagocytosis and intracellular killing of microorganisms. Macrophages are differentiated monocytes, which are one of the principal cells found to reside for long periods in the RES. These monocytes/macrophages are highly adherent, motile and phagocytic; they marshal and regulate other cells of the immune system, such as T lymphocytes; they serve as antigen processing-presenting cells. Dendritic Cells: Dendritic cells provide a link between innate and adaptive immunity by interacting with T cells in a manner to deliver strong signals for development of memory responses. Dendritic cells recognize foreign agents and pathogens through a series of pattern recognition receptors (non-specific), and are able to present antigen to both T helper and T cytotoxic cells to allow those lymphocytes to mature towards functionality. Lymphoid Cells Lymphoid cells provide efficient, specific and long-lasting immunity against microbes/pathogens and are responsible for acquired immunity. Lymphocytes differentiate into three separate lines: (1) thymic-dependent cells or T lymphocytes that operate in cellular and humoral immunity; (2) B lymphocytes that differentiate into plasma cells to secrete antibodies; and (3) natural killer (NK)

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cells. T and B lymphocytes produce and express specific receptors for antigens while NK cells do not. B Lymphocytes: B lymphocytes differentiate into plasma cells to secrete antibodies. The genesis of mature B cells from pre-B cells is antigen-independent. The activation of B cells into antibody producing/secreting cells (plasma cells) is antigen-dependent. Mature B cells can have 1-1.5 x 105 receptors for antigen embedded within their plasma membrane. Once specific antigen binds to surface Ig molecule, the B cells differentiate into plasma cells that produce and secrete antibodies of the same antigen-binding specificity. If B cells also interact with T helper cells, they proliferate and switch the isotype (class) of immunoglobulin that is produced, while retaining the same antigen-binding specificity. T helper cells are thought to be required for switching from IgM to IgG, IgA, or IgE isotypes. In addition to antibody formation, B cells also process and present protein antigens. T Lymphocytes: T lymphocytes are involved in the regulation of the immune response and in cell mediated immunity, and help B cells to produce antibody. Mature T cells express antigen-specific T cell receptors (TCR). Every mature T cell also expresses the CD3 molecule, which is associated with the TCR. In addition mature T cells usually display one of two accessory molecules, CD4 or CD8, which define whether a T cell will be a helper T lymphocyte, or a cytotoxic T lymphocyte (CTL). The TCR/CD3 complex recognizes antigens associated with the major histocompatibility complex (MHC) molecules on target cells (e.g. virus-infected cell). Development of T lymphocytes During differentiation in the thymus, immature T cells undergo rearrangement of their TCR and genes to generate a diverse set of clonotypic TCRs. Immature thymocytes are selected for further maturation only if their TCRs do not interact with self-peptides presented in the context of self-major histocompatibility complex (MHC) molecules on antigen presenting cells. T Helper Cells: T helper cells (Th) are the primary regulators of T cell- and B cell-mediated responses. They 1) aid antigen-stimulated subsets of B lymphocytes to proliferate and differentiate toward antibody-producing cells; 2) express the CD4 molecule; 3) recognize foreign antigen complexed with MHC class II molecules on B cells, macrophages or other antigen-presenting cells; and 4) aid effector T lymphocytes in cell-mediated immunity. Currently, it is believed that there are two main functional subsets of Th cells, plus multiple other helper subsets of importance. T helper 1 (Th1) cells aid in the regulation of cellular immunity, and T helper 2 (Th2) cells aid B cells to produce certain classes of antibodies (e.g., IgA and IgE). The functions of these subsets of Th cells depend upon the specific types of cytokines that are generated, for example interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by Th1 cells; IL-4, IL-6 and IL-10 by Th2 cells. Two other classes of T helper cells are thought to be involved in oral tolerance and serve as regulators for immune function. Th17 cells, characterized by IL-17 secretion, are thought to be involved as effector cells for autoimmune disease progression, and protect surfaces (skin, gut) from extracellular bacteria. Tfh cells (follicular helper T cells) also provide help to B cells enabling them to develop into antibody-secreting plasma cells. They function inside of follicular areas of lymph nodes. Finally, although no longer prevalent in the literature, a subclass called Th3 cells were historically identified as secreting IL-4 and TGF- to provide help for IgA production; they were thought to be suppressive for Th1 and Th2 cells.

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T Cytotoxic Cells: T cytotoxic cells (CTLs) are cytotoxic against tumor cells and host cells infected with intracellular pathogens. These cells 1) usually express CD8, and, 2) destroy infected cells in an antigen-specific manner that is dependent upon the expression of MHC class I molecules on antigen presenting cells. T Suppressor/ T Regulatory Cells: T suppressor cells suppress the T and B cell responses and express CD8 molecules. T regulatory cells (Tregs) also affect T cell response, with many cells characterized as CD4+CD25+, TGF- secretors. Tregs regulate/suppress other T cell activities, and help prevent development of autoimmunity. Natural Killer T Cells: Natural killer T cells (NKT) are a heterogeneous group of T cells that share properties of both T cells and natural killer (NK) cells. These cells were identified as T cells that recognize an antigen-presenting molecule (CD1d) able to bind self- and foreign lipids and glycolipids. They constitute only 0.2% of all peripheral blood T cells. The term “NK T cells” was first used in mice to define a subset of T cells that expressed the natural killer (NK) cell-associated marker NK1.1 (CD161). It is now generally accepted that the term “NKT cells” primarily refer to CD1d-restricted T cells co-expressing a heavily biased, semi-invariant T cell receptor (TCR) and NK cell markers. Natural killer T (NKT) cells should not be confused with natural killer (NK) cells. Natural Killer Cells: NK cells are large granular “innate” lymphocytes that nonspecifically kill certain types of tumor cells and virus-infected cells. NK cells share many surface molecules with T lymphocytes. These circulating large granular lymphocytes are able to kill “self” in the absence of antigen-specific receptors. NK cells are especially effective against viral infected cells, and keep the expansion of virus in check until adaptive immunity kicks in. In this regard, they also secrete interferon-gamma, which is an effective immunoregulator. NK cells can also kill via antibody-dependent cellular cytotoxic mechanisms (ADCC) via their Fc receptors. NK cells

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express a large number of receptors that deliver either activating or inhibitory signals, and the relative balance of these signals controls NK cell activity. Antigen Presenting Cells (APCs) are found primarily in the skin, lymph nodes, spleen and thymus. They may also be present throughout the diffuse lymphoid system. Their main role is to present antigens to antigen-sensitive lymphoid cells. APCs may be characterized by their ability to phagocytose antigens, location in body, and expression of Major Histocompatibility Complex (MHC) related molecules. Two main types of APCs are Dendritic Cells and Macrophages. Of note, B cells are a special class of APCs; because they have antigen-specific antibody receptors they are enabled to internalize and process targeted antigens.

Note: Immature Dendritic Cells can give rise to multiple types of “effector”' Dendritic Cell phenotypes, each of which uniquely instruct distinct T-cell fates (e.g. specific adaptive immune function, regulation, and tolerance).

Lymphoid Organs The lymphatic organs are tissues in which lymphocytes mature, differentiate and proliferate. Lymphoid organs are comprised of epithelial and stromal cells arranged either into discretely capsulated organs or accumulations of diffuse lymphoid tissue. The primary (central) lymphoid organs are the major sites of lymphopoiesis, where B and T lymphocytes differentiate from stem cells into mature antigen recognizing cells. The secondary lymphoid organs, therefore, are those tissues in which antigen-driven proliferation and differentiation take place. Historically, the primary lymphoid organ was first discovered in birds, in which B cells undergo maturation in the bursa of Fabricius, an organ situated near the cloaca. Humans do not have a cloaca, nor do they possess a bursa of Fabricius. In embryonic life, B cells mature and differentiate from hematopoietic stem cells in the fetal liver. After birth, B cells differentiate in the bone marrow. Maturation of T cells occurs in a different manner. Progenitor cells from the bone marrow migrate to the thymus where they differentiate into T lymphocytes. The T lymphocytes continue to differentiate after leaving the thymus, and are driven to do so by encounter with specific antigen in the secondary lymphoid organs.

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Primary Lymphoid Organs Thymus Gland: The lymphoid organ in which T lymphocytes are educated, mature and multiply. It is a lymphoepithelial organ composed of stroma (thymic epithelium) and lymphocytes, almost entirely of the T-cell lineage. This is where T lymphocytes learn to recognize self antigens as self, and where these cells differentiate and express specific receptors for antigen. Only 5-10% of maturing lymphocytes survive and leave the thymus. Fetal Liver and Adult Bone Marrow: Islands of hematopoietic cells in the fetal liver and in the adult bone marrow give rise directly to B lymphocytes. Secondary Lymphoid Organs

The spleen and lymph nodes are the major secondary lymphoid organs. Additional secondary lymphoid organs include the tonsils, appendix, and Peyer’s patches. Aggregates of cells in the lamina propria of the digestive tract lining may also be included in this category, as well as any tissue described as MALT (mucosa-associated lymphoid tissue), GALT (gut-associated lymphoid tissue) or BALT bronchus-associated lymphoid tissue). Last but not least, the bone marrow can serve as an important secondary lymphoid organ. In addition to being a site of B cell generation, the bone marrow contains many mature T cells and plasma cells. Figure. Distribution of lymphoid tissues in the body. Actor, Elsevier’s Integrated Immunology and Microbiology. 2012.

Lymph Node: Lymph nodes form part of the network which filters antigen from tissue fluid or lymph during its passage from the periphery to the thoracic duct. Histologically, the lymph node is composed of a B cell cortex containing primary and secondary follicles, a T cell paracortex, and a central medulla which contains cords of lymphoid tissue. Spleen: The spleen is a filter for blood, and is actively involved in the removal of dying and dead erythrocytes. There are two main types of tissue; red pulp and white pulp. The white pulp contains the lymphoid tissue, arranged around a central arteriole as a periarteriolar lymphoid sheath (PALS). The PALS is composed of T and B cell areas, and contains germinal centers. Dendritic reticular cells and phagocytic macrophages can be found in germinal centers where they work to present antigen to lymphocytes.

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Review your histology chapters dealing with Hematopoiesis and the Immune System! Table. Myeloid Leukocytes and Their Properties

Phenotype Morphology Circulating Differential Count* Effector Function

Neutrophil PMN

granulocyte 2-7.5x109/L Phagocytosis and digestion of microbes

Eosinophil PMN

granulocyte 0.04-0.44x109/L Immediate hypersensitivity (allergic)

reactions; defense against helminths

Basophil PMN

granulocyte 0-0.1x109/L Immediate hypersensitivity (allergic)

reactions

Mast Cell PMN

granulocyte Tissue Specific Immediate hypersensitivity (allergic)

reactions Monocytes monocytic 0.2-0.8x109/L Circulating macrophage precursor Macrophage monocytic Tissue Specific

Phagocytosis and digestion of microbes; antigen presentation to T cells

Dendritic Cell monocytic Tissue Specific

Antigen presentation to naïve T cells; initiation of adaptive responses

* Normal range for 95% of population, +/- 2 standard deviations

Table. Lymphoid Leukocytes and Their Properties

Total Lymphocytes 1.3-3.5x109/L Effector Function B Cell monocytic Adaptive Humoral immunity

Plasma Cell monocytic Adaptive Terminally differentiated, antibody

secreting B cell T Cell monocytic Adaptive Cell-mediated immunity Natural Killer T Cell (NKT) monocytic (rare) Adaptive Cell-mediated immunity (lipids)

Natural Killer Cell (NK) monocytic Innate Innate response to microbial or infection

Clinical Vignette - Congenital Asplenia (Case 30 in Geha and Notarangelo): Mr. and Mrs. Vanderveer had five children. Their 10 month old daughter developed a cold, followed by upper respiratory infection. The child became feverish, convulsive and died; the causative agent was Haemophilus influenza which was isolated from the throat and cerebrospinal fluid. At autopsy she was found to have no spleen. How does the lack of a spleen affect B cell function, and what implications does this have towards immune responses to infective agents? In adults? In children?

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Summary: Cells of the Immune System Immune system cells are derived from pluripotent hematopoietic stem cells. Immune responses by these cells are divided into innate (natural) and acquired categories. Acquired immunity requires recognition specificity to foreign antigens, and is subdivided, based on participation B lymphocytes (humoral) and T lymphocytes (CMI). Surface molecules on human cells may be defined according to designation of Cluster of Differentiation (CD) antigens, which are useful for identifying different cell populations. Cells of the RES provide natural immunity against microorganisms via phagocytosis and intracellular killing, recruitment of other inflammatory cells, and presentation of antigens. Leukocytes provide innate or specific adaptive immunity, and are derived from myeloid or lymphoid lineage. Myeloid cells include highly phagocytic, motile neutrophils, monocytes, and macrophages that provide a first line of defense against most pathogens. The other myeloid cells, including eosinophils, basophils, and their tissue counterparts, mast cells, are involved in defense against parasites and in the genesis of allergic reactions. Cells from the lymphoid lineage are responsible for humoral or cell mediated immunity. The major properties of the acquired immune response are specificity, memory, adaptiveness, and discrimination between self and non-self. Lymphoid cells in these categories include T and B lymphocytes and NK cells. T and B cells produce and express specific receptors for antigens while NK cells do not. Receptor specificity is related to gene rearrangement of variable region components during development, according to essential features for clonal selection. B lymphocytes secrete antibodies; their activation is antigen-dependent following which they differentiate into plasma cells. Upon interaction with T helper cells, they proliferate and switch the isotype (class) of immunoglobulin produced, while retaining the same antigen-binding specificity. B cells also process and present protein antigens; they have specific surface antigens (CD molecules) necessary for response to foreign antigens. T lymphocytes are involved in regulation of immune response and in cell mediated immunity. During thymic differentiation, immature T cells undergo rearrangement of their TCR genes to generate a diverse set of clonotypic TCRs. Immature thymocytes are selected for further maturation only if they recognize foreign antigens in the context of "self" molecules. Mature T cells usually display one of two accessory molecules. CD4+ T helper cells are the primary regulators of T cell- and B cell-mediated responses, and are further subdivided into subsets dependent upon cytokines secreted. CD8+ T cytotoxic cells (CTLs) are cytotoxic against tumor cells and host cells infected with intracellular pathogens. T suppressor cells suppress the T and B cell responses and express CD8 molecules. T regulatory cells (Treg) are helper cells that suppress other T cell activity and help prevent autoimmunity. Natural Killer cells (NK) are large granular lymphocytes that nonspecifically kill certain types of tumor cells and virus-infected cells. The NK cells are able to kill “self” in the absence of antigen-specific receptors. They kill via antibody-dependent cellular cytotoxic mechanisms (ADCC) via their Fc receptors.

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INNATE IMMUNITY and INFLAMMATION Jeffrey K. Actor, Ph.D.

713-500-5344 Objectives: (1) Introduce innate immune defense mechanisms. (2) Define chemical mediators involved in inflammation. (3) Review cell types involved in innate immune responses, and their role in inflammation. (4) Define ADCC, chemokines, and Pattern-recognition receptors. Keywords: Innate Immunity, Innate Defense Barriers, Neutrophils, Monocytes, Macrophages, Natural Killer (NK) cells, Phagocytosis, APC, ADCC, Chemokines, Complement, PRRs, Inflammasome. Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York, NY. 6th edition, 2009. Chapters 2, 10 and 11; Geha and Notarangelo. Case Studies in Immunology. Garland Publishing, New York, NY. 6th edition, 2012. Case 15. Chediak-Higashi Syndrome; Case 25. Neutropenia; Case 26. Chronic Granulomatous Disease; Case 27. Leukocyte Adhesion Deficiency. Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/innate-immunity/ Innate immune mechanisms provide the first line of defense from infectious disease. The innate immune system is comprised of components which are present prior to the onset of infection and constitute a set of mechanisms that are not specific for a particular organism. Rather, the innate components recognize classes of molecules frequently encountered on invading pathogens, so as to allow defensive measures while the specific immune response is either generated or upregulated. Innate immune components are present from birth and consist of non-specific components. The innate defensive barriers can be divided into four major categories:

1. Anatomic - skin, mucous membranes 2. Physiologic - temperature, low pH, chemical mediators 3. Phagocytic and Endocytic - phagocytose to kill and digest microorganisms 4. Inflammatory - induction of vascular fluid leakage to area of tissue damage

Anatomic Barrier. The skin and mucous membranes provide an effective barrier against microorganisms. The skin has the thin outer epidermis and the thicker underlying dermis to impede entry, as well as sebaceous glands to produce sebum. Sebum is made of lactic acid and fatty acids, which effectively reduce skin pH to between 3 and 5 to inhibit organism growth. Mucous membranes are covered by cilia which trap organisms in mucous and propel them out of the body. Physiologic Barrier. The physiologic barrier includes factors such as temperature, low pH, and chemical mediators. Many organisms can not survive or multiply in elevated body temperature. Soluble proteins such as lysozymes, interferons and complement components play a major role in innate immunity. Lysozmes can interact with bacterial cell walls; interferons alpha and beta are natural inhibitors of viral growth; complement components use both specific and non-specific immune components to convert inactive forms to active components that damage membranes of pathogens. Low pH in the stomach discourages growth.

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Phagocytic and Endocytic Barriers. Blood monocytes, tissue macrophages and neutrophils phagocytose and kill microorganisms via multiple complex digestion mechanisms. Bacteria become attached to cell membranes and are ingested into phagocytic vesicles. Phagosomes fuse with lysosomes where lysosomal enzymes digest captured organisms. Inflammatory Barriers. Invading organisms cause localized tissue damage leading to complex inflammatory responses. In 1600 BCE, Celsus described the four cardinal signs of inflammation as rubor (redness), tumor (swelling), calor (heat), and dolor (pain). Later, Galen (2nd century) a fifth sign was added; functio laesa (loss of function). Inflammatory responses lead to (1) Vasodilation causing erythema (redness) and increased temperature; (2) increased capillary permeability which allows exudates (fluid) to accumulate leading to tissue swelling (edema); and (3) influx of cells to site of tissue damage. Once cells enter area of damage, they release further chemotactic factors to call in additional cells to damaged area, leading to Chemotaxis, Activation, Margination, Diapedesis (extravasation), and finally recognition and attachment of these cells to the damaged site. - - - - - - - - - - - - - - - - - - - - - - - - - - Chemical Mediators of Inflammation

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Hageman factor: Plasma globulin (110 kD), blood clotting factor XII, which is activated

by contact with surfaces to form Factor XIIa, that in turn activates factor XI. Factor XIIa also generates plasmin from plasminogen and kallikrein from prekallikrein. Both plasmin and kallikrein activate the complement cascade. Hagemann factor is important both in clotting and activation of the inflammatory process.

Thrombin: Protease (34 kD) generated in blood clotting that acts on fibrinogen to produce

fibrin. Consists of two chains, A and B, linked by a disulphide bond. Thrombin is produced from prothrombin by the action either of the extrinsic system (tissue factor + phospholipid) or, more importantly, the intrinsic system (contact of blood with a foreign surface or connective tissue). Both extrinsic and intrinsic systems activate plasma factor X to form factor Xa which then, in conjunction with phospholipid (tissue derived or platelet factor 3) and factor V, catalyses the conversion.

Kallikrein: Plasma serine proteases normally present as inactive prekallikreins which are

activated by Hageman factor. Act on kininogens to produce kinins, to mediate vascular reactions and pain.

Plasmin: Trypsin like serine protease that is responsible for digesting fibrin in blood clots.

Generated from plasminogen by the action of another protease, plasminogen activator. The enzyme catalyses the hydrolysis of peptide bonds at the carbonyl end of lysine or arginine residues. It also acts on activated Hageman factor and on complement.

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Bradykinin: Vasoactive nonapeptide (RPPGFSPFR) formed by action of proteases on kininogens. Very similar to kallidin (which has the same sequence but with an additional N terminal lysine). Bradykinin is a very potent vasodilator and increases permeability of post capillary venules, it acts on endothelial cells to activate phospholipase A2. It is also spasmogenic for some smooth muscle and will cause pain.

Arachidonic Acid Metabolites: Inflammatory Role

Cell Types Involved in Innate Immunity The cell types involved in innate immune responses include the polymorphonuclear cells (neutrophils), monocytes and macrophages, eosinophils, and Natural Killer (NK) cells. Some of these cells are capable of killing target cells via nonspecific (non-MHC dependent) through release of lytic enzymes, perforin or TNF. Others are involved in phagocytic mechanisms that kill via intracellular processes. Neutrophils. Neutrophils are typically the first infiltrating cell type to site of inflammation. Endothelial cells increase expression of E-selectin and P-selectin which are recognized by neutrophil surface mucins (PSGL-1 or sialyl Lewisx). Chemoattractants (IL-8) trigger adhesion and subsequent diapedesis. Multiple complement components (e.g. C5a) are chemotactic for neutrophils, along with fibrinopeptides and leukotrienes. Activated neutrophils express high Fc receptors and complement receptors to allow increased phagocytosis of invading organisms. Activation of neutrophils leads to respiratory burst producing reactive oxygen and nitrogen intermediates, as well as release of primary and secondary granules containing proteases,

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phospholipases, elastases and collagenases, and lactoferrin. Pus, a yellowish white opaque creamy matter produced by the process of suppuration consists of innumerable neutrophils (some dead and dying) and tissue debris.

Figure. Cell membrane adhesion molecules and cytokine activation events associated with neutrophil transendothelial migration. Left: Weak binding of selectin ligands on the neutrophil to E-selectin on the endothelial cells. Middle: IL-1 and TNF- upregulation of E-selectin, which facilitates stronger binding. Right: The activation effects of IL-8 on neutrophils cause a conformational change in the integrins (e.g., LFA-1) to allow them to bind ICAM-1. Coico and Sunshine, 2009. Fig 11.4. Mononuclear Cells and Macrophages. Mediators such as MIP-1 and MIP-1 attract monocytes to the site of pathogenic infection. The monocytes express surface ligands which recognize ligands (VCAM-1) on endothelial cells, leading to diapedesis. Activated tissue macrophages secrete IL-1, IL-6 and TNF- which further increase expression of adhesion molecules on endothelial cells to recruit neutrophils and more monocytes. These molecules also increase release of acute-phase proteins from the liver to assist in events leading to body temperature increase. Monocytes and macrophages ingest and destroy bacteria. Multiple factors assist in preparing the particulate for engulfment and targeting for destruction, including various opsonins comprised of complement components. Phagocytes bear several different receptors that recognize microbial components and induce phagocytosis. Five such receptors on macrophages are: CD14, Toll-like receptors (such as TLR-4), the macrophage mannose receptor, the scavenger receptor, and the glucan receptor. All 5 receptors bind bacterial carbohydrates. CD14 and CR3 are specific for bacterial lipopolysaccharide (LPS). In addition, complement receptors assist in this process. Figure. Endocytosis and phagocytosis by macrophages. Phagocytosed organisms are subjected to killing by lysosomal enzymes in phagolysosomes. Killing of phagocytosed microbes is sone via ROS and NO mediated mechanisms. These same substances can also be released to kill extracellular microbes.

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Figure. Important cytokines secreted by macrophages in response to bacteria and bacterial products include IL-1, IL-6, CXCL8 (IL-8), IL-12, and TNF-a. TNF-a is an inducer of a local inflammatory response that helps to contain infections. CXCL8 is also involved in the local inflammatory response, helping to attract neutrophils to the site of infection. IL-1, IL-6, and TNF-a have a critical role in inducing the acute-phase response in the liver and induce fever, which favors effective host defense in several ways. IL-12 may also activate natural killer (NK) cells. The Inflammasome: Assembly and activation of the inflammasome is an essential process in innate immune defense. The inflammasome is a cytosolic, multiprotein platform that allows activation of pro-inflammatory caspases that cleave the precursor of interleukin-1β (pro-IL-1β) into the active form. Secretion of active IL-1β helps to initiate a potent inflammatory response. Antigen Presentation. Phagocytosed or pinocytosed antigens may then be presented to the adaptive immune system cells. Conventional Dendritic Cells, macrophages and monocytes are specifically good at presenting antigens to T lymphocytes. In addition, B cells, are also extremely good APCs. Some of the critical molecules which play a role in antigenic presentation by APCs to T cells are given in the accompanying figure.

Figure from Immunology (6th ed). 2006. Goldsby, Kindt, Osborn and Kuby. WH Freeman Publisher.

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NK Cells. NK cells are large granular lymphocytes that nonspecifically kill certain types of tumor cells and virus-infected cells, and function as both cytolytic effectors and regulators of immune responses. NK cells express a large number of receptors that deliver either activating or inhibitory signals; the relative balance of these signals controls NK cell activity. NK cells are activated upon detection of abnormalities in target cells such as the loss of antigen presentation molecules (MHC class I expression) or up-regulation of stress-induced ligands. A variety of receptors trigger the NK cytolytic activity directed toward certain tumor targets, virally infected cells, and even normal immune system constituents such as immature dendritic cells. NK cells are also important regulators of the adaptive immune system via their ability to secrete a number of cytokines in response to immune activation.

Antibody-Dependent, Cell-Mediated Cytotoxicity (ADCC). ADCC is a phenomenon in which target cells coated with antibody are destroyed by specialized killer cells. Among the cells that mediate ADCC are NK cells, macrophages, monocytes, neutrophils and eosinophils. The killing cells express receptors for the Fc portion of antibody coated targets. Recognition of antibody coated target leads to release of lytic enzymes at the site of Fc mediated contact. In the case of NK cells and eosinophils, target cell killing may involve perforin-mediated membrane damage. Coico amd Sunshine, 2009. Fig.15.1 Clinical Relevance

Clinical Vignette – Case 15. Chediak-Higashi Syndrome: Chediak-Higashi syndrome is a rare inherited disorder in which a severe immunological deficiency has been linked to deficits in NK cell function and to deficiency in chemotactic and bactericidal function for neutrophils. Thus, these individuals are more susceptible to bacterial infections. These individuals have characteristic giant lysosomes within neutrophils. Bone marrow transplantation is the only effective therapy.

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Chemokines. Chemokines are specialized cytokines that are chemotactic for leukocytes. They are small polypeptides that are synthesized by a wide variety of cell types. They act through receptors that are members of the G-protein coupled signal transducing family. All chemokines are related in amino acid sequence and their receptors are integral membrane proteins that are characterized by containing seven membrane-spanning helices.

Chemokines fall mainly into two distinct groups. The CC chemokines have two adjacent cysteine residues (hence the name "CC"). The CXC chemokines have an amino acid between two cysteine residues. Each chemokine reacts with one or more receptors, and can affect multiple cell types. Chemokines and their functions will be covered again in greater depth in the Adaptive Immunity chapter.

Properties of selected chemokines.

Chemokine Major Cell Source Cell Type Attracted

CCL2 (MCP-1) Monocytes and Macrophages,

Fibroblasts Chemoattractant for monocytes

CCL3 (MIP-1) Monocytes, T cells, Fibroblasts,

Mast cells Chemoattractant for neutrophilic granulocytes

CCL5 (Rantes) T cells, Endothelium Chemoattractant for Eosinophils and Basophils, Monocytes and Dendritic cells, and T cells

CCL11 (Eotaxin) Monocytes and Macrophages,

Endothelium and Epithelium Chemoattractant for Eosinophils

CXCL8 (IL-8) Monocytes and Macrophages,

Fibroblasts, Endothelial cells Chemoattractant for Neutrophils

An expanded list of cytokines and chemokines is provided in the Appendix.

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Complement Components. The activation of complement is an important component of innate immunity. This will be discussed in detail in further lecture. A brief introduction to complement follows:

Activation of the complement system results in the production of several different polypeptide cleavage fragments that are involved in five primary biological functions of inflammation and immunity. 1. Direct Cytolysis of foreign organisms (e.g. bacteria): Antibodies recognizing pathogenic determinants form the basis of a physical structure to which complement components interact. Specifically, complement component C1 interacts with the Fc portion of IgM and IgG (except IgG4) binding to the surface of bacteria. The binding of C1 initiates a cascade of events whereby a membrane attack complex (MAC) is built upon the cellular surface. Synthesis of the MAC structure culminates in assembly of a pore channel in the lipid bilayer, causing osmotic lysis of the cell. MAC formation requires prior activation by either the classical or alternative pathways, and utilizes the proteins C5b, C6, C7, C8, and C9. 2. Opsonization of foreign organisms. Complement components (e.g. C3b or inactivated C3b; iC3b) bind to pathogens. Interaction with receptors (CR1, CR2, CR3, and CR4) on the surface of macrophages, monocytes, and neutrophils leads to enhanced phagocytosis and targeted destruction of organisms. 3. Activation and directed migration of leukocytes. Proteolytic degradation of C3 and C5 leads to production of leukocytes chemotactic anaphylatoxin. For example, C3a is chemotactic for eosinophils. C5a is a much more potent chemokine, attracting neutrophils, monocytes and macrophages, and eosinophils. Interaction of C3a, C4a or C5a with mast cells and basophils leads to release of histamine, serotonin, and other vasoactive amines, resulting in increased vascular permeability, causing inflammation and smooth muscle contraction.

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4. Solubilization and clearance of immune complexes. One of the major roles complement plays is the solubilization and clearance of immune complexes from the circulation. First, C3b and C4b can covalently bind to the Fc region of insoluble immune complexes, disrupting the lattice, and making them soluble. C3b and C4b bound to the immune complex are recognized by the CR1 receptor on erythrocytes facilitating their transport to the liver and spleen. In the liver and spleen the immune complexes are removed and phagocytosed by macrophage-like cells. The RBCs are returned to the circulation. 5. Enhancement of humoral immune response. Coating of antigens with C3d (a breakdown product of C3) facilitates their delivery to germinal centers rich in B and follicular dendritic cells.

Clinical Relevance

Clinical Vignette – Factor I Deficiency (Case 32, Geha and Notarangelo): The alternative pathway of complement activation is important in innate immunity. Deficiency in Factor I (as well as deficiency in Factor H) affects cleavage of C3b, and therefore leads to reduced C3bi. The nonproduction of iC3b results in defective opsonization, which is critical for removing and destroying invading bacteria.

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Receptors of the Innate Immune System. Receptors of the innate immune system recognize broad structural motifs that are highly conserved within microbial species [called Pathogen-Associated Molecular Patterns (PAMPs)]. Such receptors are referred to as Pattern-Recognition Receptors (PRRs). In a similar manner, Damage/Danger-Associated Molecular Pattern molecules (DAMPs) initiate immune activity as part of the noninfectious inflammatory response. Receptor engagement leads to triggering of signal pathways that promote inflammation.

Receptors of the Innate Immune System. [Table adapted from Immunology (2002) by Goldsby, Kindt, Osborne and Kuby - W.H.Freeman, et al., NY.] TLR = Toll-like Receptor.

FIGURE 2.6. (A) Pattern recognition receptors called Toll-like Receptors (TLRs) bind to molecules with specific pattern motifs expressed by various pathogens. (Coico, 2009)

Receptor (location) Target (source) Effect of Recognition

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Link between Innate and Adaptive (Acquired) Immunity Recognition of pathogen via Pattern Recognition Receptors (PRR) or Toll-Like Receptors

(TLR) leads to activation and maturation of APCs. APCs process antigen and present to naïve T cells. Specifically, dendritic cells form a major

bridge between cells of the innate and adaptive immune responses. Presentation is accompanied by secretion of cytokines to assist development of T cell

response (e.g. Th1 maturation via presence of IL-12). Absence of internal activation signals can leads to Th2 development (MyD88 regulated).

Figure 6-4. Link between innate and adaptive (acquired) immunity. Pathogen recognition through pattern recognition receptors is an important bridge between innate and adaptive immune function. Recognition leads to activation and maturation of the presenting cell. Here, dendritic cells are depicted as primary presenting cells, which assist in dictating subsequent responses. Processed antigen is presented to naive T cells, accompanied by secretion of cytokines to assist development and maturation of T-cell phenotypic response (e.g., T helper cell-1 maturation via presence of interleukin-12). Inset box shows important Toll-like receptors and specific ligands involved in pathogen recognition. At least 15 different Toll-like receptors have been identified. A more complete list of Toll-like receptors and ligands is provided in the Appendix. Final word:

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We will see in later lectures that lymphocyte responses can be divided by specificity and function. The lymphocytes are considered “adaptive” historically. Indeed, the classical B lymphocytes and T lymphocytes we discuss later in the course will be primarily of the adaptive phenotypic groups. However, there is a population of lymphocytes that respond with “innate-like” activity. These include: T-cells, invariant natural killer T-cells, and B-1 cells. All these cell types respond quickly (1-3 days) to a limited pool of antigens at sites of infection. These cells will be defined as we discuss them. Summary: Innate Immunity Immune system cells are derived from pluripotent hematopoietic stem cells. Immune responses of the innate immune system provide natural immunity against microorganisms via phagocytosis and intracellular killing, recruitment of other inflammatory cells, and presentation of antigens. Leukocytes that provide innate immunity are derived from myeloid lineage. These cells include highly phagocytic, motile neutrophils, monocytes and tissue macrophages, eosinophils, and Natural Killer (NK) cells. These cells provide a first line of defense against most pathogens. Innate defense barriers include (1) anatomic barriers, (2) physiologic barriers, (3) Phagocytic barriers, and (4) inflammatory barriers. Damage to tissue caused by invading pathogens can lead to rubor, tumor, calor, dolor, and functio laesa. Tissue damage leads to an influx of inflammatory cells through chemotaxis, activation, margination and diapedesis. The inflammatory process is initiated and controlled via multiple chemical mediators. Neutrophils are usually the first cell type to arrive at the site of tissue damage. Activation leads to respiratory bursts and release of granules to control bacterial growth. Mononuclear cells and macrophages engulf organisms via multiple mechanisms, leading to control and destruction within intracellular phagosomes. NK cells are large granular lymphocytes that kill targets via ADCC or through lysis using perforin-induced mechanisms. Chemokines and complement components are critical for activation of innate immune functions. Defects may lead to severe clinical complications. Pattern Recognition Receptors present on innate immune system cells assist in the recognition of bacteria and virions. Recognition by PRRs of PAMPs leads to activation of multiple facets of cellular response. In a similar manner, damage/danger associated DAMPS can function to elicit innate inflammatory functions in non-infectious situations.

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A general summary chart of innate components, effectors and function: Component Effectors General Function Anatomic and

Physiologic Barriers

Skin and Mucous Membranes Temperature, Acidic pH, Lactic acid Chemical Mediators

- Physical barriers to limit entry, spread and replication of pathogens

Inflammatory Mediators

Hageman factor Thrombin Kallikrein Bradykinin Leukotrienes and Prostaglandins Complement Cytokines and Interferons Lysozymes Acute Phase Proteins and Lactoferrin

- Clotting, activation of inflammation - Protease acting to produce fibrin - Mediating vascular reactions and pain - Vasoactive nonapeptide; spasmogenic

for some smooth muscle and will cause pain

- Vasodilation and increased vascular permeability

- Direct lysis of pathogen or infected cells - Activation/Mediation of other immune

components - Bacterial cell wall destruction - Mediation of response

Inflammatory Mediators

Complement Cytokines and Interferons Lysozymes Acute Phase Proteins and Lactoferrin Leukotrienes and Prostaglandins

- Direct lysis of pathogen or infected cells - Activation of other immune components - Bacterial cell wall destruction - Mediation of response - Vasodilation and increased vascular

permeability

Cellular Components

Polymorphonuclear Cells Neutrophils, Eosinophils Basophils, Mast Cells

Phagocytic-Endocytic Cells Monocytes and Macrophages Dendritic Cells (multiple

subsets) Other Cells

NK cells

- Phagocytosis and intracellular destruction of microorganisms

- Presentation of foreign antigen to

lymphocytes - ADCC

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IMMUNOGENS AND ANTIGENS

Sudhir Paul, PhD

OBJECTIVES To learn the molecular attributes and properties of compounds which render them immunogenic and antigenic. KEYWORDS Immunogen, antigen, hapten, epitope, adjuvant. READING

Chapter 3 of the Coico, et al textbook. Geha and Notalangelo, 2012. Case Studies in Immunology, 6th Ed., 46. Hemolytic Disease of the Newborn. Multiple Myeloma (On file on Blackboard/LRC). Web Resource: https://med.uth.edu/pathology/courses/immunology/immunology/links-for-lectures/immunogens-and-antigens/

ANTIGEN OR IMMUNOGEN?

IMMUNOGEN - Agent capable of binding immune receptors AND inducing an immune response by B cells and T cells

ANTIGENS - Agent that binds with varying degrees of specificity to immune receptors (antibodies on B cells; T cell receptor on T cells)

All immunogens are antigens, but not all antigens are immunogens. IMPORTANCE OF IMMUNOGENICITY

Germfree colostrum-deprived piglets are immunologically "virgin" and extremely susceptible to microbial infection due to lack of passive maternal immunity. They are, however, highly immunologically competent as determined by their excellent immune response to various immunogens. An immunogen is the inducer of specific antibody formation. The initial step in the primary immune response is priming of multipotent uncommitted cells ("virgin" X cells) to committed monopotent cells (Y cells). Y cells proliferate and differentiate into antibody-forming cells (Z cells). Adapted from Y.-B. Kim 1975

Vaccines are the cornerstone of eradicating microbial disease – many available vaccines. See slide.

New vaccines are needed for emerging diseases. See slide. EPITOPES RECOGNIZED BY T OR B CELLS Epitopes are the three dimensional arrangements of atoms (sites) on the surface of an antigen that bind to the paratope of an antibody OR the linear peptides that bind the MHC molecules/T cell receptor. Epitopes recognized by B cells generally differ from those recognized by T cells.

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B cells can mount specific antibody responses without or with help from T cells (T-independent or T dependent cells).

PHYSICOCHEMICAL FORCES INVOLVED IN ANTIGEN-IMMUNE RECEPTOR BINDING

Antibody binding to antigen does not involve covalent chemical bonds. Instead, several weaker types of molecular interactions are utilized. Thus, the reactions are reversible. There are four kinds of forces that stabilize antigen-antibody interactions: 1. Electrostatic interactions. Usually due to the attraction between the charged

amino acid residues in proteins such as lysine, arginine, glutamic acid and aspartic acid, for instance. The number of such interactions will enhance the affinity of the interaction dramatically.

2. Hydrogen bonding. Electrostatic binding with covalent character. Example, -H atom shared by electronegative N and O atoms.

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3. Van der Waal’s forces. Attractive and repulsive forces between induced oscillating dipoles in the electron clouds of two adjacent atoms. The force is proportional to the 7th power of the distance separating two molecules. This is a weak force, but is additive, and there are many van der Waal’s contacts in antibody-antigen complexes.

4. Hydrophobic bonding. Usually involves non-polar amino acids, e.g., leucine, isoleucine.

MAJOR CLASSES OF ANTIGENS/IMMUNOGENS The following major chemical classes of compounds may be antigenic/immunogenic:

1. Proteins or glycoproteins. Most proteins or glycoproteins are excellent antigens. The greater the complexity and molecular weight, the better it is as an antigen.

2. Carbohydrates or polysaccharides. Bacterial capsules (i.e. pneumococci) are powerful antigens. The ABO blood group epitopes are carbohydrates.

3. Lipids. Are not routinely antigenic, but if used as a hapten, immune

responses can be elicited, i.e. sphingolipids. 4. Nucleic Acids. Are poorly immunogenic themselves, but as haptens are good

antigens. Antibodies to DNA are important in patients with systemic lupus erythematosus.

SEQUENTIAL AND CONFORMATIONAL EPITOPES Two general classes of epitopes can be distinguished. They are best described as they exist on protein antigens, but other classes of antigens (i.e. carbohydrates and nucleic acids) can also express antigenic/immunogenic epitopes under some circumstances.

1. Conformational (Non-Sequential) Epitopes Conformational epitopes require the native 3-dimensional configuration of the molecule to be intact for their expression. Denaturation of the molecule destroys these kinds of epitopes and antibodies specific for conformational epitopes will not bind denatured antigens. Conversely, denaturing the molecule prior to injection of the animal (cooking an egg) will alter the conformation of the molecule and the antibodies elicited that are specific for antigens on the denatured form will not react with the undenatured form of the molecule. 2. Sequential Epitopes Sequential epitopes are short stretches of amino acids (4-7 in length) which can be recognized MHC molecules in short peptides or by antibodies in short peptide

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regions within larger antigens. Thus, the only requirement is that the right sequence of amino acids is expressed.

EXAMPLE OF CLINICAL RELEVANCE

Two cases in Geha & Notarangelo, 6th edition, emphasize the influence of genetic factors in immunogenicity of infectious agents and immunogenicity of administered vaccines. Case 12—MHC Class I Deficiency—This case describes the consequences of a failure of antigen processing for protection from infectious agents. Tatiana Islayev, age 17, had been chronically ill since age 4. She had a history of repeated sinus, lung, and middle ear infections due to a variety of respiratory viruses. Her 7-year old brother Alexander had a similar history. The parents and 3 other children were healthy. Tatiana and Alexander had received oral polio vaccinations as well as DPT and BCG vaccinations and tolerated them well. WBC analysis showed a profound deficiency of CD8 T cells. Further studies showed that both Tatiana and Alexander had a nonsense mutation in their TAP-2 genes, a gene coding for a protein that transports peptide fragments into the lumen of the endoplasmic reticulum where it binds to MHC class I molecules and this complex is then transported to the surface of the cell to be recognized by a CD8 T cell. Case 8—MHC Class II Deficiency—This case illustrates a genetically acquired susceptibility to pyogenic and opportunistic infections. Helen Burns was the second child born to her parents. She had received routine polio and DPT vaccinations at 2 months of age. At 6 months of age she developed pneumonia in both lungs, accompanied by a severe cough and fever. Pneumocystis carinii was isolated from a tracheal aspirate and she was treated with pentamidine and seemed to recover fully. Since P. carinii was found (an opportunistic pathogen), severe combined immunodeficiency (SCID) was suspected. Her T cells were found incapable of responding to tetanus toxoid and her serum Ig concentrations were very low. Her CD4 T cells were very low but her CD8 cell count was normal (ruling out a diagnosis of SCID). While working up her sib and parents for possible bone marrow donation, it was found that Helen’s B cells did not express MHC Class II molecules. Her mother was selected as the best donor of bone marrow. The graft was successful and normal immune function was restored.

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REQUIREMENTS FOR IMMUNOGENICITY Four characteristics that contribute to the immunogenicity of a substance:

1. Size, dose, route Usually, compounds of less than 1,000 daltons are non-immunogenic. Compounds between 1,000 and 6,000 daltons may or may not be immunogenic. Those greater than 6,000 daltons are generally immunogenic. Intermediate dose is most immunogenic. Immunogenicity is also a function of route of administration. 2. Chemical Composition Physicochemical complexity is usually necessary for a compound to be immunogenic. Homopolymers of amino acids usually are not immunogenic (i.e. B. anthracis poly-gamma-D-glutamic acid, 50,000 daltons). 3. Foreignness Foreignness was once considered to be an absolute requirement for immunogenicity. It is now clear that certain self-components can be immunogenic to the individual. Foreignness is an excellent general guideline as to whether something might be immunogenic, but it is not a definitive requirement for immunogenicity. Particulate and denatured antigens are often more immunogenic. 4. Adjuvants/Degradability Adjuvants enhance immune responses by inducing cytokine release or antigen processing. T-dependent immunogens must be enzymatically degraded in order to be immunogenic. Peptides of D-amino acids are non-immunogenic whereas their L-isomers usually are immunogenic. Genes mapping to the Major Histocompatibility Complex (MHC) can profoundly affect the degree of immunogenicity of any substance.

HAPTENS Haptens are low molecular weight compounds that are non-immunogenic by themselves but become immunogenic after conjugation to high molecular weight carrier substances that are immunogenic. The figure below illustrates coupling non-immunogenic p-amino-arsonic acid to a carrier to make it immunogenic.

Clinical Relevance—The hapten concept has been adapted to modern vaccine technology with great success. There are several vaccines licensed that are based on covalently coupling isolated epitopes to carrier molecules, usually tetanus toxoid. Hapten-type vaccines for pneumococcus and for Haemophilus are currently available. Several others are in development. Such an approach provides a much safer type of vaccine compared to using whole immunogen molecules or killed viral preparations.

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MULTIVANT ANTIGENS Macromolecules usually have multiple unique or repeat epitopes. The former type of immunogens induce heterogenous immune responses (mixtures of antibodies or T cells directed to individual antigens). The latter type of immunogens are often T-independent. Both types of antigens can form large complexes with multiple antibodies, a phenomenon that can cause pathological immune complex deposition, particularly in the kidney. IMMUNOLOGIC SPECIFICITY AND CROSS-REACTIVITY The forces mediating antigen-antibody recognition allow for a high degree of specificity. That is, antibodies specific for one epitope or hapten can easily distinguish that epitope or hapten from other similar structures. However, this specificity is not absolute because antibodies specific for one epitope can bind with structurally similar, but non-identical epitopes although with a lower affinity. Specificity and cross-reactivity can be distinguished by inspecting the following table reporting antibody reactivity with various structurally defined carbohydrate epitopes:

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CROSS-REACTIVITY Cross reactivity refers to the situation where the cell receptor or antibody can react with two molecules because a) they share one or more identical epitopes or b) the epitope in question is similar enough in sequence or in shape to bind to the receptor with a weaker, yet functional, affinity. Examples: 1. Toxoids—Antibodies elicited with toxoids react with native toxins (Clinical Application—vaccination with tetanus toxoid and with diphtheria toxoid). 2. ABO Blood Group Antigens—Antibodies elicited by certain environmental carbohydrate antigens react with the human A or B blood group antigens. 3. There are 4 strains of the flavivirus that causes Dengue. The virus infects cells of the monocyte-macrophage lineage. Infection with one strain elicits antibodies reactive with a common epitope on all 4 strains. Upon infection with a different strain, the antibody to the common epitope reacts by cross-reaction and facilitates phagocytosis by macrophages, thus helping the virus gain entry and the second infection is typically much more severe than the first due to this cross-reactivity. 4. Yet another example of cross-reactivity is the ability of antibodies to bacterial antigens to attack host tissues, causing autoimmune disease.

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ADJUVANTS Definition: An adjuvant is a substance, which when mixed with an immunogen, enhances the immune response against the immunogen. The adjuvant itself is not usually immunogenic. Examples of Immunologic Adjuvants

1. Freund’s Complete Adjuvant. This is a mixture of a petroleum based oil, an emulsifying agent and killed Mycobacteria. A water-in-oil emulsion is formed with microdroplets of antigen solution surrounded by the oil. This works by slowly releasing antigen over a long period of time while inducing a delayed hypersensitivity reaction. It is used experimentally, but not in humans.

2. Lipopolysaccharide (LPS). Is experimental 3. Muramyldipeptide. Is experimental 4. Synthetic Polynucleotide (Poly AU). Is experimental 5. Aluminum Hydroxide (alum precipitate). Is used in humans.

Functions to enhance the ingestion and eventual processing of antigen. 6. Cytokines. Are experimental

Currently, adjuvant research is a high priority research area for enhancing the immunogenicity of the new genetically engineered vaccines. Aluminum hydroxide is currently the only FDA licensed adjuvant in the US. There are several new adjuvants in phase III clinical trials but the FDA has not yet licensed any of these. Some other adjuvants are licensed in other countries, but are not available in the US. Adjuvant MF59 is licensed in Europe, but not in the US. MF59 consists of stable droplets (<250 nm) of the metabolizable oil squalene and two surfactants, polyoxyethylene sorbitan monooleate and sorbitan trioleate, in an oil-in-water emulsion.

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SUMMARY 1. Immunogens elicit immune responses; antigens bind to antibodies, lymphocyte

receptors or MHC molecules. 2. The properties of foreignness, size, chemical complexity and degradability all

contribute to the degree of immunogenicity. 3. Haptens are low molecular weight substances that only become immunogenic upon

covalent coupling to an immunogenic molecule. Haptens are models for epitopes. 4. Major classes of antigens include carbohydrates, lipids, nucleic acids and proteins

or glycoproteins. 5. Linear epitopes require only the primary linear structure to be intact but

conformational epitopes require that the 3D integrity of the molecule is intact. 6. Antibody binding to antigen does not utilize covalent interactions—multiple weak

interactions stabilize the binding such as electrostatic interactions, hydrogen bonding, hydrophobic interactions and Van der Waal’s forces.

7. Immunologic specificity is dependent on stereochemical positioning or spatial arrangement of chemical groupings and on the chemical nature of the group (mass and charge).

8. Immunologic cross-reactions can be due to antigens sharing identical epitopes or having epitopes with similar but non-identical chemical groupings.

9. Adjuvants enhance the magnitude of the resulting antibody response when mixed with an immunogen before injection. Aluminum hydroxide (alum) is the only licensed adjuvant for human use in the USA. Several others are currently in phase III clinical trials and some other adjuvants (i.e. MF59) are licensed in foreign countries.

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IMMUNOLOGY Antibody Structure and Function

Dr. Keri C. Smith

OBJECTIVES Develop an understanding of how the structural and molecular features of antibody molecules mediate both the protective and the pathologic functions of the different classes of antibodies. KEYWORDS Immunoglobulin (Ig), isotype, allotype, idiotype, opsonin. READING Chapter 4 in the Coico et al textbook, 2009. Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/antibody-structure-and-function-iii/ INTRODUCTION The production of circulating antibodies is one of the major functions of the immune system. Antibodies belong to the general class of glycoproteins called globulins, due to their property of being insoluble in half-saturated ammonium sulfate solutions. Subsets of antibodies have been discovered and they are now known collectively as immunoglobulins, abbreviated Ig. Studies of the molecular structure of the various Ig have clarified many of the properties such as specificity, cellular reactivity, complement fixation, placental transfer and anaphylactic activity after mast cell activation. ISOLATION AND CHARACTERIZATION Ig are found in large quantities in blood serum. The bulk of Ig migrates in the gamma region when subjected to electrophoresis at pH 8.2. Tiselius and Kabat proved in 1939 that the gamma region contained most of the antibodies in an immunized animal:

46

The shape of the gamma peak, being rather broad, suggested very early that the antibody population was a heterogeneous collection of molecules with slightly different charges. This heterogeneity was a major early obstacle in attempts to determine the structure of antibody molecules and relate structure to function. This problem was partially solved by the discovery of myeloma proteins that are homogeneous Ig produced by cells in a type of cancer called multiple myeloma, a cancer of plasma cells. STRUCTURE OF LIGHT AND HEAVY CHAINS Enzymatic fragmentation and chemical reduction studies showed the basic 4-chain structure of the predominant Ig, originally called gamma globulin. Pepsin and papain fragments of Ig were used, along with studies of the reductive products to establish the 4-chain structure with each molecule consisting of 2 identical heavy chains and 2 identical light chains. Amino acid sequences then were used to further distinguish constant portions of the chains from variable portions. The domain concept, hinge region and carbohydrate locations were determined

.

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Light Chains Studies of Ig from many species showed that nearly all species studied had two types of Light chains, called κ and λ. The difference between the two types of light chains is in the amino acid sequence of the constant region domain. The overall ratio of the two Light chain types varies between species (mice have 95% of their Ig with κ chains whereas human Ig has 60% κ and 40% λ). Heavy Chains There are 5 different classes or ISOTYPES of heavy chains. Each class of Heavy chain has a characteristic amino acid sequence that distinguishes it from the other four classes but all five classes have significant percentages of amino acid sequence similarities. The five Heavy chain Ig classes are IgG, IgA, IgM, IgE and IgD. The different Heavy chains corresponding to their class are given Greek letter designations: γ, α, μ, ε and δ. In many species, there are two or more subclasses of some heavy chains that differ from one another by only a few amino acids. Humans have 4 subclasses of the IgG isotype called IgG

1, IgG

2, IgG

3, and

IgG4. IgA has two subclasses IgA

1 and IgA

2.

Domains Amino acid sequence studies of Ig shows that there is regularity to the structure in which there are disulfide-bridged loops of approximately 60 amino acids for each 100-110 amino acids. This is the case for both Heavy and Light chains. These are called domains. There are two domains on both κ and λ Light chains and either 4 or 5 domains on heavy chains. The amino acid sequences in the first domain on both Light and Heavy chains are highly variable from molecule to molecule, and are referred to as the V

L or V

H

domains, respectively. The other Light chain domain is constant in its amino acid sequence for the κ or λ type of chain and is referred to as the C

L domain.

The constant domains of Heavy chains are numbered from the amino terminal end toward the carboxy terminal end as C

H1, C

H2, C

H3 and (for IgM and IgE) C

H4.

The following shows some specific biological functions carried out by Ig domains. DOMAIN FUNCTIONS OF HUMAN IgG ------------------------------------------------------------------------------------------------------------- Domain(s) Function V

H + V

L Antigen Binding

CH1 + C

L Spacer between antigen-binding and effector functions

CH2 Binding C1q

Control of catabolic rate C

H3 Interaction with Fc receptor on macrophage/monocyte

CH1 + C

H3 Bind to Protein A

Interact with Fc Receptors on placental syncitiotrophoblast, neutrophils, and cytotoxic K-cells

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Hinge Region IgG, IgA and IgD genes each have an exon coding for a short span of amino acids that occupy the space between the C

H1 and C

H2 domains. This segment is rich in Cys and Pro

and permits significant flexibility between the two Fab arms of the antibody and the area is called the hinge region, accordingly. Since this stretch is open to solvent, it is highly susceptible to protease cleavage. Variable Region or Variable Domain Kabat and Wu developed their variability plot to try to predict which amino acids in the Variable regions actually contacted antigen. They found that the greatest variability in sequence between molecules occurred at 3 distinctive regions in Light and Heavy chains. These were then called hypervariable regions. They were separated by sequences called framework regions. It has been formally proven that the amino acids comprising the hypervariable regions are the contact residues for antigen. Since they form the region of structural complementarity for Ag epitopes, they are termed complementarity-determining regions (CDRs).

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In this plot the term VARIABILITY is defined as the ratio of the number of different amino acids at a given position to the frequency of the most common amino acid at that position. For example, in the original research at position 7, 63 different light chains had been sequenced and Serine occurred 41 times so its frequency was 41/63 or 0.65. In all, 4 amino acids were found at position 7. Thus the Variability value was 4/0.65 or 6.15. IMMUNOGLOBULIN VARIANTS Isotypes The five major classes of Ig (IgG, IgA, IgM, IgE, IgD) are isotypic variants of immunoglobulins or isotypes. Structural variations in the heavy chains distinguish the isotypes from one another. The structural elements that define one isotype are the same for each species. There are subclasses of IgG and IgA.

TABLE 4.2. Important Differences Between Human IgG Subclasses

IgG1 IgG2 IgG3 IgG4

Occurrence (% of total IgG) 70 20 7 3

Half-life 23 23 7 23

Complement binding + + +++ —

Placental passage ++ ± ++ ++

Binding of monocytes +++ + +++ ±

ALLOTYPES Definition: Ig allotypes are defined as structural variants of the constant regions of L or H chains of Ig that are coded by germ line genes. These differences are detected serologically. Specific antibodies are available for analyzing these differences from one individual to another. The differences are usually due to a single amino acid difference in the light or heavy chain. Sometimes is it more than one amino acid difference. The best examples of allotype differences in human Ig are the three allelic variants of human kappa light chains.

Allotype Amino acid differences

Km(1) Valine @ 153, Leucine @ 191

Km(1,2) Alanine @ 153, Leucine @ 191

Km(3) Alanine @ 153, Valine @ 191

Mendelian co-dominant autosomal allelic genes code Allotypes. There are 4 subclasses of human IgG and two subclasses of IgA (described earlier). Importance: Allotypes are used forensically in cases of disputed parentage or in analysis of blood. The allotypic variation has no influence on antibody specificity.

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Clinical Vignette

IDIOTYPES Idiotypes are epitopes found in V regions of antibody molecules. They are defined serologically. The idiotypic epitopes (Idiotopes) are thought to be located near or even within the paratope site on the antibody. It is clear that several different kinds of anti-idiotypic antibodies exist based on the type of idiotope that they recognize. The two most common are shown in he following diagram:

Results of Pediatric vaccine trials published in 1985 and 1986 show some correlation between severity of Hemophilus influenzae type b infections and the IgG2 allotype of the patient (Ambrosino et al, J. Clin. Invest. 75, 1935-1942, 1985; Granoff et al, J. Infect. Dis. 154, 257-264, 1986). Briefly, they showed that children with the G2m(23) allotype have higher levels of immunity than the G2m(23) negative children and Km(1) allotype is more protective in black children than other L chain allotypes.

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Summary of the properties of human immunoglobulin isotypes.

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Schematic structures for the 5 major isotypes of immunoglobulins are shown below:

STRUCTURAL FEATURES OF IgG Important Structural Features of the human IgG isotype include:

a) Two domains in the L chain and 4 domains in the H chain. b) The Inter-H chain disulfides hold the two large halves together in the area called

the HINGE region (see above) due to its flexibility. L→H disulfides covalently connect the L chains to the H chains.

c) The single carbohydrate moiety is located in the C

H2 domain. This carbohydrate

may have important structural and/or functional properties. The general function of carbohydrate on the Ig’s as well as other glycosylated polypeptides seems to be that it plays a role in cellular transport and in secretion.

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BIOLOGIC PROPERTIES OF IgG AGGLUTINATION AND PRECIPITATE FORMATION IgG antibodies can cause agglutination of particulate antigens like bacteria. With soluble antigens such as toxins, IgG antibodies can form insoluble precipitates. The mechanism will be discussed in the lecture on Antigen-Antibody reactions. Both types of reactions make the complexes easier for macrophage scavenger cells to ingest and destroy the antigen. PLACENTAL PASSAGE In humans, IgG is selectively passed through the placenta (but NOT IgG

2) to provide passive

immunity to the fetus. This begins in the 3rd

or 4th

month of pregnancy in humans. Resistance of the neonate to most common infections is almost exclusively via this passive IgG. Active transfer is mediated by the Fc region of IgG molecules. While this passive immunity is crucial for neonatal protection, some transferred antibodies can be destructive. Erythroblastosis fetalis, hemolytic disease of the newborn, is mediated by the passage of anti-Rh (D) antibodies of the IgG class across the placenta in an Rh (D)-negative mother carrying an Rh (D) positive fetus. She can be sensitized to the Rh antigen by a previous pregnancy. These antibodies can destroy fetal red blood cells and cause disease that can vary from mild to fatal, depending on the maternal titer of anti-Rh (D).

OPSONIZATION IgG is a powerful opsonizing antibody (from the Greek opsonin, to prepare for eating). The antibody reacts with microbial epitopes and its Fc region is then efficiently bound by specific Fc receptors on macrophages and/or polymorphonuclear cells. The net effect is engulfment of the bacteria into the phagocyte. The figures on the following page illustrate both the mechanism of opsonization. ANTIBODY DEPENDENT CELL MEDIATED CYTOTOXICITY (ADCC) Certain large granular lymphocytes [also called Natural Killer (NK) cells] have Fc receptors on their surface and when antibodies bind cellular antigens (for instance on tumor cells) NK cells can then bind to the antibody via the Fc portion. The NK cell can kill the tumor cells due to the secretion of substances by the NK cell that are cytotoxic to the tumor cells. Only

Case 46—Hemolytic Disease of the Newborn—Mrs. Waymarsh was 31 and pregnant for the third time. Her blood type was A, Rh-negative. Her husband was also type A, but Rh-positive. Her first child was born healthy

and she was given RhoGAM following delivery. During her 2nd

pregnancy, she developed a 1:16 indirect Coombs titer of 1:16 and a healthy girl was induced at 36 weeks and she was again given RhoGAM. Five years later in the third pregnancy, her Coombs titer was 1:16 at 14 weeks and bilirubin was found in increasing amounts in the amniotic fluid . A low fetal hematocrit (6.2%, normal is 45%) was detected in fetal blood. 85 ml of type O, Rh-negative packed RBC were transfused into the umbilical vein. At 30.5 weeks another transfusion of 75 ml was given, and at 33.5 weeks 80ml was transfused. At 34.5 weeks, labor was induced and a normal female infant was born.

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IgG antibodies are implicated in this type of activity and the importance of ADCC in host defense or tissue damage is still controversial.

COMPLEMENT ACTIVATION IgG antibodies are efficient activators of the Complement system. Complement will be covered in a separate lecture. Briefly, many antigen-antibody interactions trigger a series of enzymes collectively known as Complement. Some of the by-products of these reactions can act as opsonins and other components are chemotactic (attract phagocytic cells). IgG antibody activation of Complement can have profound biological effects, some positive and some negative. Details will be presented in the lecture on Complement. BACTERIAL IMMOBILIZATION Motile bacteria can have their movement arrested by IgG and IgM antibodies by cross-linking their flagella or clumping them via their flagella. The antibody can function in this regard like handcuffs in stopping the waving of flagella. The result is that the bacteria are less invasive and less efficient in spreading through tissue. VIRAL NEUTRALIZATION Most viruses utilize some form of cellular receptor for initial binding that results in the virus gaining entry into the cell or moving its DNA or RNA into the cell. IgG and IgM antibodies specific for those structures on the virus that bind to the cell receptors will inhibit or eliminate initial binding to the cell, thereby protecting the cell from viral entry. The binding of IgG also facilitates phagocytosis of the organism. TOXIN NEUTRALIZATION Bacterial toxins usually are toxic to cells because the toxin binds to specific cellular receptors to gain entry to the cell and then toxic effects occur intracellularly. The strategy that the immune system employs to protect the animal from toxins is to make a variety of antibodies specific for many different epitopes on the toxin to immobilize it in the form of an antigen-antibody aggregate and stop the toxin from reaching the cell receptor. The Ab-Ag aggregates can be easily phagocytosed and the toxins degraded and rendered non-toxic by acid proteases in the phagosomes. The problem is in finding a way to use the toxins to trigger an immune response without killing the host with toxin. This has been done with diphtheria and tetanus

55

toxins very successfully by treating the toxins with formaldehyde. Formaldehyde treatment eliminates the toxic properties of the molecules without significantly affecting the antigenic makeup of them (review the section on Cross-reactions in the lecture and chapter on Immunogens and Antigens). These TOXOIDS then can be used to make vaccines that will elicit a strong immune response with no toxic effects.

STRUCTURAL FEATURES OF IgM IgM antibodies are called macroglobulin antibodies because of their high molecular weight. Important structural features of IgM include:

a) the molecules are polymers of 5 four-peptide subunits each bearing an extra CH domain as compared to IgG or IgA molecules.

b) polymerization of the subunits into a pentamer depends upon the presence of J

chain whose function may be to stabilize the Fc sulfhydryl groups during Ig synthesis so that they remain available for cross-linking the five subunits.

c) the free molecule assumes a “star” or “wagon wheel” shape in free solution, but

when bound to antigens on membranes it adopts a “crab-like” shape. d) the net paratope valency is 10, but with larger antigens, the effective valency falls

to 5 and this is attributed to steric restriction due to the lack of flexibility in the molecule.

e) the hinge region is not nearly as flexible as the hinge in IgG. f) the IgM antibodies tend to be of relatively low affinity as measured against haptens

but, because of their multivalency, they bind with high avidity to antigens with multiple epitopes.

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g) there are 5 distinct CHO moieties, compared to one in IgG. h) there is an intersubunit cysteine bridge in the CH3 domain bridging the 4-chain

subunits in the molecule. i) the Complement binding site is in the CH3 domain.

BIOLOGIC PROPERTIES OF IgM Most of the IgM is found in intravascular spaces and the concentration in serum is low in normal circumstances. IgM does not pass the placenta so elevated levels of IgM in the fetus are indicative of congenital or perinatal infection. It is the first isotype to appear in serum after vaccination with most antigens (T-dependent antigens) and elevated levels of IgM in adults is also indicative of recent antigen exposure AGGLUTINATION IgM antibodies are highly efficient in aggregating or agglutinating particulate antigens such as bacteria or red blood cells. ISOHEMAGGLUTININS The IgM population of antibodies includes the natural isohemagglutinins, the naturally occurring antibodies reactive with the red blood cell antigens of the ABO series. These antibodies are thought to be elicited by bacteria that have carbohydrate epitopes similar to, or identical to, the antigens of the A or B blood group. The following table shows the kinds of isohemagglutinin antibodies normally found in patients with the various blood groups: Blood Type Isohemagglutinin Normally Present A Anti-B B Anti-A AB None O Anti-A and Anti-B COMPLEMENT ACTIVATION Its pentameric form coupled with the correct amino acid sequence for binding complement make IgM the most efficient isotype on a mole/mole basis for complement activation. It has been shown experimentally that the binding of one IgM antibody on the surface of a red blood cell is sufficient to lyse the cell but that in excess of 100 IgG antibodies are needed to lyse a red blood cell. STRUCTURAL FEATURES OF IgA. IgA appears selectively in the sero-mucous secretions such as saliva, tears, nasal fluids, sweat, colostrum and secretions of the lung, genitourinary and gastrointestinal tracts where it has the job of defending the exposed external surfaces of the body against attack by micro-organisms from the environment. It is present in these fluids as a dimer stabilized against proteolysis by combination with another protein, secretory component, which is the cleaved portion of the polymeric Ig receptor (PIgR) that allows for transport across the epithelial cell layer (See figure below). sIgA is a single peptide of molecular weight 60,000 Important structural features of the IgA molecule include:

57

a) A larger number of disulfides compared to IgG b) Two intrachain disulfides on the H chain in the CH2 and CH3 domains providing for intermolecular bonding and J chain binding, respectively. c) Three sites of glycosylation

Subclasses of IgA. Two subclasses of IgA exist. They are designated IgA1 and IgA2. The momomeric form of IgA1 is the major subclass found in serum, whereas dimeric IgA1 and IgA2 are found equally in mucosal secretions. The IgA1 and IgA2 subclasses differ in the length of their hinge regions, and the IgG2 sublcass contains a disulfide bond at the C terminus of the light chain

BIOLOGIC PROPERTIES OF IgA Serum IgA I is monomeric and has no known biological function and a short (5.5 day) half-life. Secretory IgA (sIgA) is always in the dimeric form Most IgA is transported to epithelial surfaces (see figure below) where it functions as a first-line barrier of protection for these sensitive surfaces.

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IgA ROLE IN MUCOSAL INFECTIONS One of the primary roles of IgA on mucosal surfaces is to protect these surfaces from infection. LOTS of sIgA is made in the intestine – estimated 3 g/day, also found in nasal passages, saliva, and breast milk. IgA ROLE IN BACTERICIDAL ACTIVITY IgA antibodies will not fix Complement. The IgA molecule has bactericidal activity for gram-negative organisms, but only in the presence of lysozyme that is normally present in the same secretions where IgA is found. The secretory component that remains attached to dimeric IgA may provide some protection against bacteria. IgA ROLE IN VIRICIDAL ACTIVITY IgA is an effective viricidal agent, preventing attachment of viral structures to specific cellular receptors and, due to its multivalency in the dimeric form, is an efficient agglutinator of viruses. PASSIVE IMMUNOTHERAPY sIgA can be transferred from mothers’ breast milk to the intestinal tract of the infant. Provides protection against pathogens (i.e. rotavirus, cholera) STRUCTURAL FEATURES OF IgD. IgD is only found in trace quantities in serum. Its primary biological role is in triggering of lymphocytes. It is co-expressed on the surface of certain subsets of lymphocytes along with IgM. Important structural features of the IgD molecule include:

a) IgD is not synthesized and secreted by mature plasma cells. b) The constant region of the δ chain is comprised of 383 amino acids making it

longer than γ and α chains, but shorter than μ and ε which have 4 constant region domains.

c) The hinge region of IgD is the largest of any Ig hinge with 64 amino acids in two

structurally distinct segments of about 30 residues each. This allows for maximum flexibility in contacting antigen.

BIOLOGIC PROPERTIES OF IgD IgD is nearly non-detectable in serum. IgD is found on the surface of mature B cells that have not been antigen stimulated and is thought to be involved in maturation of these cells. This will be discussed in more detail in the lecture on “Biology of B and T Cells”. STRUCTURAL FEATURES OF IgE. IgE molecules are normally only present in serum in trace amounts. The Fc part of the IgE molecule has a recognition site for IgE receptors on the surface of mast cells. When IgE

59

antibodies of a given specificity encounter their antigen while bound to the surface of mast cells, degranulation of the mast cells ensues with large amounts of histamine and other vasoactive amines are released that have dramatic physiological consequences. IgE can be thought of as the “allergy” or reaginic antibody. IgE is responsible for the symptoms of hay fever and of extrinsic asthma when patients with atopic allergy come into contact with the allergen, e.g. grass pollen, ragweed pollen, penicillin etc.

Important structural features of IgE include:

a) the large number of carbohydrate moieties. b) the 5 distinct domains in the H chain (reminiscent of IgM). The cytotropic regions

appear to be the CH2 and C

H3 domains.

BIOLOGIC PROPERTIES OF IgE IgE IN HYPERSENSITIVITY REACTIONS Type I hypersensitivity is mediated by IgE antibodies and occurs when an IgE response is directed against innocuous antigens, such as pollen. The resulting release of pharmacological mediators such as histamine by the IgE-sensitized mast cells produces an acute inflammatory reaction with symptoms such as asthma or rhinitis. The most characteristic manifestation of these types of reactions is hay fever; swollen eyes, runny nose, etc. However, if sensitization is systemic and antigen is introduced IV (bee or wasp sting, injection of antibiotics, ingestion of allergen orally) the symptoms can proceed rapidly through hives to cardiac arrest. Mast cells and their circulating counterpart the basophil display a high affinity receptor for the Cε2:Cε3 junction area in the Fc region of the IgE molecule. Local or even distant production of IgE molecules specific for any antigen can then coat these cells with specific antibody molecules of the IgE class. Arrival of antigen then causes:

a) cross-linking of the receptors b) the breakdown of phosphatidyl inositol to inositol triphosphate c) generation of diacylglycerol d) an increase in intracytoplasmic free calcium.

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Since inhibitors of methyltransferases and serine esterases inhibit all these events and mediator release, it is assumed that activation of these enzymes by receptor bridging is the initial event. Phospholipase c activation generates both IP3 (which mobilizes intracellular Ca2+), and diacylglycerol which activates protein kinase c. The biochemical cascade produces membrane-active “fusogens” such as lysophosphatidic acid that may facilitate granular membrane fusion and degranulation, and the series of arachidonic acid metabolites formed by the cyclooxygenase and lipxygenase pathways.

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CLINICAL VIGNETTES

IgE IN PARASITIC INFECTIONS Elevated levels of IgE have been detected during the course of infection with certain parasites. For example, infections with ascaris (a roundworm) routinely raises the detectable levels of IgE antibodies more than other isotypes and immunization with ascaris antigens selectively induces the formation of IgE antibodies. It is possible that IgE originally arose in evolution as a specific means of protection from parasites. The degranulation process releases histamine, heparin, eosinophil and neutrophil chemotactic factors, and platelet activating factor, while leukotrienes B4, C4 and D4, prostaglandins and thromboxanes are all newly synthesized. When there is a massive release of these mediators, their bronchoconstrictive and vasodilatory effects predominate and become life threatening. The figures below summarize the triggering mechanisms and effects of IgE-antigen crosslinking on mast cells. Typical immune responses in humans, as defined by detection of antibodies in serum specific for the injected immunogen, have 4 clearly defined phases. Each phase correlates with known mechanisms involved in immune responses. Primary Response

1. Latent or Lag phase is long 2. Exponential Production phase-moderate rate 3. Steady state reached at modest Ab concentrations 4. Declining phase

The first clinical vignette is on reserve in the LRC. It is a story entitled “How it feels to die” written by a former editor of Life Magazine. Briefly, he self-prescribed some penicillin tablets when he woke up one morning. The story describes his experience with severe anaphylactic shock. It describes his treatment by his family physician who (lucky for him) lived right next door. She had the right things with her to treat him and he lived. Case 49—Acute systemic Anaphylaxis—A life-threatening immediate hypersensitivity reaction to peanuts. John was a healthy 22 month-old who developed swollen lips while eating a cookie containing peanut butter. A month later he ate another of the same kind of cookie. He started to vomit, became hoarse, had difficulty breathing, started to wheeze and developed a swollen face. He was rushed to the ER but became lethargic and lost consciousness en route. Upon arrival at the ER his blood pressure was 40/0 (normal 80/60), pulse was 185 (normal 80-90) and respiration was 76 (normal 20). Epinephrine, saline, anti-histamine, and corticosteroid were administered. Within one hour he was responsive and after an overnight hospital stay including further epinephrine and anti-histamine treatments, he was discharged. The parents were instructed to avoid giving him any foods containing peanuts or peanut extract. Further tests were scheduled in the Allergy Clinic.

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Secondary (Anamnestic) Response

1. Lag phase is shorter (sometimes MUCH shorter!) 2. Exponential Production phase is steeper 3. Higher concentrations of antibody are detectable (sometimes MUCH higher!) 4. Production of antibody continues for a longer time (Slower declining phase)

In anamnestic responses, class switching from IgM to IgG production is much quicker and antibodies of the IgA and IgE class are more likely to be detected than in primary responses. Affinity maturation of the IgG produced is usually detected, that is, the IgG produced after secondary immunization has a higher affinity for antigen than IgG synthesized during the primary response. THE IMMUNOGLOBULIN SUPERFAMILY Structural features found in Immunoglobulin heavy and light chains are also seen in other proteins, frequently in membrane bound glycoproteins. Due to the structural similarities, these proteins are classified as members of the Immunoglobulin Superfamily. One of the more important members of the Ig superfamily is the T cell receptor. It will be discussed in detail in a subsequent lecture.

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ANTIBODY ENGINEERING Presents a way to make “custom made” antibodies for therapeutic use. “Recapturing in recombinant form the naturally developed diversity of antibodies” Single chain Fv (ScFv): V domains of expressed antibodies linked with a flexible peptide. VL and VH domains are cloned into a plasmid vector, then the recombinant protein is secreted by bacteria. Can also be expressed as Fab fragments containing full length light chains linked by disulfide bonds to the VH-CH1 fragment of the heavy chain. Half life is extremely short (hours). “Humanized” antibodies: V domains of mouse antibodies against a specific target are cloned into vectors encoding the constant regions of human antibodies. Resulting antibody may be specific for antigen, and the potential for antigenicity is decreased. Immunotoxins: conjugate antibodies with toxins to be able to specifically bind to target cells and deliver toxin to a specific area (minimizes side effects).

Case—Multiple Myeloma (posted on Blackboard), a malignancy of terminally differentiated B lymphocytes. Isabelle Archer was a 55 yo housewife who began to experience excessive fatigue. Routine blood tests showed an elevated sedimentation rate. The elevated sed rate prompted measurement of her serum Ig levels. IgG was 3790 mg/dl (normal is 600-1500), IgA was 14 mg/dl (normal is 150-250) and Igm was 53 mg/dl (normal is 75-150). Electrophoresis screening of her serum showed a monoclonal protein in the gamma region which was shown to be IgG with kappa light chains. Over the next 3 years her IgG level rose to 6312 mg/dl and she experienced destruction of the second thoracic vertebral body with extrusion of a plasmacytoma. She was treated with hemotherapy and radiation and when her IgG level rose to 8200 mg/dl she was treated with cyclophosphamide, etoposide and decadron, which lowered her IgG level to 6000 mg/dl. She now remains fully active requiring occasional blood transfusions for anemia and complains at times of bone pain. Her serum IgG is stable at 6200 mg/dl. The electrophoresis result, shown here, shows normal serum in lane 1, 3 and 5, Mrs. Archer’s serum in lanes 2, 4 and 6. Lanes 1 & 2 are stained just for protein, lanes 3 and 4 were stained with anti-lambda antiserum and lanes 5 and 6 were stained with anti-kappa antiserum.

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CH

2 C

H3

CL

VL

CH1

VHC

DRC

DR

CD

R

CD

RCD

R

CD

R

CH

2 C

H3

Fv

CDR affinityengineering

Complementactivation

Fc receptorbinding

VL-VHcombinatorial diversity

C domainvectors

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SUMMARY (Structural Features) 1. Antibodies are collectively defined as Immunoglobulins (Ig). Were originally found in

the globulin fraction of serum and migrate in electrical fields in the gamma region. 2. The basic Ig unit consists of 2 Light chains and 2 Heavy chains, all covalently bound via

disulfide bridges. Domains of the molecule responsible for unique biological functions are also disulfide bonded.

3. There are two types of Light chains in humans, κ and λ, distinguished by different amino

acid sequences in the constant domain. 4. There are five classes or isotypes of heavy chains, γ, α, μ, δ, and ε corresponding to the

heavy chains of IgG, IgA, IgM, IgD, and IgE, respectively. 5. The hinge region in Ig allows for flexibility of the molecule. 6. The variable region or domain contains the short stretches of amino acid sequences that

make contact with antigen. These areas are called hypervariable regions or complementarity-determining regions of the molecule.

7. Distinguishing features of IgG molecules include 4 domains on each of the two γ heavy

chains and two domains on the light chains, inter-H chain disulfide bridges between the H chains, a single carbohydrate moiety in the C

H2 domain. The IgG subclasses differ

structurally, in the H chains. It is normally the most abundant Ig. 8. Distinguishing features of IgM molecules include 5 domains on each of the two μ chains,

the soluble form of IgM is a pentamer of 4-chain subunits with a J chain attached with a total paratope valency of 10 giving it high avidity. There are 5 separate carbohydrate moieties on each H chain, the Complement binding site is in the CH

3

domain. 9. Distinguishing features of IgA molecules include 4 domains on each of the two α chains, a

larger number of disulfides compared with IgG, three sites of glycosylation, two subclasses of IgA exist (IgA1 and IgA2) and two allotypic forms of IgA2.

10. Distinguishing features of IgD include 4 domains on each of the two δ chains with an

extended (64 amino acid) hinge region. Like IgM and IgA, IgD is more highly glycosylated than IgG. IgD is found in only trace amounts in serum, most is bound to surfaces of mature lymphocytes.

11. Distinguishing features of IgE include 5 domains on each of the two ε chains. Like IgM,

IgA, and IgD, IgE is more highly glycosylated than IgG. The CH2 and C

H3 domains are

cytotropic for mast cells. Only trace amounts are found in serum (except in some allergy patients) as most is attached to mast cells.

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12. Allotypes are structural variants of L or H chains. Are germ line encoded Mendelian autosomal codominant genes. Usually consist of one or a very few amino acid differences.

13. Idiotypes are epitopes found in the V regions of specific antibodies. The idiotype

defining each antibody specificity is different.

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SUMMARY (Biological Properties) 1. Class and subclass differences among the Ig’s for mediating biological functions such as

placental transfer, complement fixation, half life, location of function (secretions, intravascular) depend on structural features of the molecules.

2. Antibodies can be either protective or destructive. 3. IgG antibodies efficiently agglutinate or precipitate antigens, cross the placenta,

opsonize bacteria for phagocytosis, mediate ADCC, neutralize bacterial toxins, immobilize motile bacteria and neutralize viruses.

4. IgA is transported to epithelial surfaces in a dimerized form and protects such surfaces

from infection. IgA is uniquely bactericidal only in the presence of lysozyme, is virucidal by preventing attachment of viruses to cell receptors.

5. IgM is concentrated intravascularly and does not pass the placenta. It is a highly effective

agglutinator of particulate antigens due to its pentameric structure, is the principle isohemagglutinin (barrier to blood transfusion), and is highly efficient for fixing complement.

6. IgD is found on the surface of mature antigen sensitive B cells, only in trace amounts in

serum and has no significant known protective properties. 7. IgE causes allergies. Binds to the surface of mast cells and then cross-linking by antigen

triggers the mast cell to secrete several pharmacologically active substances that cause the symptoms of allergies. Functionally protective in parasitic infections.

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Antibodies

Table 3-1. Classes of Antibody Isotypes and Functional Properties*

Immunoglobulin Class Isotype IgM IgD IgG IgE IgA Structure Pentamer Monomer Monomer Monomer Monomer,

dimer

Heavy chain designation

μ δ γ ε α

Molecular weight (kDa) 970 184 146-165 188 160 × 2

Serum concentration(mg/mL)

1.5 0.03 0.5-10.0 <0.0001 0.5-3.0

Serum half-life (days) 5-10 3 7-23 2.5 6

J chain Yes No No No Yes

Complement activation Strong No Yes, except IgG4

No No

Bacterial toxin neutralization

Yes No Yes No Yes

Antiviral activity No No Yes No Yes

Binding to mast cells and basophils

No No No Yes No

Additional properties Effective agglutinator of particulate antigens, bacterial opsonization

Found on surface of mature B cells, signaling via cytoplasmic tail

Antibody-dependent cell cytotoxicity

Mediation of allergic response, effective against parasitic worms

Monomer in secretory fluid, active as dimer on epithelial surfaces

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COMPLEMENT Rick A. Wetsel, Ph.D.

Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York, NY. 6th edition, 2009. Chapter 13; Geha and Notarangelo. Case Studies in Immunology. Garland Publishing, New York, NY. 6th edition, 2012. Case 32. Factor I Deficiency; Case 33. Deficiency of C8. Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/complement/ WHAT IS COMPLEMENT? Complement is a system of more than 30 serum and cell surface proteins that is involved in numerous functions in inflammation and immunity. In conjunction with specific antibodies, it acts as the primary humoral defense system against bacterial and viral infections. Complement activity is heat labile and can be destroyed by heating serum to 56O C 30 minutes (which inactivates the C3 and C4 proteins as well as other complement components). Most of the complement proteins in the serum are produced by liver hepatocytes. C3 is the most abundant serum complement protein with a normal range of 1.0 to 1.5 mg/ml in healthy individuals. Some of the complement components (e.g. C3, factor B) are acute phase proteins and can increase in concentration two to three fold. Many of the complement proteins have shared sequences, indicating that they evolved by gene duplication and recombination. The complement genes are scattered throughout the human genome; the genes for the proteins C4, C2, and factor B are located within the Major Histocompatibility Complex on chromosome 6. COMPLEMENT FUNCTIONS Activation of the complement system results in the production of several different polypeptide cleavage fragments that are involved in five primary biological functions of inflammation and immunity. 1. Cytolysis of foreign organisms (e.g. bacteria) 2. Opsonization and phagocytosis of foreign organisms 3. Activation of inflammation and directed migration of leukocytes 4. Solubilization and clearance of immune complexes 5. Enhancement of humoral immune response

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THE COMPLEMENT CASCADES Complement activation involves the sequential activation of complement proteins, either by protein-protein interactions or by proteolytic cleavage. At each step, the number of protein molecules activated increases, amplifying the reaction. This sequential reaction is call the complement cascade. Many complement proteins are present as zymogens which are activated either by conformational changes or by proteolytic cleavage by other complement proteins. Activation of these zymogens results in specific serine protease activities that are capable of cleaving other complement proteins, producing the complement cascade. ACTIVATION Complement activation is initiated by the presence of antigen-antibody complexes (Classical Pathway), foreign cell surfaces (Alternative Pathway), or by mannose on pathogenic organisms (Lectin Pathway). FIGURE 1. OVERVIEW OF COMPLEMENT CASCADES

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Space for notes:

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CLASSICAL PATHWAY ACTIVATION -Primarily by IgG and IgM immune complexes -IgM > IgG3 > IgG1> IgG2 -IgG4, IgA, IgD, and IgE do not activate Activation of the classical pathway requires the local reaction of antibodies with two or more antigenic sites. These Ag-Ab complexes may consist of a single IgM molecule bound to two or more antigenic sites, or two or more human IgG molecules (IgG1, IgG2, or IgG3) bound to epitopes. Such a complex could (for example) occur on a bacterial cell surface or in an aggregate of antibodies with soluble antigens. Ag-Ab reaction causes conformational changes in CH2 of IgG and CH3 of IgM, permitting the binding of C1q. Binding of two or more arms of C1q causes conformational changes that lead to cleavage and activation of the bound zymogens C1r and C1s. FIGURE 2. Bridging of two membrane-bound IgG molecules by the C1 component. Binding distorts the C1 molecule and triggers activation.

Activated C1s can cleave C4 and C2 into large (C4b and C2b) and small (C4a and C2a) fragments. C4 is cleaved first, and approximately 1% binds to a nearby surface via a covalent linkage. C2 can complex with surface bound C4b and can be cleaved by C1s. The resulting C4bC2b complex is the classical pathway C3 convertase, and has the ability to specifically cleave C3 into large (C3b) and small (C3a anapylatoxin) fragments. C3 is the most abundant complement protein and plays a pivotal role in complement activation. Many molecules can be cleaved into C3b and C3a. Cleavage results in exposure of the labile thiolester bond in C3b, permitting some to bind covalently to proteins and carbohydrates on cell surfaces. The C3a anaphylatoxin is released into the blood and mediates many important inflammatory activities that will be discussed

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later. Some of the C3b binds to C4bC2b to form C4b2b3b, which is the C5 convertase. This complex by the C2b protease subunit will cleave C5 into big (C5b) and small (C5a anaphylatoxin) subunits. C5a is released into the blood and as C3a mediates many important inflammatory activities. C5a on a molar basis is 100 times more potent than C3a. FIGURE 3. The classical pathway of complement activation generates a C3 convertase that deposits large numbers of C3b molecules on the surface of the pathogen.

FIGURE 4. Cleavage of C3 and C4 exposes a thiolester bond that causes the resulting large fragments, C3b and C4b, to bind covalently to nearby molecules on bacterial or viral surfaces.

The classical pathway of complement can also be activated by the serum mannose binding lectin complex (MBL-MASP). This complex is structurally similar to the C1 complex. However, instead of binding to immune complexes it binds to directly to polysaccharides on gram-negative bacteria. The mannose binding lectin is C1q-like in structure and the MBL associated proteases (MASP) are similar to C1r and C1s. MBL-MASP on binding bacterial surfaces can cleave C4 and C2 thereby activating the remainder of the classical pathway.

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ALTERNATIVE PATHWAY ACTIVATION The main difference between the classical and alternative pathways is that the initiation of the classical requires an activating substance. The alternative pathway, by contrast, runs continuously and spontaneously at low levels in the blood plasma. The alternative pathway activation occurs when C3b binds to a surface that lacks inhibitors that block complement activity, such as most bacterial cell surfaces. Certain plastic surfaces, like those initially used in heart-lung machines and dialysis machines, also activate the alternative pathway with obvious deleterious effects. Because antibody is not necessary, the alternative pathway represents an innate immune response and can react as soon as bacteria enter the body. The alternative pathway is also important in amplifying reactions initiated by the classical pathway. The low level activation of C3 that allows the alternative pathway to be activated is called the tick-over model. The thiolester bond in C3 is spontaneously hydrolyze at low rates yielding a C3b-like [C3(H2O)] molecule that now has a binding site for factor B exposed. The bound factor B is attacked by factor D, which cleaves it into Ba and Bb fragments. The Ba fragment is released, while the Bb fragment remains noncovalently associated with C3(H2O), forming an initial C3 convertase. The Bb subunit of this convertase has serine protease activity specific that can now specifically cleave additional C3 molecules into C3a and C3b fragments. If a activator surface is nearby, such as a bacterial surface, then the newly formed C3b molecule can covalently attach and bind factor B. The bound factor B is cleaved by factor D and the surface-bound C3 convertase (C3bBb) then attacks another native C3 molecule and so on. The activating surface (bacteria) thus accelerates a reaction that in its absence occurs at a slow rate. FIGURE 5. THE TICK-OVER MODEL

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LYTIC PATHWAY-FORMATION OF THE MEMBRANE ATTACK COMPLEX (MAC) After the C3 convertase cleaves C3 to generate C3b, the next step in either the classical, lectin, or alternative pathways is the binding of C3b to the C3 convertase complex, changing it to a C5 convertase, which catalyzes the proteolytic cleavage of the C5 protein. Cleavage of C5 to C5a and C5b represents the first step of the lytic pathway. The small C5a fragment is released into the blood and is the most potent complement anaphylatoxin. The large C5b molecule binds proteins C6 and C7. The complex C5b67 has hydrophobic regions that permit it to insert into the lipid bilayer nearby cell membranes. Subsequent binding of C8 permits some leakage of cell contents, causing slow lysis. This process is accelerated by binding of multiple C9 molecules, which assemble to form a protein channel through the membrane. C9 is analogous to perforins produced by cytolytic T cell and NK cells. C5b6789 is called the Membrane Attack Complex (MAC). MAC formation is an important mechanism for eliminating bacteria resistant to intracellular killing by phagocytes, such as Neisseria species. FIGURE 6. FORMATION AND REGULATION OF THE MEMBRANE ATTACK COMPLEX (MAC)

REGULATION OF COMPLEMENT ACTIVATION Complement activation is a tightly regulated series of reactions, that without control would result in the inappropriate activation on normal host cells. This would result in excess inflammatory mediators and by direct lysis of host cellular membranes by MAC.

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This does not normally happen because there exist several complement inhibitors in serum as well as on the surface of host cells. C1-inhibitor (C1INH) serum protein binds to activated C1r,C1s, removing it from C1q C4-binding protein (C4BP) serum protein that binds C4b displacing C2b; co-factor for C4b cleavage by factor I Complement Receptor 1 (CR1;CD 35) Binds C4b displacing C2b, or C3b displacing Bb; cofactor for I Factor H (H) serum protein binds C3b displacing Bb; cofactor for I Decay Accelerating Factor (DAF;CD55) Membrane protein displaces Bb from C3b and C2b from C4b Membrane Cofactor Protein (MCP;CD46) membrane protein that promotes C3b and C4b inactivation by I CD59 (Protectin) Prevents formation of MAC on homologous cells. Widely expressed on membranes S Protein (Vitronectin) serum protein binds C5b-7 prevents insertion into membrane Clusterin (SP-40-40) serum protein binds C5b-7 prevents insertion into membrane Complement Receptors There are several characterized complement receptors that are involved in binding complement activation and degradation products. They are expressed on various cell

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types and are involved in mediating many of the biological functions attributed to complement. TABLE II DISTRIBUTION AND FUNCTION OF RECEPTORS FOR COMPLEMENT PROTEINS ON SURFACE OF CELLS

C5a Receptor (C5aR;CD88) is a seven transmembrane G-protein coupled receptor expressed primarily on neutrophils and macrophages. Also found on hepatocytes and various tissue epithelial cells. Causes smooth muscle contraction, histamine release from mast cells and vasodilation. Will modulate the hepatic acute phase response. It also is a potent chemoattractant for neutrophils, monocytes, macrophages, and eosinophils. C3a Receptor (C3aR) also seven transmembrane receptor. Tissue distribution currently being worked out. Causes smooth muscle contraction, histamine release from mast cells, and vasodilation. Chemoattractant for eosinophils but not neutrophils.

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BIOLOGICAL FUNCTIONS OF COMPLEMENT 1. CYTOLYSIS OF FOREIGN ORGANISM BY C5B-9 MAC COMPLEX 2. OPSONIZATION AND PHAGOCYTOSIS C3b, C3bi is coated on microorganisms (opsonization) Receptors for C3b (CR1) and C3bi (CR3) on macrophages and neutrophils can then bind the complement coated bacteria facilitating the phagocytosis reaction

3. ACTIVATION OF INFLAMMATION AND CHEMOTAXIS OF LEUKOCYTES BY COMPLEMENT ANAPHYLATOXINS (C3A, C4A, C5A)

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All three peptides mediate: 1. smooth muscle contraction 2. histamine release from mast cells and 3. increase vascular permeability. C5a on binding C5aR mediates chemoattraction of neutrophils, monocytes, macrophages, and eosinophils. C3a is a chemoattractant for eosinophils but not neutrophils. 4. SOLUBILIZATION AND CLEARANCE OF IMMUNE COMPLEXES One of the major roles complement plays is the solubilization and clearance of immune complexes from the circulation. First, C3b and C4b can covalently bind to the Fc region of insoluble immune complexes, disrupting the lattice, and making them soluble. C3b and C4b bound to the immune complex is recognized by the CR1 receptor on erythrocytes facilitating their transport to the liver and spleen. In the liver and spleen the immune complexes are removed and phagocytized by macrophage-like cells. The RBCs are returned to the circulation. Individuals deficient in the early complement components cannot make C3b and C4b. They are therefore predisposed to immune complex diseases such as systemic lupus erythematosus.

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5. ENHANCEMENT OF THE IMMUNE RESPONSE CR2 (CD21) is expressed on B-cells and follicular dendritic cells. This receptor binds the C3d fragment of C3. C3b coated on antigens will be broken down eventually to C3d and C3c fragments by factor I. The C3c fragment is released into the blood with no know function. The C3d fragment remains covalently bound to the antigen. Coating of antigens with C3d facilitates their delivery to germinal centers rich in B and follicular dendritic cells. CR2 also is part of the B-cell co-receptor complex. Binding of C3d coated antigens to CR2 leads to signaling through CD19. Animals deficient in C3 have an impaired immune response to T dependent antigens.

COMPLEMENT DEFICIENCIES AND ASSOCIATED ABNORMALITIES Human deficiencies in many of the complement proteins have been described. These deficiencies are usually attributable to inherited mutated genes. Genetic deficiencies in classical and alternative pathway components, including C1q, C1r, C4, C2, properdin, and factor D have all been described. C2 deficiency is the most common of the complement deficiencies. Deficiencies in the early classical pathway proteins predispose individuals to the development of systemic lupus erythematosus (SLE). The reason for this is not completely clear, but it is at least partly do to the inability of these individuals to clear immune complexes readily. Because of its central importance in killing bacteria, homozygous C3 deficiency can be lethal, especially in young children if it is not diagnosed. Deficiencies in the terminal components predispose these individuals to recurrent bacterial infections with Neisserial species.

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Table III. Complement Deficiencies and Associated Diseases

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Dear Immunology students, There is confusion between designation of the correct term used for the complement C3 convertase, with discrepancies between the lecturer and what is printed in the Coico, 2009 text. We will use the nomenclature provided by the lecturer and use what is listed in the syllabus. Here is our understanding. The nomenclature for complement has undergone revision so that the large, target-bound fragment is consistently given the 'b' designation, while the small, soluble fragment is called 'a'. Thus over the last 10 years texts have begun to reverse 2a and 2b, which were initially named incorrectly by this convention. According to the current (but not universally accepted) nomenclature, the C3 convertase is C4b2b and C4a and C2a are the released fragments; Coico, et al. apparently have not yet adapted this change. You will see other opinions in nomenclature, primarily from older texts that have not adopted the new naming structure. Therefore, due to a change in nomenclature in order to maintain the a=smaller and b=larger scheme, the correct C3 Convertase is C4b2b.

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SUMMARY

1. The complement system is a group of <30 serum proteins and cell surface molecules that act as important part of the overall immune system.

2. The activities of complement include: 1) cytolysis of foreign organisms, 2)

opsonization and phagocytosis of foreign organisms, 3) activation of inflammation and directed migration of leukocytes, 4) solubilization and clearance of immune complexes, and 5) enhancement of humoral immune response.

3. The complement cascade is a series of reactions involving complement proteins.

It can be divided into two phases: activation and lysis (MAC formation). Activation involves protein-protein interactions and proteolytic cleavage, whereas the lytic pathway involves protein-protein interactions.

4. There are three activation pathways: the classical, lectin, and alternative.

Classical pathway activation requires antigen-antibody complexes (containing IgM or certain IgG subclasses). The Lectin pathway is a newly described pathway that activates the classical pathway independent of antibody. The Mannose Binding Lectin complex is substituted in place of C1 and recognizes polysaccharides on bacterial surfaces. The alternative pathway is an innate system in which complement components react directly with foreign substances. Classical pathway activation involves the proteins C1,C4,C2, and C3, and the alternative pathway utilizes the proteins C3, B, D, and P.

5. Complement activation results in the release of anaphylatoxins (C3a,C4a, and

C5a). They are important mediators of inflammation, causing recruitment and activation of neutrophils, macrophages, and other cell types. Activation also produces cleavage products (C3b, C3bi, and C4b) which serve as opsonins, enhancing phagocytosis. The C3d cleavage fragment is involved in enhancing the immune response.

6. MAC formation produces a channel in the cytoplasmic membrane of bacteria or

other cells, leading to cell lysis. It requires prior activation by either the classical or alternative pathways, and utilizes the proteins C5b, C6, C7, C8, and C9.

7. The complement system is regulated by several inhibitors, including C1 inhibitor,

Factor H, C4 binding protein, CD 59, and Decay Accelerating Factor.

8. Deficiencies in complement components result in increased susceptibility to bacterial infections and can lead to autoimmune diseases, including systemic lupus erythematosus.

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STUDY PROBLEMS (you should feel confident about the topic if you can answer the following): 1. From memory, draw the classical, lectin, and alternative pathways of complement. 2. Understand how each is activated. 3. Name 5 biological functions mediated by complement activation. 4. Name the complement activation products that mediate each function. 5. What are the three complement anaphylatoxin peptides? 6. How does complement clear immune complexes from the circulation? 7. What complement activation products bind covalently to cell surfaces? 8. Know how the complement activation pathways are affected if a certain component is missing. 9. If a patient is missing a particular complement protein, what disease(s) are they

predisposed? 10. On the figure shown on the next page, assign the activation fragments responsible for that function. Study Problem 11.

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Complement Component Table I

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Actor. 2012. Figure 6.6. Activation of complement through the classic pathway (antigen-antibody complexes), the alternative pathway (recognition of foreign cell surfaces), or the lectin pathway (or mannose-binding pathway) promotes activation of C3 and C5, leading to construction of the membrane attack complex..

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GENERATION OF ANTIBODY DIVERSITY Steven J. Norris, Ph.D.

Recommended Reading: Actor, 2012, Chapter 3. WebResource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/genetic-basis-of-ab-structure/ I. General principles 1. Ag-binding diversity results from differences in the Variable domains due to –

Multiple V gene segments V(D)J joining Random assortment of H, L

chains Junctional/insertional diversity Somatic mutation

2. Functional diversity is due to differences in the Constant domains

IgM C’ fixation IgG C’ fixation, opsonization

through Fc receptors IgA secreted Ig IgE allergic reactions

Changes in these Ig isotypes occur through isotype switching 3. Generation of Ag-binding diversity occurs during B or T cell development before

exposure to antigens; isotype switching occurs in B cells after antigen exposure. 4. Both the generation of Ag-binding diversity and isotype switching involve DNA

rearrangement. 5. Each B cell and all of its progeny produce only one type of heavy and light chain V

region, and thus have a single antigen specificity. This specificity can be ‘fine tuned’ by point mutations (so-called somatic hypermutation) that occurs after antigen exposure.

Development of Ag-binding Diversity (VDJ rearrangement) (IgM/IgD producing B cells) Exposure to Ag + T cell cytokines results in isotype switching (B cells change expression from IgM to other isotypes)

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II. The heavy chain Ig locus and VDJ rearrangement 1. The heavy chain Ig locus is on chromosome 14 in humans. It encodes the IgM and IgD

heavy chains ( and , respectively) that are expressed by B cells initially, as well as the other heavy chain isotypes (1, 2, 3, 4, 1, 2, and ) that are expressed after antigen exposure. In all cells except B cells, it is found in the so-called germline configuration, i.e. the same arrangement that is found in ova and spermatozoa; this germline form of the locus is hence passed from one generation to the next.

2. In the germline form, the Ig heavy chain locus contains multiple V, D, J , and C gene

segments:

Abbreviation: Meaning: Number: Size: Function: V “Variable” ~50 ~95 aa D “Diversity” ~20 ~3-6 aa Form part of Variable Domain J “Joining” ~6 ~13 aa C “Constant” 9* ~110 aa

per Domain Form the Constant Regions

*One for each heavy chain isotype; each constant region contains 3 or 4 C domains (see figure on proceeding page)

3. Stem cells in the bone marrow or fetal liver are stimulated to differentiate into B cells. These B cell precursors (pro-B cells) are then stimulated to undergo VDJ rearrangement. The pro-B cells begin to express Rag-1 and Rag-2, which direct the recombination of the Ig genes in B cell precursors and the TCR genes in T cell precursors. In B cells, the first step is D-J joining, in which the DNA between randomly selected D and J regions is looped out and the intervening sequence is deleted (see diagram). (This process involves the recognition of 7-bp and 9-bp sequences next to the D and J regions; the same type of recognition occurs in all Ig and TCR VDJ rearrangements.)

4. Following D-J joining, a similar looping out and deletion mechanism occurs between

the V and D regions, resulting in V-D joining. The resulting contiguous V,D, and J gene segments have no intervening introns and form the Variable Region Exon. If the

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rearrangement is successful (i.e. does not contain any stop codons or frameshifts), chains (IgM heavy chains) are produced. Further rearrangement of the Ig heavy chain loci stops, a process called allelic exclusion. However, many VDJ rearrangements are unsuccessful, in which case the second copy of chromosome 14 undergoes rearrangement. If this recombination is unsuccessful, the cell undergoes apoptosis and dies.

Coico et al., 2009 Fig. 6.3. V(D)J rearrangement. V-J joining in the V locus is shown

5. After successful rearrangement, the

chain is transported to the surface of the cell along with the surrogate light chain proteins, VpreB and 5. The presence of this complex on pre-B cells triggers the initiation of light chain rearrangement.

Coico., 2009, Fig. 7.2. (A) IgM-like receptor on Pre-B cells; (B) Surface IgM on mature B cells.

6. In mature B cells, the (IgM) and (IgD) heavy chains can both be expressed on the same cell by the alternative splicing of RNA as shown in Fig. 6.4 (preceding page).

III. Light chain rearrangement. 1. In humans and most other mammals, there are two light chain loci called kappa () and

lambda (). These are located on two different chromosomes. The germline arrangement is similar to that of the heavy chain locus, except there are no D gene segments. Also, for the kappa locus there is only one constant region, whereas the lambda locus has multiple constant regions, each with its own J gene segment.

2. Once successful heavy chain rearrangement occurs, the pre-B cell proceeds with

kappa gene rearrangement. In this case, randomly selected V and J segments in one chromosome join together to form the Variable Region Exon; no D segments are involved.

3. If a functional kappa chain is produced, V(D)J rearrangement stops and the cell

becomes a immature B cell that expresses only IgM on its surface. The surface IgM will be anchored by a hydrophobic ‘tail’, and will look like the molecule shown in Fig.

Clinical vignette – B cell maturation, from Geha and Notarangelo, “Case Studies in Immunology”. Case 1 X-linked Agammaglobulinemia – medical student Bill Grignard has normal T cell

function, but has almost no B cells or antibodies due to a defect in signaling at the pre-B cell stage.

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7.2 (see above). Later, the cell can coexpress both IgM and IgD and thus become a mature B cell.

4. If the first rearrangement does not produce a kappa chain, then the second ‘sister’

chromosome will undergo rearrangement. If that is unsuccessful, then the two lambda loci will rearrange one after the other. If those are nonproductive, the cell will undergo apoptosis and die, as was the case for the heavy chain locus.

5. The end result of this process is an immature B cell expressing only IgM with either

kappa or lambda light chains. As the cell leaves the bone marrow, it begins to express both IgM and IgD and thus becomes a mature B cell. This difference is important because immature B cells are more readily tolerized (made nonresponsive to antigen) than are mature B cells.

6. The steps of B cell development are summarized in the following figure (Coico, 2009,

Fig. 7.1). B cell tumors may be ‘frozen’ at different stages of this maturation process; the less mature tumor types tend to be more aggressive and to have a poorer prognosis.

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IV. Mechanisms of Ag-binding diversity It is estimated that each individual is capable of producing B and T cells with 1015 to 1018 different antigen binding sites, each with a different (but perhaps overlapping) antigen specificity! As a result, there are very few compounds that will not induce an immune response, even if they are synthetic and have never been found in nature previously. Equally amazing is that most of the diversity is generated during V(D)J rearrangement, prior to antigen exposure. Thus each individual randomly produces a huge “repertoire” of B and T cells, only a small proportion of which (<1%) will respond to any one antigen or infectious agent. How is this Ag-binding diversity created? As described in the Antibody Structure and Function lecture, the portions of the Variable Region that participate in antigen binding are called Complementarity Determining Regions or CDRs for short. All differences in antigen binding are thus due to differences in these sequences. Two of the CDRs (CDR1 and CDR2) are ‘hard-wired’ into the V gene segment, and thus depend upon the V segment selected during rearrangement. CDR3 consists of the junction of the V, D, and J gene segments and hence has a high degree of variability. The CDRs of both the heavy and light chain participate in the formation of the antigen binding pocket or paratope. V D J

Coico et al., 2009, Fig. 4.4 Coico et al., 2009, Fig. 4.5 There are 5 major mechanisms for generating antibody diversity in humans: 1. Availability of multiple V gene segments – as indicated above, there are ~50 V gene

segments in the heavy chain locus, and about 40 V gene segments each in the kappa and lambda light chain loci. Thus there are about 50 heavy chain and 80 light chain sequences encoding CDR1 and CDR2.

2. Combinatorial diversity (different VDJ and VJ combinations) - the V, D, and J

regions in heavy chains (and the V and J regions in light chains) are selected randomly during V(D)J rearrangement (“joining”). For example, one cell could express a heavy chain with VH3, DH1, and JH5, while another could express VH43, DH3, JH1; Thus 50 x 20 x 6 or 6000 different heavy chain VDJ loci can occur. A lesser degree of variation can occur in the light chain recombinations, because there are no D regions; there are ~200 and ~160 different VJ combinations in kappa and lambda, respectively. This combinatorial diversity affects the sequence of CDR3.

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3. Assortment of heavy and light chains. Because the heavy and light chain loci

recombine independently, each B cell will contain a different combination of H and L chains. This raises the total number of possible V, D, and J combinations to ~2 x 106.

4. Junctional and insertional diversity. The recombination between, say, D and J

segments is not precise, i.e. may occur a few base pairs in one direction or the other. This ‘sloppiness’ causes differences in the amino acid sequence and leads to junctional diversity.

Insertional diversity results from the activity of terminal deoxynucleotide transferase (TDT), an enzyme that is expressed during heavy chain rearrangement. TDT adds nucleotides randomly at the V-D and D-J junctions. Both junctional and insertional diversity affect CDR3. All of the above mechanisms occur during B cell development, before antigen exposure.

5. Somatic hypermutation – the V regions of the antibody heavy and light chain genes undergo a >10,000 higher rate of mutation than ‘regular’ DNA. This somatic hypermutation occurs only after antigen stimulation. Some of these mutations increase the affinity of antibody for antigen, and those B cells expressing antibody with higher affinity will be selectively stimulated, increasing the proportion of high affinity antibody in secondary responses. This process is called affinity maturation.

Altogether, these mechanisms produce almost an endless variety of antibody specificities.

V. Isotype switching Isotype switching (also called class switching) results in the changing of the isotype of antibody expressed by a given B cell, e.g. from IgM to IgG3. Here are some features of isotype switching. 1. The constant region gene expressed is always the one immediately downstream of the

V region (exception: both IgM and IgD can be expressed by ‘niave’ B cells that have not been stimulated by antigen).

2. The V region does not change during isotype switching; therefore the same antigenic specificity is retained.

3. Isotype switching results when antigen-stimulated B cells receive a cytokine signal from T helper cells. For example, IL-4 stimulates B cells to switch to IgE or IgG1.

4. Switching involves the deletion of intervening DNA between specific recombination sites called switch regions (see figure below). Because the intervening DNA is lost, the B cell cannot ‘switch back’ to an isotype that has already been deleted.

5. The V region and C regions are transcribed together, and RNA splicing and translation results in expression of the ‘new’ isotype.

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VI. Membrane vs. secreted Ig expression 1. B cells express only membrane-bound immunoglobulins. 2. Plasma cells express only secreted immunoglobulins. 3. The difference between membrane vs. secreted Ig is the presence or absence of a

hydrophobic ‘tail’ at the carboxy terminus of each heavy chain. 4. Regulation of membrane vs. secreted Ig expression is due to alternative splicing of

the RNA transcript. 1. Membrane-bound form – RNA splicing results in retention of the M exons, which

encode the hydrophobic amino acid region that anchors the Ig in the membrane. 2. Secreted form – the RNA transcript is cleaved before the M exons, resulting in a

readily secreted, hydrophilic form of the heavy chain. 5. Differentiation of a B cell into a plasma cell results in this change. VII. Regulation of Ig expression. 1. After VDJ joining, the promoter 5’ to the V region is brought within a few thousand base

pairs of the enhancer element between the J region and the constant region. The enhancer greatly increases the rate of transcription, increasing Ig production.

2. Ig production is further increased in the differentiation of B cells into plasma cells. VIII. The immunoglobulin gene superfamily How did this fantastic mechanism evolve in the first place? 1. Antibodies are present in some form in all vertebrates, but are not found in invertebrate

animals. However, vertebrates and invertebrates express a large number of closely related cell surface proteins, which are collectively called the immunoglobulin gene superfamily. It is thought that antibodies and T cell receptors evolved from cell surface receptors used for other functions, such as cell-cell interactions.

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2. Domain structure. All members of the immunoglobulin (Ig) gene superfamily contain structures called Ig domains. An example of the two domains found in Ig light chains is shown below. Domains have the following features:

Primary amino acid sequence similarity (often only 20-30 percent) About 100-110 amino acids in length Beta-pleated sheet structure Commonly have an intrachain disulfide bond

3. Members of the Ig gene superfamily are

important in both immunologic and non-immunologic systems, and include the following: Immunoglobulins T cell receptors Major histocompatibility class I and II

proteins Many receptors specific for

leukocytes (e.g. CD3, CD4, and CD8)

Many additional cell-surface receptors not exclusively involved in the immune system (e.g. intercellular adhesion molecule 1 [ICAM1], neuro-cellular adhesion molecule [NCAM], and chorioembryonic antigen [CEA]) Coico et al., Fig. 4.14

4. The ‘Big Bang’ theory – John Marchalonis at University of Arizona and others have

proposed that the genes encoding recombinase proteins RAG1 and RAG2 were obtained by horizontal transfer of DNA from fungi or bacteria, resulting in the sudden acquisition of the antibody system in early vertebrates.

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SUMMARY – GENERATION OF ANTIBODY DIVERSITY

1. Generation of antigen-binding diversity results from V(D)J recombination during B cell development and somatic mutation after antigenic stimulation. Functional diversity of antibodies results from isotype switching, allowing expression of the 9 different Ig isotypes. Each B cell and all of its progeny express only one heavy chain and one light chain V region sequence, and thus all have the same antigenic specificity.

2. During B cell development, rearrangement of the heavy chain locus occurs first. D-J recombination is followed by V-D recombination, resulting in formation of the V domain exon comprised of V, D, and J gene segments. This process requires pairing of 7-bp and 9-bp sequences, after which the intervening DNA is ‘looped out’ and deleted permanently from the chromosome.

3. Light chain rearrangement occurs through a similar process, in which randomly selected V and J gene segments in the kappa or lambda light chain loci are joined.

4. V(D)J recombination proceeds through a hierarchy of heavy chain locus kappa locus lambda locus. Functional heavy and light chains must be produced, or the developing B cell undergoes apoptosis and dies.

5. There are five sources of antibody diversity: 1) presence of multiple V gene segments; 2) Combinatorial diversity, resulting from random recombination of V, D, and J segment combinations; 3) junctional and insertional diversity, resulting in changes in the V-D and D-J junctions; 4) co-expression of different H and L chain pairs; and 5) somatic hypermutation.

6. Isotype switching occurs after antigenic stimulation and requires cytokines produced by T cells. DNA is deleted between switching regions, so that a different constant region gene is juxtaposed close to the V domain exon. Expression of different Ig isotypes results.

7. B cells express only the membrane-bound form of Ig, whereas plasma cells express only the secreted form. This results from differential termination of heavy chain transcription.

8. Ig gene expression is upregulated by enhancer elements and other factors; expression by plasma cells is much higher than in B cells.

9. Immunoglobulins and T cell receptors are members of the immunoglobulin gene superfamily, which apparently first evolved a large set of cell surface receptors.

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THE ROLE OF THE MHC IN THE IMMUNE RESPONSE Jeffrey K. Actor, Ph.D.

713-500-5344

Objectives: (1) Understand genetic organization of the major histocompatibility complex. (2) Present an overview of differential processing of antigens in the MHC class I and class II pathways. (3) Discuss MHC restriction as related to presentation of antigens. (4) Present an overview of disease association with MHC type. (5) Introduce CD1 non-peptide lipid presentation.

Keywords: HLA, H-2, Class I, Class II, 2-microglobulin, APC, Polymorphism, CD1 Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York, NY. 6th edition, 2009. Chapter 8; Geha and Notarangelo. Case Studies in Immunology. Garland Publishing, New York, NY.6th edition, 2012. Case 8: MHC Class II Deficiency. Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/role-of-mhc-in-the-immune-response/ The Major Histocompatibility Complex (MHC) is a locus on a chromosome comprised of multiple genes encoding histocompatibility antigens that are cell surface glycoproteins. MHC genes encode both class I and class II MHC antigens. These antigens play critical roles in interactions among immune system cells; class I participates in antigen presentation by macrophages to CD8+ lymphocytes (CTL), class II molecules participates in antigen presentation by macrophages to CD4+ lymphocytes (T helper). MHC genes are very polymorphic. The locus also encodes a third category of MHC genes, those of the class III type. The class III MHC molecules include complement proteins, tumor necrosis factor, and lymphotoxin. In man, the MHC locus is designated as HLA (Human Leukocyte Antigen). MHC molecules gain their name because they were first identified as the targets for rejection of grafts between individuals. When organs are transplanted across MHC locus differences between donor and recipient, graft rejection is prompt. In 1980 the Nobel Prize was awarded to Baruj Benacerraf, Jean Dausset and George D. Snell, for their work involving the major histocompatibility complex and rejection of skin grafts using inbred strains of lab mice. In mice, the MHC locus is designated as H-2. It has since been determined that the function of the MHC is the presentation of antigen fragments (epitopes) to T cells. Organization and Structure of the MHC Genes and Gene Products MHC molecules are organized into 3 classes. Class I molecules are found on all nucleated cells. The class II molecules are found on B-cells and macrophages. Class III genes encode for various soluble proteins that include certain complement components. Human MHC: The HLA locus in humans is found on the short arm of chromosome 6. The class I region consists of HLA-A, HLA-B, and HLA-C loci and the class II region consists of the D region which is subdivided into HLA-DP, HLA-DQ, and HLA-DR subregions. Class I molecules are important in presentation of intracellular antigen to CD8+ t cells as well as for effector functions of target cells. Class II molecules are important in the induction of an immune response, since antigen-presenting cells must complex an antigen with the class II molecules to present it in

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the presence of cytokines to CD4+ lymphocytes. Class III molecules encoded by genes located between those that encode class I and class II molecules include complement components.

Coico and Sunshine, 2009. Fig. 8.1 Polymorphism of Class I and Class II MHC genes Each chromosome 6 encodes three class I molecules B, C and A and three class II proteins DR, DP and DQ. All six of these MHC molecules show a high level of allotypic polymorphism, i.e. certain regions of the molecules differ from one person to another. The chance of two unrelated people having the same allotypes at all twelve sets of genes that encode MHC molecules is very small. There is a 25% chance of having identical genes that encode MHC molecules with each sibling, but only six of the twelve sets of genes that encode MHC molecules are inherited from each parent. MHC class I and class II molecules that are not possessed by an individual are seen as foreign antigens upon transplantation and are dealt with by the recipient's immune system accordingly. The highly polymorphic class I and class II MHC products are central to the ability of T cells to recognize foreign antigen and the ability to discriminate "self" from "non-self". The Class I MHC molecules are each somewhat different from one another with respect to amino-acid sequence, and all three are co-dominantly expressed in the membrane of every nucleated cell in an animal - but, depending on the organ involved, at different levels of expression (as high as 5 x 105 molecules per cell on lymphocytes). The term "co-dominantly expressed" means that each gene encoding these proteins on each parental chromosome of the diploid cells is expressed. MHC class I molecules expressed on progeny (F1) cells match maternal or paternal class I molecules since only the subunit genes exhibit species-specific polymorphisms. MHC class II molecules expressed on F1 cells include homologous and heterologous dimer mixtures since both and subunit genes exhibit species-specific polymorphism. Homologous dimers match class II molecules expressed on either parental cell type while heterologous dimers are unique to the F1 genotype and are functionally non-equivalent to parental class II molecules. Structure of the MHC Class I Molecule Each class I locus codes for a transmembrane polypeptide of molecular weight approximately 45 kDa, containing three extracellular domains (1, 2, 3). The molecule is expressed at the cell surface in a noncovalent association with an invariant polypeptide called 2-microglobulin (2M)

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of 12 kDa. 2M is a member of the Ig superfamily, the complex of class I and 2M appears as a four-domained molecule with the 2M and 3 domain of class I juxtaposed near the cell surface membrane.

Figure. View of MHC class I showing how a T cell receptor interacts with the class I molecule/2-M with peptide bound in the peptide binding groove.

Figure. Schematic representation of an intact class I antigen in the plasma membrane. MHC class I showing the association of a class I molecule with 2M.

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Structure of the MHC Class II Molecule Class II molecules have 2 transmembrane polypeptide chains ( and , 30-34 and 26-29 kDa respectively); the peptide-binding site is shared by the two domains furthest from the cell membrane. The overall structure of the peptide-binding site is very similar for both class I and class II MHC molecules; the base is made of -pleated sheet, as in an immunoglobulin domain – the sides of the groove that holds the peptide are -helices. Peptides bind within the allele specific pockets defined by the 2 transmembrane polypeptide chains, where they are presented to the TCR for recognition. The extracellular domain shows variability in amino acid sequences, yielding grooves with different shapes. These grooves cradle the processed antigen for interaction with the T cell receptor. The CD4 molecule assists in the recognition process, and binds to the invariant portion of the MHC class II molecules. Like class I genes, class II genes also exhibit polymorphism with multiple allelic forms expressed. In humans, allelic forms are designated different from the mouse. For examples, human class II genes are given numbers such as HLA-D4 or HLA-D7.

Figure. View of MHC class II showing how a T cell receptor interacts with the class II molecule with peptide bound in the peptide binding groove.

Figure. Schematic representation of an intact class II antigen in the plasma membrane. MHC class II showing the two chain class II molecule.

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MHC Class III Molecules: Class III HLA genes encode complement components that show no structural similarity to either class I or class II molecules. These genes, along with genes encoding tumor necrosis factor (TNF), separate HLA class II and class I genes on the chromosome. MHC and Antigen Presentation There are two major classes of presented antigen (Ag) called endogenous and exogenous Ag. MHC class I presents endogenous Ag epitopes to CD8+ T cells and MHC class II present exogenous Ag epitopes to CD4+ T cells. All nucleated cells are capable of presenting MHC class I, but only specialized cells present Ag epitopes on MHC class II. These are macrophages, dendritic cells and B cells. When exogenous Ag enters the body it is phagocytosed, digested and the resulting fragments are presented on MHC class II. When the CD4+ T cell receptor binds Ag-MHC class II it is activated to proliferate and secrete cytokines which in turn activate the other immune competent cells to generate humoral and/or cellular immunity. When CD8+ T Cell (CTL) receptor binds Ag-MHC class I it is activated to produce and secrete toxin that kills the cell to which it is bound. The cells that ingest, digest and present exogenous Ag epitopes on MHC class II are called antigen presenting cells (APCs), and the process of ingestion, digestion and presentation is called antigen processing and presentation. All nucleated cells can display MHC class I, but only APCs display MHC class II. CD8+ T Cell recognize Ag-MHC class I and CD4+ T Cell recognize Ag-MHC

class II. Antigen is recognized in conjunction with proteins of the major

histocompatibility complex (MHC). Different antigen degradation and processing pathways produce MHC-peptide complexes where "endogenous" peptides associate with class I molecules and "exogenous" peptides associate with class II molecules.

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Endogenous (cytoplasmic) antigen processing and MHC class I presentation: MHC class I molecules bind peptide fragments derived from proteolytically degraded proteins endogenously synthesized by a cell. Small peptides are transported into the endoplasmic reticulum where they associate with nascent MHC class I molecules before being routed through the Golgi apparatus and displayed on the surface for recognition by cytotoxic T lymphocytes. MHC class I molecules bind small antigenic peptides that are 8-10 amino acid residues in length.

Coico and Sunshine, 2009. Fig. 8.7.

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Exogenous (endosomal) antigen processing and MHC class II presentation: MHC class II molecules bind peptide fragments derived from proteolytically degraded proteins exogenously internalized by "antigen presenting cells," including macrophages, dendritic cells, and B cells. The resulting peptide fragments are compartmentalized in the endosome where they will associate with MHC class II molecules before being routed to the cell surface for recognition by helper T lymphocytes. MHC class II molecules bind larger antigenic peptides usually 13-18 amino acid residues in length (but may be longer). Like class I molecules, class II MHC molecules are synthesized in the RER. The class II and chains reside there as a complex with an additional polypeptide called the invariant chain (Ii). The invariant chain blocks the groove of the class II molecule and prevents endogenous antigens from binding there. The MHC/invariant chain complex is transported to an acidic endosomal or lysosomal compartment that contains a degraded antigen peptide. The invariant chain comes off the complex, exposes the groove of the class II molecule, and allows the antigen peptide to slip into the groove. The class II/antigen peptide complex is then transported to the surface of the APC where it is available for interaction with CD4 TH cells.

FIGURE 8.5. Processing of exogenous antigen in MHC class II pathway, (Ii = invariant chain; CLIP = fragment of Ii bound to MHC class II groove.)

Coico and Sunshine, 2009. Figure 8.5.

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Coico and Sunshine, 2009. Table 8.1.

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The role of the MHC in Thymic Education The education process by which T cells in the thymus learn to recognize antigenic peptides in the context of self-MHC molecules is a two-step process involving both positive and negative selection. Step 1: Initially, immature thymocytes within the thymic cortex express low

levels of TCR, but high levels of both CD4 and CD8 (double-positive cells). They interact with thymic epithelial cells that express high levels of both class I and class II MHC molecules. Thymocytes with moderate affinities for these self-MHC molecules are allowed to develop further, while thymocytes with affinities too high or too low for self-MHC are induced to die by apoptosis. The thymocytes that survive are said to have been "positively selected" through their interaction with self-MHC.

Step 2: The positively selected thymocytes then begin to express high levels of TCR, some of which recognize self components other than self-MHC. These cells must be deleted to prevent autoimmune destruction of healthy host tissues. Negative selection is the elimination of T cells reactive with self components other than the MHC. Negative selection occurs in the deeper cortex, at the corticomedullary junction, and in the medulla of the thymus. The thymocytes interact with antigen processed and presented by interdigitating cells and macrophages. Only thymocytes that fail to recognize self antigens are allowed to survive and proceed along the maturation process, with the remainder undergoing apoptosis. Eventually, T cells that survive the negative selection process lose either CD4 or CD8, becoming "single positive" cells. Fewer than 5% of thymocytes survive selection and leave the thymus to take up residence in the secondary lymphoid organs.

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Role of MHC in activation of T lymphocytes The binding between the TCR and the MHC/antigen peptide complex is highly specific and acts as the first signal to induce T cell activation. T cells do not respond to either self-MHC alone or to free peptide. Activated T cells differ from resting T cells in that they proliferate and secrete lymphokines and/or lytic substances. The affinity of the TCR for the MHC/antigen complex is often too low to fully activate the T cell; there are numerous accessory molecules that increase avidity between the T cell and APC by performing an adhesive function. Cytotoxic T lymphocytes: CTLs are able to kill target cells directly by inducing apoptosis. Nucleases and other enzymes activated in the apoptotic process may help destroy the viral genome, thus preventing the assembly of virions and potential infection of other cells. CTL induce apoptosis only in the target cell; neighboring tissue cells are not affected. Two mechanisms for induction of apoptosis have been identified: Preformed perforins are released at the target cell surface which generate

transmembrane pores in the target cell, through which a second protein, granzyme, can gain entry to the cytosol and induce the apoptotic series of events.

Apoptitic signaling via membrane-bound molecules can occur via Fas on the target cell surface and Fas ligand on the CTL surface. The processes of antigen recognition, CTL activation and delivery of apoptotic signals to the target cell can be accomplished within 10 minutes. The apoptotic process in the targeted cell may take 4 hours or more and continues long after the CTL has moved on to interact with other tissue cells.

T helper lymphocytes: The initial interaction between T lymphocytes and the APC is mediated by adhesion molecules. Interactions occur between LFA-1, CD-2 and ICAM-3 on the T-cell, and with ICAM-1, ICAM-2, LFA-1 and LFA-3 on the APC. These molecules synergize in binding of lymphocytes to the APCs. This transient binding allows the T-cell to sample the large numbers of MHC molecules on the surface of the APCs for their specific peptide. If a T-cell recognizes its peptide ligand bound to MHC, signaling via the T-cell receptor complex is induces more conformational changes, eventually leading to the production of T-cell cytokines. T cells require co-stimulation through binding of the CD-28 ligand with the CD-80/CD-86 ligand of the APC. T-dependent B cell activation: B cells can also specifically take up antigen via binding through their surface Ig. This is internalized, broken down to peptides and the peptides are presented on the B cell surface held in the peptide binding grooves of MHC class II molecules. If this B cell interacts with a primed T cell that recognizes the peptide/class II complex on the B cell then the T cell may transiently express accessory ligands. This results in cells going into cell cycle and the secretion of cytokines. For the B cell, this action, in concert with correctly released T cell cytokines, will drive isotype switching as well as maturation of the B lymphocyte into a plasma cell.

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Association of Disease with MHC Haplotype Particular MHC alleles are associated with better protection against certain infections. Certain alleles are associated with a greater chance of developing autoimmunity. Some diseases are distinctly more common in individuals with a particular MHC allele or MHC haplotype. Diseases with a strong association with certain MHC alleles include insulin-dependent diabetes and Graves' disease. Expression of HLA-DR4 is associated with rheumatoid arthritis. Nearly 90% of people with ankylosing spondylitis carry the HLA-B27 allele. Expression of HLA-DR2 is associated with multiple sclerosis. The Association of HLA serotype with susceptibility to autoimmune disease will be covered in the AutoImmunity Lecture. An updated list of genetic polymorphisms associated with increased susceptibility to disease will be presented during that lecture. It is hypothesized that in some cases MHC molecules serve as receptors for the attachment and entry of pathogens into the cell; this makes individuals with a certain HLA type more susceptible to infection by a particular intracellular pathogen using that HLA molecule as a receptor. Alternatively, an infectious agent might possess antigenic determinants that resemble MHC molecules (molecular mimicry). Such resemblance might allow the pathogen to escape immune detection because it is seen as "self," or it may induce an autoimmune reaction. Because MHC molecules differ in their ability to accommodate different peptides, some individuals who express certain MHC genes may lack the ability to present microbial epitopes capable of inducing protective T cell responses. Finally, there may simply be no T cells capable of recognizing a particular MHC/antigen combination, leading to a "hole in the T cell repertoire."

Coico and Sunshine, 2009. Figure 8.6.

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Clinical Vignette - The case of Helen Burns (Case 8 in Geha and Notarangelo): Helen Burns was 6 months old when she developed pneumonia, caused by the opportunistic pathogen Pneumocystis carinii. Helen was tested for severe combined immunodeficiency; it was found that Helen's T cells could be stimulated with mitogen (phytohemagglutinin), but could not respond to specific antigenic stimuli. It was further established that Helen had low overall immunoglobulin levels and decreased CD4 cells. Her CD8 cell counts were within normal range. Helen's white blood cells were examined for expression of MHC Class I and Class II molecules. She was diagnosed with MHC Class II deficiency. A bone marrow transplant was performed using Helen's mother as a donor. The graft was successful and immune function was restored.

How does MHC Class II deficiency selectively affect CD4 T cell function, and what implications does this have towards immune responses to infective agents?

Geha and Notarangelo, 2012. Figure 8.3.

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Summary: Role of HLA in the Immune System MHC molecules are organized into 3 classes. Class I molecules are found on all nucleated cells. The class II molecules are found on B-cells and macrophages. Class III genes encode for various soluble proteins that include certain complement components. Human class I region genes consists of HLA-A, HLA-B, and HLA-C loci and the class II region genes consists of the D region which is subdivided into HLA-DP, HLA-DQ, and HLA-DR subregions. All MHC molecules show high allotypic polymorphism.

Class I molecules are important in presentation of Ag epitopes to CD8+ T cells as well as for effector functions of target cells. Each class I locus codes for a transmembrane polypeptide containing three extracellular domains (1, 2, 3), which is expressed at the cell surface in a noncovalent association with 2-microglobulin (2M). All nucleated cells express Class I molecules. Class II molecules present antigen in the presence of cytokines to CD4+ lymphocytes. Class II molecules have 2 transmembrane polypeptide chains. Peptides bind within the allele specific pockets defined by the 2 transmembrane polypeptide chains, where they are presented to the TCR for recognition.

Different antigen degradation and processing pathways produce MHC-peptide complexes where "endogenous" peptides associate with class I molecules and "exogenous" peptides associate with class II molecules. MHC class I molecules bind small antigenic peptides that are 8-10 amino acid residues in length; MHC class II molecules present slightly larger peptides.

T cells in the thymus learn to recognize antigenic peptides in the context of self-MHC molecules by a two-step process involving both positive and negative selection.

The binding between the TCR and the MHC/antigen peptide complex acts as a signal to induce T cell activation. T cells do not respond to either self-MHC alone or to free peptide. Accessory molecules increase avidity between the T cell and APC by performing an adhesive function.

CTLs recognize Ag in the context of MHC class I, and kill target cells directly by inducing apoptosis. They release preformed perforins at the target cell surface to generate transmembrane pores in the target cell, through which a second protein, granzyme, gains entry to the cytosol to initiate an apoptotic series of events. CTLs can also deliver apoptitic signals via surface bound molecules.

T helper lymphocytes recognize Ag and MHC class II on the APC in a manner mediated by adhesion molecules. The recognition is specific and requires co-stimulation through ligand interactions on the APC. Activation of the T helper cell leads to specific cytokine release. B cells are good antigen presenters to T cells.

Certain MHC alleles are associated with a greater chance of protective immune responses to pathogens, as well as towards developing autoimmunity. Some diseases are distinctly more common in individuals with a particular MHC allele or MHC haplotype. Possible reasons for this are discussed.

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T CELL RECEPTOR: Structure and Genetic Basis

Jeffrey K. Actor, Ph.D. 713-500-5344

Objectives: (1) Present an overview of the T receptor structure and organization of the gene loci encoding for the T cell receptor chains; (2) explain mechanisms underlying generation of T cell receptor diversity; (3) examine the stages in thymic selection of T lymphocytes; and (4) compare and contrast the T cell receptor with the B cell receptor. Keywords: T cell receptor (TCR). Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York, NY. 6th edition, 2009. Chapter 9; Geha and Notarangelo. Case Studies in Immunology. Garland Publishing, New York, NY. 6th edition, 2012. Case 7: Omenn Syndrome. Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/the-t-cell-receptor-structure-and-genetic-basis/ The acquired immune response is subdivided, based on participation of two major cell types. B lymphocytes originate in the bone marrow, and synthesize/secrete antibodies. This is termed humoral immunity. T lymphocytes mature in the thymus, and secrete immunoregulatory factors following interaction with antigen presenting cells; this is termed cellular immunity (CMI). Lymphocyte Biology Lymphoid cells provide efficient, specific and long-lasting immunity against microbes/pathogens and are responsible for acquired immunity. This lecture will primarily examine the biology of two classes of lymphocytes: (1) thymic-dependent cells or T lymphocytes that operate in cellular and humoral immunity; and (2) B lymphocytes that differentiate into plasma cells to secrete antibodies. T and B lymphocytes produce and express specific receptors for antigens. The major properties of the acquired immune response are specificity, memory, adaptiveness, and discrimination between self and non-self. All of these properties are related to the random selection of variable region components during the development of both B cells and T cells. The lymphatic organs are tissues in which lymphocytes mature, differentiate and proliferate. The primary (central) lymphoid organs are those in which B and T lymphocytes mature into antigen recognizing cells. In embryonic life, B cells mature and differentiate from hematopoietic stem cells in the fetal liver. After birth, B cells differentiate in the bone marrow. Maturation of T cells occurs in a different manner. Progenitor cells from the bone marrow migrate to the thymus where they differentiate into T lymphocytes. The T lymphocytes continue to differentiate after leaving the thymus, and are driven to do so by encounter with specific antigen in the secondary lymphoid organs.

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The secondary lymphoid organs are those tissues in which antigen-driven proliferation and differentiation take place. The spleen and lymph nodes are the major secondary lymphoid organs. Additional secondary lymphoid organs include the tonsils, appendix, and Peyer’s patches. Aggregates of cells in the lamina propria of the digestive tract lining may also be included in this category, as well as any tissue described as MALT (mucosa-associated lymphoid tissue), GALT (gut-associated lymphoid tissue) or BALT bronchus-associated lymphoid tissue). T Lymphocytes: T lymphocytes are involved in regulation of immune response and cell mediated immunity. They provide necessary factors to help B cells produce antibody. Mature T cells express antigen-specific T cell receptors (TCR). Every mature T cell expresses the CD3 molecule, which is associated with the TCR. The TCR/CD3 complex recognizes antigens associated with the major histocompatibility complex (MHC) molecules on target cells (e.g. virus-infected cell). The TCR is also expressed on the cell surface in association with co-receptor or accessory molecules (CD4 or CD8).

The structure of the T-cell receptor (TCR) complex showing the predominant form of the antigen-binding chains, and , and the associated signal transduction complex, CD3 (, , and chains) plus (zeta) or eta) or (theta. (-) and (+) represent electrostatic interactions.

T Cell Receptor: The TCR is a transmembrane heterodimer composed of two disulfide-linked polypeptide chains. T lymphocytes of all antigenic specificities exist prior to contact with antigen. Each lymphocyte carries a TCR of only a single specificity. T Lymphocytes can be stimulated by antigen to give rise to progeny with identical antigenic specificity. Lymphocytes reactive with “self” are deleted or inactivated to ensure that no immune response is mounted against self components. The vast majority of T lymphocytes express alpha [] and beta [] chains on their surface. Cells that express gamma [] and delta [] chains comprise only 5% of the normal circulating T cell population in healthy adults. Each chain (, or ) represents a distinct protein with approximate molecular weight of 45 kDa. An individual T cell can express either an or a heterodimer as its receptor, but never both. The TCR recognizes antigen in the form of peptides which are bound in the groove on MHC molecules (reviewed in detail in lecture: Role of MHC in Immune Response). The interactions

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between heterodimers create three hypervariable regions called complementarity determining regions (CDRs 1, 2, and 3).

The interaction of TCR, MHC, and peptide. The complementarity determining regions (CDRs) of the TCR V regions and peptide bound in the peptide-binding groove of an MHC class I molecule are depicted. [Based on the crystal structure described by K. C. Garcia et al. (1998): Science 279: 1166.]

The T cell receptor genes are closely related members of the immunoglobulin gene superfamily. Each chain consists of a constant (C) and a variable (V) region, and is formed by a gene-sorting mechanism similar to that found in antibody formation. The repertoire is generated by combinatorial joining of variable (V), joining (J), and diversity (D) genes, and by N region diversification (nucleotides inserted by the enzyme deoxynucleotidyl-transferase). Unlike

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immunoglobulin genes, genes encoding TCR do not undergo somatic mutation. Thus there is no change in the affinity of the TCR during activation, differentiation, and expansion. TCR-CD3-complex: The TCR heterodimer is tightly associated with six independently encoded CD3 subunits (, , , , and ) required for efficient transport to the cell surface. CD3 subunits possess long intracellular tails and are responsible for transducing signals upon TCR engagement. Genes Coding for T-Cell Receptors: Genes which code for the T cell receptor and the mechanisms used to generate TCR diversity are similar to those of immunoglobulins. The TCR V, D, and J genes are mixed together in a more complicated manner than found for

immunoglobulin genes. and uses only V and J gene segments. and use V, D, and J gene segments. There are many more V and V genes (50-100) than V and V genes (5-10) present in germ

line. The and chain genes are mixed together in one locus. The genes encoding the chain are

entirely located between the cluster of V and J gene segments. The top and bottom rows show germline arrangement of the variable (V), diversity (D), joining (J), and constant (C) gene segments at the T-cell receptor and loci. During T- cell develop-ment, a V-region sequence for each chain is assembled by DNA recombination. For the chain (top), a V gene segment rearranges to a J gene segment to create a functional gene encoding the V domain. For the chain (bottom), re-arrangement of a D, a J, and a V gene segment creates the functional V-domain exon.

Geha and Notarangelo, 2012. Figure 7.1. Order of TCR Gene Rearrangement: The earliest cell entering the thymus has its TCR genes in the germ line configuration

(unrearranged). Both and chain genes then begin to rearrange, more or less simultaneously. If the chain genes rearrange successfully, then chain genes also start to rearrange. If both

and genes rearrange functionally, no further gene rearrangement takes place and the cell remains a T cell.

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If and/or rearrangements are not functional, then gene rearrangement continues followed by gene rearrangement. In this manner, a product appears, and the cell becomes an T cell.

The Process of Recombination: Recombination of V, D, and J gene segments is coordinated by recombinase-activating genes RAG-1 and RAG-2. The enzymes recognize specific DNA signal sequences consisting of a heptamer, followed a spacer of 12 or 23 bases, and then a nonamer. If either RAG gene is impaired or missing, homologous recombination events are abolished. This gives rise to severe combined immunodeficiency (SCID). Mutations which result in partial enzymatic activity can also occur, and can give rise to immunodeficiency diseases. An example of such disorder is Omenn Syndrome, discussed in detail in the Case Studies in Immunology (Geha and Notarangelo, chapter 7) text.

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Generation of T-Cell Receptor Diversity: The overall level of diversity is greater for T cell receptors than that for immunoglobulins. This is primarily due to additional junctional diversity in possible TCR gene rearrangements. Most of the variability in the TCR occurs within junctional regions encoded by D, J and N nucleotides. This is the region that corresponds to the CDR3 loops that form the center of the antigen binding sites. So, while the center of the binding site is highly variable, the remaining portion of the heterodimer is subject to relatively little variation.

Immunoglobulins T cell : Receptors Number of V gene pairs ~2 - 3.4 x 106 5.8 x 106 Junctional diversity ~3 x 107 ~2 x 1011 Total Diversity ~1014 ~1018

Development of T lymphocytes During differentiation in the thymus, immature T cells undergo rearrangement of their TCR and genes to generate a diverse set of clonotypic TCRs. Immature thymocytes are selected for further maturation only if their TCRs do not interact with self-peptides presented in the context of self-major histocompatibility complex (MHC) molecules on antigen presenting cells. Different signals lead to the alternate developmental outcomes of maturation or apoptosis (positive versus negative selection). Positively selected thymocytes undergo alternate commitment to either the T killer or T helper lineages, which correlate precisely with a cell's TCR specificity towards MHC class I or II molecules, respectively. Lineage commitment is marked phenotypically by the loss of expression of one of the co-receptor molecules, CD8 or CD4. Immature thymocytes express both co-receptors (double positive), while T killer or T helper cells express only CD8 or CD4, respectively (single positive CD8+ or CD4+). Figure. Changes in surface molecules of thymocytes at different stages of maturation.

The majority of peripheral blood T lymphocytes express the and form of the TCR. In healthy adults, less than 5% express a heterodimer comprised of the and chains. Virtually all the cells that express the TCR- are CD4+CD8- (T helper) or CD4-CD8+ (T cytotoxic or T suppressor). Almost all cells expressing TCR- are CD4-CD8- (double negative). While the TCR- expressing lymphocytes are known to function as helper and cytotoxic cells, the function of the TCR- cells is not well understood.

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No interaction =

CD4+CD8+ cell DEATH

Interaction = Pos Selection

MHC + self

MHC + non-self

++, CD4+CD8+ cell interacts with Thymic epithelial cell

++, CD4+CD8+ cell

interacts with interdigitating cell

High affinity interaction =

DELETION

Low affinity interaction =

SURVIVALCommitment CD4+ or CD8+

No interaction =

CD4+CD8+ cell DEATH

Interaction = Pos Selection

MHC + self

MHC + non-self

No interaction =

CD4+CD8+ cell DEATH

Interaction = Pos Selection

MHC + self

MHC + non-self

++, CD4+CD8+ cell interacts with Thymic epithelial cell

++, CD4+CD8+ cell

interacts with interdigitating cell

High affinity interaction =

DELETION

High affinity interaction =

DELETION

Low affinity interaction =

SURVIVALCommitment CD4+ or CD8+

Low affinity interaction =

SURVIVALCommitment CD4+ or CD8+

Figure. Main stages in the development of a T lymphocyte.

Figure. Main stages in Thymic Selection. T Helper Cells: T helper cells (Th) are the primary regulators of T cell- and B cell-mediated responses. They 1) aid antigen-stimulated subsets of B lymphocytes to proliferate and differentiate toward antibody-producing cells; 2) express the CD4 molecule; 3) recognize foreign antigen complexed with MHC class II molecules on B cells, macrophages or other antigen-presenting cells; and 4) aid effector T lymphocytes in cell-mediated immunity. Currently, it is believed that there are two main functional subsets of Th cells, plus other helper subsets of importance. T helper 1 (Th1) cells aid in the regulation of cellular immunity, and T helper 2 (Th2) cells aid B cells to produce certain classes of antibodies (e.g., IgA and IgE). The functions of these subsets of Th cells depend upon the specific types of cytokines that are generated, for example interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by Th1 cells; IL-4, IL-6 and IL-10 by Th2 cells. Two other classes of T helper cells are thought to be involved in oral tolerance and serve as regulators for immune function. Th3 cells secrete IL-4 and TGF- and provide help for IgA production, and have suppressive properties for Th1 and Th2 cells. Th17

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cells, characterized by IL-17 secretion, are thought to be involved as effector cells for autoimmune disease progression. T Cytotoxic Cells: T cytotoxic cells (CTLs) are cytotoxic against tumor cells and host cells infected with intracellular pathogens. These cells 1) usually express CD8, and, 2) destroy infected cells in an antigen-specific manner that is dependent upon the expression of MHC class I molecules on antigen presenting cells. T Suppressor/ T Regulatory Cells: T suppressor cells suppress the T and B cell responses and express CD8 molecules. T regulatory cells also affect T cell response, with many cells characterized as CD4+CD25+, TGF- secretors. T Cells: Not all T cells express TCRs. An alternative is to express chains of the TCR. Generally, cells lack CD4, although some cells do express CD8. The functions of cells are not well understood. T cells can function in the absence of MHC molecules. They home to the lamina propria of the gut, and are thought to assist in protection against microorganisms entering through epithelium at mucosal surfaces. Their range of response to antigens is limited. expressing cells have been found to be active towards mycobacterial antigens and heat shock proteins. They have the ability to secrete cytokines like their counterparts. Natural Killer T Cells: Natural killer T cells (NKT) are a heterogeneous group of T cells that share properties of both T cells and natural killer (NK) cells. The majority of these cells recognize an antigen-presenting molecule (CD1d) that binds self- and foreign lipids and glycolipids. They constitute only 0.2% of all peripheral blood T cells. The term “NK T cells” was first used in mice to define a subset of T cells that expressed the natural killer (NK) cell-associated marker NK1.1 (CD161). It is now generally accepted that the term “NKT cells” refers primarily to CD1d-restricted T cells coexpressing a heavily biased, semi-invariant T cell receptor (TCR) and NK cell markers. Natural killer T (NKT) cells should not be confused with natural killer (NK) cells. - - - - - - - - - - - - - - - - - - - - - - - - - - Comparison of the B cell and T cell receptors:

Both BCRs and TCRs share these properties: they are integral membrane proteins they are present in thousands of identical copies exposed at the cell surface they are made before the cell ever encounters an antigen they are encoded by genes assembled by the recombination of segments of DNA allelic exclusion ensures only one receptor with a single antigenic specificity they demonstrate N region addition during gene rearrangement they have a unique binding site this site binds to a portion of the antigen called an antigenic determinant or epitope the binding, like that between an enzyme and its substrate depends on complementarity of

the surface of the receptor and the surface of the epitope the binding occurs by non-covalent forces (again, like an enzyme binding to its substrate) successful binding of the antigen receptor to the epitope, if accompanied by additional

"signals", results in: 1. stimulation of the cell to leave G0 and enter the cell cycle

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2. repeated mitosis leads to the development of a clone of cells bearing the same antigen receptor; that is, a clone of cells of the identical specificity.

BCRs and TCRs differ in:

their structure the genes that encode them the type of epitope to which they bind TCRs do not somatically mutate TCRs do not undergo isotype switching TCR gene recombination exhibits far greater junctional diversity than Ig genes TCRs are never secreted from the T cell

Summary: T Lymphocytes T lymphocytes are involved in regulation of immune response and in cell mediated immunity. Every mature T cell expresses CD3, which is associated with the TCR. During thymic differentiation, immature T cells undergo rearrangement of their TCR and genes to generate a diverse set of clonotypic TCRs. Immature thymocytes are selected for further maturation only if they recognize foreign antigens in the context of MHC molecules. Mature T cells usually display one of two accessory molecules. CD4+ T helper cells are the primary regulators of T cell- and B cell-mediated responses, and are further subdivided into functional subsets dependent upon cytokines secreted. CD8+ T cytotoxic cells (CTLs) are cytotoxic against tumor cells and host cells infected with intracellular pathogens. T suppressor cells suppress the T and B cell responses and express CD8 molecules. Summary: T Cell Receptor: Structure and Genetic Basis Mature T cells express antigen-specific TCR in a complex with CD3 molecules. The TCR is a disulfide-linked heterodimer composed of either or chains. T cells express either or chain heterodimers, but never both.

Clinical Vignette - Omenn Syndrome (Case 7 in Geha and Notarangelo): Patients with Omenn syndrome demonstrate severe immunodeficiency characterized by the presence of activated, anergic, oligoclonal T cells, hypereosinophilia, and high IgE levels. There is a body of evidence to indicate that the immunodeficiency manifested in patients with Omenn syndrome arises from mutations that decrease the efficiency of V(D)J recombination. These individuals bear missense mutations in either the RAG-1 or RAG-2 genes that result in partial activity of the two proteins. In many cases, amino acid substitutions map within the RAG-1 homeodomain and decrease DNA binding activity, while others lower the efficiency of RAG-1/RAG-2 interaction.

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T cell receptor genes are closely related members of the immunoglobulin gene superfamily and derive part of their structural diversity form recombination of different V, D, and J gene segments. The mechanisms for T cell receptor gene switching are similar to those of immunoglobulin genes, but T cell receptor genes do not have somatic mutations. chains of the TCR have only V and J segments, and join to chains. chains of the TCR have genes for V, D, and J segments. The process of recombination is coordinated by recombinase-activating genes RAG-1 and RAG-2. If rearrangements are unsuccessful on both chromosomes, chains join to chains to give phenotypic T cells. chains have only V and J segments; chains have V, D, and J segments.

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ADAPTIVE IMMUNE RESPONSE I and II

Jeffrey K. Actor, Ph.D. MSB 2.214, 713-500-5344

Required Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York, NY. 6th edition, 2009. Chapters 7, 9, 10, 11. Geha and Notarangelo. Case Studies in Immunology. Garland Publishing, New York, NY. 6th edition, 2012. Case 47. Toxic Shock Syndrome.

Supplemental Reading for Cancer Immunology: Coico and Sunshine, 2009. Chapter 19. Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/adaptive-immune-response/ OBJECTIVES

1. Distinguish between innate and adaptive immune responses on the basis of antigen specificity, HLA restriction and memory.

2. Understand role of immune mediators including cytokines, chemokines, costimulatory and adhesion molecules in the development of adaptive immune responses.

3. To describe the various effector and regulatory functions of T and B cells. 4. To demonstrate the molecular events associated with T cell and B cell activation. 5. Compare and contrast effector cells in cytotoxic mediated immunity.

a. Describe the concepts of tumor antigens. b. Describe the effectors mechanisms in tumor immunity.

6. To develop a practical understanding of mechanisms and clinical relevance of T - dependent and - independent antibody responses.

LEARNING OBJECTIVES: KEY WORDS cytokine, T cell receptor, B cell receptor, helper T cell, cytotoxic T

lymphocyte, NK cell, NKT cell, T-dependent antibody, T-independent antibody response.

I. OVERVIEW OF THE IMMUNE RESPONSE

The acquired immune response is subdivided, based on participation of two major cell types. B lymphocytes originate in the bone marrow, and synthesize/secrete antibodies. This is termed humoral immunity. T lymphocytes mature in the thymus, and secrete immunoregulatory factors following interaction with antigen presenting cells; this is termed cellular immunity (CMI).

A. Purpose – maintain homeostasis B. Discriminates

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Self from nonself (foreign, effete) Pathogenicity – ability to cause disease Intracellular vs. extracellular pathogen

C. Begins in utero D. Remembers previous encounters E. Goal is proper specificity, intensity, and duration

The major properties of the acquired immune response are specificity, memory, adaptiveness, and discrimination between self and non-self. All of these properties are related to the random selection of variable region components during the development of B cells and T cells. The essential features for clonal selection of these cells include: B and T lymphocytes of all antigenic specificities exist prior to contact with antigen. Each lymphocyte carries specific surface molecules (immunoglobulin or T cell

receptor) of only a single specificity. Lymphocytes can be stimulated by antigen under appropriate conditions to give rise

to progeny with identical antigenic specificity. Lymphocytes potentially reactive with “self” are deleted or inactivated to ensure that

no immune response is mounted against self components.

II. MOLECULAR COMPONENTS

A. Cytokines When confronted with above challenge, the host immune response will determine appropriate degree of antigen-specific cell mediated vs. humoral response. In order to accomplish this, various regulatory networks controlled by cytokines are activated.

1. Physical and Biological Properties small mol weight peptides and glycopeptides produced by a variety of cell types

accessory cells leukocytes somatic cells – endothelium, fibroblasts, etc.

short plasma half lives (makes determining levels clinically difficult) modulate immune/inflammatory responses by stimulating/inhibiting

various cell populations (inflammatory, epithelial, fibroblast) one cell type can make multiple cytokines and a single cytokine can be

made by a variety of cell types redundancy – multiple cytokines can have the same biological activity

pleomorphism/pleotropic – the same cytokine can have multiple activities depending upon the target cell, concentration and/or presence of other cytokines

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action can be endocrine, paracrine and/or autocrine

2. Classification – Biological Activity Interferons (,, – interfere with viral replication but also have

immunomodulatory properties Colony Stimulation Factors – support growth of WBC elements of bone

marrow; mediate various inflammatory reactions Tumor Necrosis Factors – produce hemorrhagic necrosis of tumors in

mice; major mediator of inflammation and is elevated in sepsis syndrome

Chemokines – groups of molecules that mediate chemotaxis of various inflammatory cells

Interleukins – various immunoregulatory functions between (inter) various leukocyte (leukin) populations

AN EXPANDED LIST OF CYTOKINES, INTERFERONS AND CHEMOKINES ARE INCLUDED IN THE APPENDIX.

III. T LYMPHOCYTES T lymphocytes are involved in the regulation of the immune response and in cell mediated immunity, and help B cells to produce antibody. Mature T cells express antigen-specific T cell receptors (TCR). Every mature T cell expresses the CD3 molecule, which is associated with the TCR. In addition mature T cells usually display one of two accessory molecules, CD4 or CD8. The TCR/CD3 complex recognizes antigens associated with the major histocompatibility complex (MHC) molecules on target cells (e.g. virus-infected cell). Important T cell markers

Surface Markers of T cells. Additional markers include: CD45RO, Leukocyte common antigen for memory T cells (activated). CD45RA, Leukocyte common antigen for naive T cells (resting). Coico and Sunshine, 2009. Figure 9.4.

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TCR-CD3-complex: The TCR heterodimer is tightly associated with the CD3 co-receptor made up of independently encoded subunits (, , , and two chains). The CD3 complex is required for efficient transport of the TCR to the cell surface. CD3 subunits possess long intracellular tails and are responsible for transducing signals upon TCR engagement with MHC presented antigen.

A. T Helper cells T helper cells (Th) are the primary regulators of T cell- and B cell-mediated responses. They 1) aid antigen-stimulated subsets of B lymphocytes to proliferate and differentiate toward antibody-producing cells; 2) express the CD4 molecule; 3) recognize foreign antigen complexed with MHC class II molecules on B cells, macrophages or other antigen-presenting cells; and 4) aid effector T lymphocytes in cell-mediated immunity. 1. Paired Interactions between the APC and CD4+ T cell

(Immunological synapse)

Antigen receptor MHC II : TCR - antigen binding MHC II : CD4 molecule - the co-receptor

Costimulatory pairs - second signals CD40:CD40L (CD154) CD28/CTLA-4:B7 (CD80, CD86)

Adhesion molecules CD58(LFA-3):CD2 CD54(ICAM-1):CD11a/CD18 (LFA-1)

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2. The TH1/TH2 paradigm The TH1/TH2 paradigm was first proposed by Mossman and Coffman to explain the differential effects of T cell help – i.e. T cells helping B cells and T cells helping other T cells. These cells were distinguished functionally rather than morphologically by the differences in cytokine patterns that they produced.

Differential production of specific cytokine patterns by subpopulations of CD4+ cells

TH1 help other T cells develop immunity against intracellular pathogens

(mostly T cell-mediated)

TH2 help B cells (and other WBC) develop immunity against extracellular pathogens (mostly through IgE, mast cells and eosinophils

Currently, it is believed that there are multiple functional subsets of Th cells. In addition to the ones mentioned above: Th17 cells, characterized by IL-17 secretion, are thought to be involved as effector cells for autoimmune disease progression, and protect surfaces (skin, gut) from extracellular bacteria. Tfh cells (follicular helper T cells) provide help to B cells in germinal areas enabling them to develop into antibody-secreting plasma cells; they function inside of follicular areas of lymph nodes. The functions of all the subsets of Th cells depend upon the specific types of cytokines that are generated, for example interferon-gamma (IFN-gamma) by Th1 cells and IL-4 by Th2 cells, and IL-17 by Th17 cells.

Dendritic Cell

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Regulatory T cells (Treg) represent subpopulations of T helper cells that trigger suppressive activities following engagement of their T cell receptor with presenting antigen occupying the MHC on an antigen presenting cell. They typically secrete molecules such as TGF-β, which function to suppress other T helper cell type activity. They usually express CD4 and CD25 on their cell surface, and express the transcription factor Foxp3.

T-cell Regulation: Treg receive a signal via CTLA-4 which induces their suppressive activity. Treg may also receive a signal triggering their suppressive activity following interaction with an MHC class II molecule. Treg may then suppress the activation of CD4+ T cells by secreting TGF- (and IL-10). FIGURE 12.4. From Coico, 2009.

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3. Intracellular Events – receptor mediated transcription of cytokine genes by a sequence of molecular events

FIGURE. Intracellular events in CD4+ T-cell activation. The result of activation events is enhanced transcription and increased stabilization of IL-2 mRNA. Coico, 2009.

MHCII/peptide binds TCR TCR activates CD3 CD3 transduces activation signal across membrane Tyrosine kinases (Fyn, Lck) activated by CD45 Fyn, Lck cluster with ITAMs and phosphorolate them ZAP-70 (another tyrosine kinase) binds to ITAMs Activated ZAP-70 binds to phospholipase c- (PLC-) PLC- splits PIP2 into DAG and IP3

DAG activates PKC >>>>> NF- (transcription factor) IP3 increases iCa++ >>> activated calcineurin >>> NF-AT

Transcription factors enter nucleus and bind to chromosomes Upregulate T cell activation genes (cytokine, cytokine receptors) Upregulate adhesion molecules on surface to promote further

activation events.

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4. Other events of T cell activation

Decreased expression of selectins molecules to allow homing to lymph nodes

Requirement for multiple signals from APC to activate cell

Function of costimulatory pairs – promote the T cell activation process CD40: CD154(CD40L) CD80/CD86 (B7.1,B7.2):CD28

CD80 binding upregulates TH1 CD86 binding upregulates TH2 CD28 binding upregulates IL-2 production Lack of CD28 binding induces tolerance

CD80/86:CTLA-4 downregulates IL-2 production negative activation signal – tolerance induces memory cell formation

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B. Role of Cytotoxic Cell-Mediated Immunity in Host Defense

Host defenses against extracellular infectious agents (e.g., bacteria, protozoa, worms, fungi) typically utilize (1) Antibody, (2) Complement, and/or (3) activated Phagocytes. However, these mechanisms are not adequate for defense against intracellular infectious agents (an infectious agent that invades a host cell). Therefore a different defense system is required. The mechanisms used are those referred to as cytotoxic cell mediated immunity.

Induction of helper function for cytotoxic cell mediated immunity. In many cases, first CTL encounter with antigen must have help from Helper T cells. The helper cells must recognize antigen presented by MHC Class II molecules on an APC (antigen presenting cell) (dendritic cell or macrophage). The activated Th1 cell secretes IL-2 and IFN-, which activates CTLs. Activation of Th1 cells also triggers the activation of NK cells and macrophages which then target specific cells.

Generation of CD8+ T cells effector cells and target cell killing. (A) dendritic cells activate CD8+ T cells directly. (B) One pathway for CD4+ T cells to activate CD8+ T cells. (C) Target cell killing by a CD8+ effector T cell. Coico and Sunshine, 2009. Fig 10.10.

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Effector cells in Cytotoxic Cell Mediated Immunity. Both innate and adaptive cells play a role in cytotoxic cell mediated immunity. The major cell players and their properties are listed and summarized in the table below.

CTLs – Antigen specific and MHC Class I restricted. i. CTLs express CD8. ii. CTLs kill their targets by using Perforin, Granzymes, Cytokines,

Fas and Fas ligand.

NK cells - nonspecific (they do not use a T cell receptor). i. Morphologically large granular lymphocytes (LGLs); ii. Non-T and non-B lymphocytes lacking surface CD3, CD4, CD8 and

CD19. They do not express immunoglobulins or TCRs. iii. NK cells express CD16 and CD56. iv. NK cells kill by releasing perforin, granzymes and cytokines (IFN-

and TNF).

Lymphokine activated killer cells (LAK cells) are i. Morphologically LGLs. ii. Non-T non-B lymphocytes. iii. Reaction –nonspecific.

NK-ADCC

i. antibody-dependent cellular cytoxicity (ADCC). ii. Have Fc receptors (CD16) that recognize Fc portion of IgG.

Table 1: Effector Cells in Cytotoxic Cell Mediated Immunity

Effector Cell CD markers Effector Molecules

MHC recognition

Antigen recognition

CTL TCR,CD3,CD8,CD2 Perforin, cytokines (TNF-β, IFN-)

required Class I

specific TCR

NK cell CD16,CD56, CD2 Perforin, cytokines (TNF-β, IFN-)

no nonspecific

NK cell ADCC

CD16,CD56, CD2 Perforin, cytokines (TNF-β, IFN-)

no specific IgG

LAK cell CD16,CD56, CD2 Perforin, cytokines (TNF-β, IFN-)

no nonspecific

Macrophage CD14 TNF-α, enzymes, NO, O radicals

no nonspecific

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Cytotoxic cells (CTLs) directly kill tumor cells and host cells infected with intracellular pathogens. These cells 1) usually express CD8, and, 2) destroy infected cells in an antigen-specific manner that is dependent upon the expression of MHC class I molecules on antigen presenting cells.

1. General Considerations

Adaptive host defense against intracellular pathogens CD8+ CTL is MHC I restricted Is affected by TH1 cells which are also antigen-specific but MHC II

restricted 2. Development of CTL

TCR interacts with MHC I – antigen complex o In association with CD8 o Also involves costimulatory molecules

IL-2R upregulated o IL-2 from Th cells cause clonal proliferation o IFN causes activation of CTL

3. Killing of Target cells by CTL IFNupregulates perforin formation

o Perforins form transmembrane channels that kill target o Similar to complement-mediated lysis

IFNupregulates granzyme formation o Serine proteases

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o Pass to target through the perforin-induced channels o Activate target cell apoptosis

Fas/FasL (CD95/95L) interaction

o FasL expression on T cell upregulated in activated CTL o Initiates apoptosis in target through formation of capsases

CTL releases “doomed” target to kill more target cells if available As response is regulated, CTLs themselves undergo apoptosis Remnant is antigen-specific memory CTL

Table 2: Cytotoxic Products of Activated CTLs

Cytotoxic Product

Effect on Target cell

Perforins

TNF-

Fas ligand

Nucleases Serine proteases

- Polymerize in the membrane of the target cell to form poly-perforin channels that allow cytosol to leak out and toxic molecules to enter the cell.

- Degrades proteins in cell membrane

- Initiates apoptosis

- Degrades DNA and RNA in the cell - Degrade proteins in the cell membrane

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Cell-Mediate Responses to Tumor Cells Many concepts discussed to date also apply to protection against tumor cell development. Please refresh these concepts by visiting Chapter 19 of the Coico and Sunshine text, beginning on page 303. This will be revisited later in the semester.

Effector mechanisms in tumor immunity Effector Mechanism Comment

B cells and antibodies (ADCC, CDC) Role in immunity– poorly understood

T cells (cytolysis, apoptosis) Virally- and chemically–induced tumors

NK cells (cytolysis, apoptosis, ADCC) Tumor cells not expressing MHC class 1 alleles- rejected by NK cells

LAK cells (cytolysis, apoptosis) Anti tumor response- to adoptive transfer to LAK cells

Macrophages and neutrophils Activated– by using bacterial products

Cytokines (apoptosis, recruitment of inflammatory cells)

Using adoptively transferred tumor cells- eg: GM-CSF

Limitations of effectiveness of immune responses against tumors Tumor Related Mechanisms of Escape Related Mechanisms of Escape

Failure of tumor to provide a suitable antigenic target or an effective immune response;

- lack of tumor antigen

- lack of MHC class 1 -deficient antigen processing

-antigen modulation

-antigenic masking of tumor

-resistance of tumor to tumoricidal pathways

-lack of co-stimulatory signals

-production of inhibitory cytokines -shedding of tumor antigens

Failure of host to antigenic tumor cells: -immuno-supression or immuno- deficiency

-deficiency in inducing

apoptosis and cell death

signaling - infections or old age

- deficiency in tumor antigen

presentation by host APC

- failure of host effector cells to reach the tumor (eg: stromal barrier) - failure of host to kill variant tumor cells

- T reg hindrance to tumor immunity

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C. Recognition of “Different” Antigens by T cell receptors 1. Presentation of Lipids and Glycolipids

Natural Killer T Cells: Natural killer T cells (NKT) are a heterogeneous group of T cells that share properties of both T cells and natural killer (NK) cells. These cells primarily recognize an antigen-presenting molecule (CD1d) that binds self- and foreign lipids and glycolipids*. They constitute only 0.2% of all peripheral blood T cells. The term “NK T cells” was first used in mice to define a subset of T cells that expressed the natural killer (NK) cell-associated marker NK1.1 (CD161). It is now generally accepted that the term “NKT cells” generally refers to CD1d-restricted T cells co-expressing a heavily biased, semi-invariant T cell receptor (TCR) and NK cell markers. Natural killer T (NKT) cells should not be confused with natural killer (NK) cells. [*Note: a very small population of NKTs have been identified that are classically restricted] Upon activation, NK T cells are able to produce large quantities of interferon-gamma, IL-4, and granulocyte-macrophage colony-stimulating factor, as well as multiple other cytokines and chemokines (such as IL-2 and TNF-alpha). NKT cells seem to be essential for several aspects of immunity because their dysfunction or deficiency has been shown to lead to the development of autoimmune diseases (such as diabetes or atherosclerosis) and cancers. NKT cells have recently been implicated in the disease progression of human asthma. The clinical potential of NKT cells lies in the rapid release of cytokines (such as IL-2, IFN-, TNF- α, and IL-4) that promote or suppress different immune responses.

CD1- antigen presenting molecules present lipid and glycolipids derived from microbial antigens to T cells.

These molecules are non-MHC restricted and nonpolymorphic.

They are distinct from MHC class I and II. Similar structure to MHC class I, having three extracellular domains and expressed in association with 2 microglobulin on APC.

Binds hydrophobic region of lipid with polar bound by TCR.

Binds to a variety of T cells including NK1.1 (CD4+) cells. (NKT cells).

o Induces NK1.1 to secrete large amounts of IL-4 o May be important in generating TH2 activities

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Overall, the CD1 molecules bind antigen in a deep, narrow hydrophobic pocket, with ligands interacting via hydrophobic interactions rather than hydrogen bonding.The role of CD1 in pathogenesis has not yet been fully determined. 2. Superantigens

Activate T cells expressing a specific Vsegment as part of TCR Presented by Class II molecules on MHC but not in peptide groove Several organisms have components that function as superantigens

o Staphylococcus o Rabies virus

Activate large numbers of T cells (possible mechanism for toxic shock syndrome)

•Superantigens bind directly to T-cell receptors and MHC, without processing.

•Usually involves direct interaction to V region of TCR.

VDJ

V

J

CC

VDJ

V

J

CC

VDJ

V

J

CC

VDJ

V

J

CC

VDJ

V

J

CC

VDJ

V

J

CC

SUPERANTIGENS

3. Mitogens Polyclonal activators of T cells by activating widespread mitosis Derived from plant lectins

Phytohemagglutinin (PHA) concanavalin A (conA) pokeweed mitogen (PWM)

Other mitogens Endotoxin (lipopolysaccharide) – mouse B cells, human

monocytes/macrophages AntiCD3 – polyclonal T cell activator

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VI. B CELL ACTIVATION AND FUNCTION B Lymphocytes: The genesis of µ and delta chain-positive, mature B cells from pre-B cells is antigen-independent. B cell development is characterized by recombinations of immunoglobulin H and L chain genes and expression of specific surface monomeric IgM molecules. At this stage of development, B cells are highly susceptible to the induction of tolerance. Cells bearing only monomeric IgM are referred to as immature. These cells may undergo deletion (death by apoptosis), anergy (long term inactivation, or receptor editing (reactivation via V-D-J gene recombination). Once these cells acquire IgD molecules on their surface, they become mature B cells that are able to differentiate after exposure to antigen into antibody-producing plasma cells. Mature B cells can have 1-1.5 x 105 receptors for antigen embedded within their plasma membrane. The activation of B cells into antibody producing/secreting cells (plasma cells) is antigen-dependent. Once specific antigen binds to surface Ig molecule, the B cells differentiate into plasma cells that produce and secrete antibodies of the same antigen-binding specificity. If B cells also interact with T helper cells, they proliferate and switch the isotype (class) of immunoglobulin that is produced, while retaining the same antigen-binding specificity. This occurs as a result of recombination of the same Ig VDJ genes (the variable region of the Ig) with a different constant (C) region gene such as IgG. T helper 2 cells are thought to be required for switching from IgM to IgG, IgA, or IgE isotypes. The generation of memory B cells is associated with class switching; this process occurs in the spleen or lymph node. In addition to antibody formation, B cells also process and present protein antigens. After the antigen is internalized it is digested into fragments, some of which are complexed with MHC class II molecules and then presented on the cell surface to CD4+ T cells. B cells secrete antibody upon antigenic stimulation, a multi-step process involving interactions with T cells. B cells express many surface molecules which assist in the process of antibody production through delivery of various activation signals. Some of these costimulatory molecules are depicted in the figure below. Fc receptors are important in "feedback" mechanisms to deliver negative signals to the cell.

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Surface Markers of human and murine peripheral B cells. Remember that B cells carry the HLA-D (and I-A/I-E), class II restricted major histocompatability marker, as well as have specific receptors for complement receptors. Coico and Sunshine, 2009. Figure 7.7. A. T cell - B cell cooperation

T dependent antigens Require CD4+ help for B cells to make antibody Must be to same antigen but different epitopes (linked recognition)

B cell epitope - hapten T cell epitope - carrier

T – B interactions For primary response, requires APC (dendritic cell the best) For secondary response, no APC necessary Requires cytokines for B cell growth (IL-4), proliferation (IL-6)

Isotype switch from IgM to o IgG (IFN o IgA (IL-5) o IgE (IL-4,13)

External Ag on B cell bound by surface IgM Internalized, processed and presented via MCH II to TCR in

association with CD4 molecule Costimulation (CD40:CD154; CD28:CD80/86) Adhesion (CD58:CD2; ICAM-1:LFA-1; CD72:CD5) Result is cytokine production by T cell that binds via receptor to B cell

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Coico and Sunshine. 2009. Figure 10.9.

B. T independent Responses

Do not need T cell help to make antibody Antigen is typically polymerized molecules (such as polysaccharides) Only generate IgM responses Do not generate memory

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C. B Cell Activation Pathways Surface IgM is crosslinked CD19,21,81 are coreceptors for BCR Tyrosine kinases activated (Lyn, Fyn, Blk, Lck) Phosphorylates the ITAMs of Ig/Ig molecules associated with surface Ig Syk then activated

Activated Syk activates PLC-which splits PIP2 into DAG and IP3 DAG>>PKC>>>multiple kinases IP3 >>calcineurin

Both pathways activate transcription factors – NF-, NF-AT Result in nucleus is upregulation of cytokine receptor and Ig genes

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SUMMARY The major properties of the acquired immune response are specificity, memory,

adaptiveness, and discrimination between self and non-self. Lymphoid cells in these categories include T and B lymphocytes. T and B cells

produce and express specific receptors for antigens. Receptor specificity is related to gene rearrangement of variable region components during development, according to essential features for clonal selection.

Cytokines are small molecular weight glycopeptides with a variety of cellular origins and functions, both effector and regulatory.

Helper T cells (TH) provide assistance to B cells to make antibody and other T

cells to become cytotoxic by production of specific cytokines, expression of co stimulatory and adhesion molecules molecular mechanisms involving transcription factors

Cytotoxic T lymphocytes (CTL) are for host defense against intracellular

pathogens and induce death of the target cells by various mechanisms

T cell antigen receptors can be activated by a variety of molecules such as proteins, lipids/glycolipids, superantigens and mitogens

B cells make antibodies, the quantity and isotype of which is dependent upon

whether the T cell is involved (T –dependent) or not (T-independent) and relates to both the nature of the antigen and the underlying immunological capabilities of the host

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ANTIGEN-ANTIBODY INTERACTIONS, IMMUNE ASSAYS, EXPERIMENTAL

SYSTEMS Keri C. Smith, PhD

MSB 2.218, Keri.C.Smith@uth.tmc.edu

OBJECTIVES The objective of these lectures is to learn how the exquisite specificity of antibodies can be used in the clinical laboratory for diagnostic assays that measure either antibodies or antigens and review experimental systems that will be discussed later in the course. KEYWORDS Affinity, agglutination, prozone, zeta potential, precipitation, immunoelectrophoresis, radial immunodiffusion, nephelometry, radioimmunoassay, ELISA. READING Chapter 5 of the Coico et al textbook, 2009. Case 46 in Case Studies in Immunology, 6th Ed. Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/antibody-structure-and-function-iii/ INTRODUCTION It is clear that antibodies play a major role in protection from a variety of diseases, toxins, viruses, parasites, etc. In addition, once antibodies have been made, they can be used for a variety of diagnostic assays in the laboratory to detect the presence of absence of a particular antigen or bacterium or virus in a sample. For instance, the use of antibodies specific for red blood cell antigens has made routine transfusions possible. The reaction of antigen with its homologous antibody is a two-stage phenomenon. The initial or primary binding reaction can occur invisibly. The secondary manifestation of that interaction is dependent on several factors such as:

a) Isotype of the antibody b) Valence of antigen c) Form (particulate or soluble) of the antigen

The type of assay used depends vitally on these factors. For example, determination of a patient’s red blood cell type is done using intact red cells and so the assay called agglutination is used. The kinds of assays used to detect soluble antigens such as growth hormone cannot be used for red cell typing because of the particulate nature of the red cell. Review of Figure 5.1 on page 60 in the textbook will demonstrate many of the features of antigens and of antibodies and fragments of antibodies that can dictate design of specific assays. It is clear that valency of both antigen and antibody can be important. Please review this figure thoroughly.

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PRIMARY INTERACTIONS BETWEEN ANTIBODY AND ANTIGEN

Antigens and antibodies interact as the result of multiple weak, non-covalent reactions. You should now review these interactions from the “Immunogens and Antigens” lecture. Due to the relative weakness of these forces, Ab-Ag reactions can be readily dissociated by:

a) low or high pH b) by high salt concentrations c) by chaotropic ions.

ASSOCIATION CONSTANT The strength of the primary interaction between one paratope and its epitope can be precisely measured by using the law of mass action since the reaction is noncovalent. The binding of an antigen univalent epitope such as a free hapten (H) to a paratope can be represented by the equation:

Ab + H AbH

The association constant is then defined by the expression:

K= [AbH]/[Ab][H] The K value represents the intrinsic association constant or the Affinity for monoclonal antibodies and will represent an average association constant for polyclonal antibodies AFFINITY AND AVIDITY Definition: The intrinsic association constant, the reaction between a single paratope and its epitope, is termed the affinity. Affinity measurements cannot account for the overall efficiency of binding because having more paratopes/molecule will enhance the overall efficiency of binding since each paratope on each molecule is identical in its affinity. Thus, antigen multivalency enhances antibody-binding efficiency. This enhancement due to antigen multivalency is called avidity. Affinity and avidity are illustrated in the following figure:

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SECONDARY INTERACTIONS BETWEEN ANTIBODY AND ANTIGEN.

AGGLUTINATION REACTIONS Definition: The term agglutination infers aggregation of insoluble particles. Aggregation of red blood cells or bacterial cells is routinely used for estimation of the concentration of antibodies in a serum taken from a patient or experimental animal.

Definition: The term titer is used to describe the highest dilution of that serum that will agglutinate a standard amount of the cells (i.e. 50 ul of a 1% suspension).

PROZONE-Agglutination reactions can sometimes exhibit the phenomenon of prozone. This occurs because very high concentrations of antibodies can totally saturate all epitopes on each cell added so that no cross linking occurs. As the concentration of antibodies is lowered by dilution in succeeding tubes, the numbers of cellular epitopes and antibodies then reach a ratio where effective agglutination occurs. ZETA POTENTIAL-An electrical potential between two like charged particles prevents them from physically associating. The short distance between Fab arms of IgG molecules may not overcome this repulsion, but the larger IgM molecule might be sufficiently large to overcome zeta potential. The high sialic acid density on the surface of red cells is difficult to overcome and the size, coupled with the multivalency, of IgM makes it more efficient as an agglutinator of red cells. COOMBS’ TEST-The Coombs’ test can overcome zeta potential by using a second layer of antibodies to bridge cells. If the red cell is coated with IgG antibodies, an

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antiglobulin antiserum can be added (Definition: a serum containing antibodies specific for the Fc region of IgG) and it can then cross-link the IgG antibodies previously bound to the cell thereby agglutinating the red cells. This assay is described in Figure 5.2 in the textbook.

Direct Coombs’ Test In this assay, patient blood that is suspected of having antibodies already bound to the red cell (i.e. blood from a baby at risk for Erythroblastosis fetalis) is mixed with the antiglobulin serum and positive agglutination is diagnostic for the presence of anti-Rh antibodies bound to the red cells. Indirect Coombs’ Test This is to detect the presence in serum of a non-agglutinating antibody. For example, serum from a pregnant patient suspected of having circulating IgG anti-Rh antibodies is mixed with Rh+ red cells, then the antiglobulin is added. Positive agglutination is then diagnostic for the presence of anti-Rh in patient serum, indicating that the fetus is at risk for erythroblastosis fetalis.

Clinical Vignette: Review Case 46—Hemolytic Disease of the Newborn. Indirect Coomb’s titers were used as a principal diagnostic tool in this case.

PASSIVE AGGLUTINATION-Passive agglutination is a way to use the extraordinary sensitivity of agglutination assays to detect antibodies specific for soluble antigens such as thyroglobulin to help diagnose Hashimoto’s disease, for example. In this assay, purified soluble thyroglobulin is attached to something particulate such as micro-latex beads or red cells. Then sera containing suspected antibodies specific for thyroglobulin can be titered in a standard agglutination format. If red blood cells are used as the particle, the assay is usually called passive hemagglutination to acknowledge the red cell as the carrier of the antigen.

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PRECIPITATION REACTIONS Precipitation Reaction in Solution (Fluid Phase Reactions) Antigen-Antibody reactions that result in the formation of visible precipitation of the reactants are classed as secondary manifestations of Ag-Ab reactions. In general, this reaction can be utilized to determine if there is a yes/no response for antibody binding to an antigen (in other words, it is qualitative, rather than quantitative). Understanding the Ab-Ag interactions that lead to this reaction is important, as the immune complexes formed are also found in vivo and can contribute significantly to pathology.

In the precipitation reaction, various amounts of soluble antigen are added to a fixed amount of serum containing antibody. As illustrated in the figure to the left, when small amounts of Ag are added, Ab-Ag complexes are formed with excess Ab, and each molecule of Ag is bound by Ab and cross-linked to other Ab molecules. When enough Ag is added, ALL of the antigen and antibody complex and fall out as precipitate (the zone of equivalence). When an excess of Ag is added only small Ag-Ab complexes form (no crosslinking) and the precipitate is reduced.

This reaction is affected by the number of binding sites that each Ab has for antigen, and the maximum number of Abs that can be bound by an antigen or particle at one time. This is defined as the valence of the antigen or antibody (see figure to the left) and valence of Ab and Ag has to be > 2 or precipitation will not occur. NEPHELOMETRY Nephelometry is a widely used methodology for accurately measuring quantities of the Ig classes in serum. Obviously, dramatic increases or decreases in quantities of these could contribute to diagnosis of numerous diseases. In this assay, proteins in the sample react with specific antibody (e.g. an anti-IgE antibody). The mixture is placed in a tube and inserted into the Nephelometer. When light passes through the suspension that contains aggregated particles, a portion of the light is scattered. The scattered light is measured and compared with stored standards. Thus, this is a quantitative method using liquid-phase precipitation principles. It can be applied to measuring any soluble substance provided specific antisera are available.

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PRECIPITATION REACTIONS IN GELS When we want a simple yes/no answer to determine if antigen or antibody is present, it can be helpful to slow down the rate of diffusion in a gel matrix that holds the precipitate in the gel web so that it is effectively immobilized for visualization either directly or with the aid of various staining methods. Several qualitative and quantitative methods are in wide use in medicine today for analysis of numerous hormones, enzymes, toxins, and for analysis of the products of the immune system itself. RADIAL IMMUNODIFFUSION In this reaction, a known antibody or an antigen is infused into the gel matrix. The test sample (either a suspected antigen or an antibody) is placed in the center of the gel. As the unknown sample diffuses into the surrounding agar, a precipitation reaction will occur if there is a positive Ab-Ag interaction. Since precipitation happens only at the zone of equivalence, a ring will form some distance away from the high concentration of antigen at the center (see drawing below). So, more antigen means that more diffusion has to occur to overcome the antigen excess at the center. We can take advantage of this to use radial immunodiffusion as a quantitative assay as well – if we compare the diameter of rings formed from various known quantities of antigen, we can generate a standard curve and figure out how much antigen is in an unknown sample based on the diameter of the ring it forms on the agar.

The technique of doing radial immunodiffusion and some typical results are described in Fig. 5.6, pg. 65 in the textbook.

OUCHTERLONY DOUBLE DIFFUSION ASSAY The Ouchterlony Assay was developed by Orjan Ouchterlony in the 1950’s and is still in widespread use. It has two important features.

a) it is inexpensive to use. b) it can be used to compare the relatedness of two antigens (Antigenically, are

they totally different, are they the same, or only similar?). The assay is called a Double Diffusion assay because both the antigen and antibodies are diffusing. It is a qualitative assay – either there is a reaction or there isn’t. Instead of infusing the agar with antibody or antigen, the Oucterlony assay is run by placing antibodies and antigens in separate, but close by, well. The molecules in each well then diffuse slowly into the agar in a radial fashion (diffusion in a circular fashion with an ever-increasing radius). Thus, antigen and antibody slowly diffuse toward one another. A positive result will be that a thin opaque precipitate line or band will form in the agar at right angles to a line connecting the centers of the two wells and it will usually

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be symmetrical, extending the same distance either side of the line connecting the well centers. The presence of a line is a qualitative assay for the presence of either antibody in the antiserum (using a standard antigen solution) or for the presence of antigen (using a standard antiserum). See Figure 5.5 in the Textbook.

The most widespread use of the Ouchterlony technique is for comparison of antigens. It has also been used in forensic medicine and in a variety of diagnostic assays. Study note: The three patterns of reactions (identity, non-identity, and partial identity) described in Fig. 5.5 on pg. 66 are important to understand IMMUNOELECTROPHORESIS Immunoelectrophoresis is a variation of the Ouchterlony double diffusion in gel technique. It is designed to analyze complex protein mixtures containing many different antigens. Electrophoresis separates proteins according to size (which corresponds to their mobility in the electric field) within the gel matrix. A mix of antibodies specific for the proteins is then added to a trough cut in the agar. The individual proteins and their specific antibodies will diffuse toward one another, and lines of precipitate form for each Ab-Ag interaction. This method and typical results are shown in Fig. 5.7, pg. 67 in the textbook.

Immunoelectrophoresis is a qualitative assay. It is also used in medical research for following the different steps of a purification protocol to show the disappearance of unwanted proteins when purification of one component from a mixture is desired. WESTERN BLOTS The mechanisms that underlie immunoelectrophoresis form the basis for the more commonly used Western Blot. Instead of relying on a precipitation reaction that is manifested as a line in a gel, this assay uses labeled antibodies to visualize binding. Also

Clinical Relevance: The medical diagnostic use of immunoelectrophoresis is for diagnosis of conditions where certain proteins are suspected of being absent (e.g. hypogammaglobulinemia) or of being overproduced (e.g. Multiple Myeloma). It is usually used as a first screening test, followed by quantitative tests.

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called immunoblotting-a mixture of antigens is separated by size via electrophoresis on a gel, and in an additional step the proteins are transferred onto a medium such as nitrocellulose that binds proteins tightly. Specific antibodies that have an enzyme covalently attached are incubated over the nitrocellulose. Substrate for the enzyme is added, turns colors when enzyme is present, and the colored line shows that the antigen was present. See textbook, Fig. 5.8, page 68

SOLID-PHASE IMMUNOASSAYS There are a group of assays in which the antigen or the antibody is coated on the surface of a plastic microplate and sensitive indicators such as radioactivity or enzymatic action are used to detect the presence of Ag or of Ab. There are 5 of these assays classified in two groups according to the types of antigens being analyzed: soluble or cellular.

A. SOLUBLE ANTIGENS RADIOIMMUNOASSAY--There are many different formats for doing radioimmunoassay (RIA). The example below describes a clinical test to detect Hepatitis B virus protein in patient plasma.

a) Antibody specific for Hepatitis B antigen (HBsAg) is first coated onto the surface of plastic plates and the excess is removed by rinsing out the wells with buffer solutions.

b) the remaining plastic surface is then blocked by adding an irrelevant protein solution and washing

c) A mix of patient serum and a known quantity of radiolabeled HBsAg is incubated in the wells. This leaves the plate with Ab bound to the Ag that is, in turn, bound (noncovalently) to the plastic.

d) Detection of the amount of radiolabeled antigen that binds in the presence of patient serum compared to a known control allows us to quantify the amount of HBsAg in the blood sample. This is a binding competition assay – less signal means that more unlabeled (patient) HBsAg is present and bound to the antibody. If no HBsAg is contained in the patient sample, only radiolabeled Ag binds and the signal is higher.

Clinical Correlation—HIV infections are frequently diagnosed by doing Western Blots of patient’s serum for content of antibodies specific for various HIV antigens. See Figure 5.8 in the Coico book. See Case 10—Acquired Immune Deficiency Syndrome (AIDS).

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ENZYME LINKED IMMUNOADSORBENT ASSAY (ELISA) The ELISA assay is quite similar to the RIA except that the indicator reagent used in ELISA is not radioactive. Instead, the binding antibody is coupled to an enzyme molecule that converts added substrates to a colored product that can be detected spectrophotometrically due to the color change. The assay is performed as shown at left.

Clinical Relevance: Current Laboratory RIA Assays The specific assays are to quantitate the amounts of specified antigens in body fluids such as blood or urine. Assays are available for measuring Renin, Gastrin, Parathyroid Hormone, Growth Hormone, Urine Microalbumin, Vitamin B12 and Folate. Memorial-Hermann Hospital currently is phasing out the radioimmunoassay laboratory.

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Clinical Vignette

ELISPOT assays Variation of the ELISA method. Incubate with cells instead of soluble antibody. The # of spots after addition of detection antibody and precipitable substrate = the number of cells secreting a specific antibody, thus can be used to determine the frequency of antigen specific B cells. Also used for T cell assays (e.g. the number of T cells producing a cytokine, as illustrated to the left). Often used in biomedical research.

B. CELLULAR ANTIGENS IMMUNOFLUORESCENCE

It is sometimes of diagnostic value to determine if a particular antigen is found on or in the cells of a particular tissue. In this case, assays are needed that can be

Clinical Relevance: There are a large number of commercial ELISA kits available for diagnostic purposes. Currently the Memorial-Hermann Hospital Laboratories offer ELISA assays for Hepatitis antigen, HIV and HTLV antigens. Specific assays are also available for detection of antibodies in patient’s serum for Hepatitis A virus, Hepatitis B surface antigen, Hepatitis B core antigen, Hepatitis C virus, cardiolipin, H. pylori, and for HIV types 1 and 2. Another ELISA assay is available for detection of antibodies to Human T-lymphotropic virus type I.

This is the case of a woman who contracted the AIDS virus from a blood transfusion and transmitted it to her fetus later. Antibodies specific for the gp120 HIV antigen were measured in the infant using an ELISA. The mother and father were also tested and were found to have anti-gp120 antibodies by ELISA.

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performed directly on biopsies of tissue and seen using a microscope. The method originally developed by Albert Coons and his colleagues at Harvard involves covalent attachment of fluorescent organic compounds to specific antibodies that then can be used to detect the antigen in question. The fluorescent compounds excite at different wavelengths. This is a highly sensitive and specific assay, and cells individual cells can be stained with up to 12 different compounds.

1. Direct Immunofluorescence-The antibody specific for the antigen in question is directly labeled with the fluorophor and used to identify the antigen.

2. Indirect Immunofluorescence-This is similar to the Coombs’ reaction discussed earlier (review that if necessary). It is a two step method in which the unlabeled antibody specific for the antigen in question is reacted first with the tissue and the excess antibody is washed away. Then the slide is flooded with a fluorescent anti-Ig (preferably Fc specific). This method has the advantage that it is significantly more sensitive than the Direct method.

IMMUNOHISTOCHEMISTRY is a similar technique. Instead of fluorescent labels, the detection antibodies are labeled with enzymes such as horseradish peroxidase or alkaline phosphatase (these are also used in ELISA). Addition of substrate then colors the membranes of the cells expressing the antigen of interest. FLUORESCENCE ACTIVATED CELL SORTING (FACS) ANALYSIS FACS analysis is used to identify, and sometimes purify, one cell subset from a mixture of cells. The technique and a diagram of the instrument are on page 69, Fig. 5.12. This is an extremely effective tool to identify and/or isolate specific cell subsets. The organic fluorescent compounds attached to the detection antibodies are excited by different fluorescent wavelengths, and all emit at different wavelengths as well, allowing for specific detection of the markers. Current instrumentation can detect up to 15different antigens on one cell (though most investigators use 4 colors at most).

Sorting of cells can also be accomplished using antibodies coupled to magnetic beads (magnetic activated cell sorting, or MACS). The cells are then placed over a magnetized column, and any cells with labeled antibody bound to them can be isolated from the unbound population.

Clinical Relevance: Immunofluorescence, using the indirect format, is used in clinical laboratories for screening patient’s sera for anti-DNA antibodies in suspected cases of systemic lupus erythematosus.

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MULTIPLEX ANALYSIS A relatively new technology has been developed that combines aspects of an ELISA with the sensitivity of flow cytometry. These Multiplex Bead Arrays rely on the engineering of microspheres internally “coded” with two fluorescent dyes. Combinations of the dyes can be used to generate up to 100 individual “bead sets” each of which can be coated with specific antibody. Mutiple bead sets may be incubated with sample (plasma or cell culture supernatant) in a well, and 96 well plates can be used for this analysis – allowing for the measurement of multiple parameters in multiple samples. The antibody-coated beads bind to their specific antigen target (e.g. cytokines), then biotin-labeled secondary detection antibodies are used to “sandwhich” the antigens bound on the beads. A reporter molecule, streptavidin-phycoerythrin (SA-PE) then indicates each complex. In a method similar to that used in flow cytometry, the beads are passed single file through a laser beam, complexes are identified by the fluorescence of PE, and the internal fluorochromes unique to each bead emit specific signals that are detected by digital processors. In this manner, up to 100 different analytes can be detected in a single sample, making this a powerful tool for use in screening patient responses in different disease conditions.  

LYMPHOCYTE FUNCTION ASSAYS Lymphocyte function can be compromised in certain diseases or can occur as a result of a genetic abnormality. A diagnosis can be confirmed in many cases if it is known whether or not the B or T cells are normal, if the existing B cells can make antibodies, or if the T cells can produce the correct cytokines.

Clinical Relevance: FACS can measure CD4+ cell numbers in AIDS patients to follow disease progression. FACS was used in Case 5—MHC Class I Deficiency to measure peripheral blood lymphocytes

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Mitogen Activation—Lipopolysaccharides can cause polyclonal stimulation of B cells in vitro. This activation is accurately measured by incorporation of radioactive nucleosides. Several lectins, including concanavalin A and phytohemagglutinin are effective T cell mitogens. Pokeweed mitogen stimulates polyclonal activation of both B and T cells. Numerous assays can measure antibody production by stimulated B cells (i.e. ELISA). Cytotoxicity assays measure the ability of cytotoxic T cells or NK cells to kill radioactive target cells that express a specific antigen for which the cytotoxic T cells may be sensitive. MONOCLONAL ANTIBODIES AND T CELL HYBRIDOMAS

Due to cross reactivity of antibodies and the need for more controllable assays it is sometimes of great advantage to have a homogeneous antibody preparation that is specific for only a single epitope and with high affinity. Since polyclonal antibody mixtures consist of a multitude of antibodies specific for different epitopes on even simple antigens like tetanus toxoid, and the fact that different subpopulations of antibodies with different affinities exist even in the subset specific for a single epitope, significant cross reactions can occur when using polyclonal antibodies for analytical assays. This can lead to misinterpretation of results occasionally. Kohler and Milstein developed a method for making murine antibodies that are monoclonal, that is, all antibodies are derived from a single precursor plasma cell so that all the antibodies in the preparation are identical and derived from the same original clone. The method is outlined in the figure to the left and the specific details of the hybridoma technology are covered in the textbook. (See Figure 5.13, Coico and Sunshine, 2009). The same general method is also used for making T

cell hybridomas. GENETICALLY ENGINEERED ANTIBODIES

Clinical Relevance: T cell hybridomas are valuable for the large-scale production of several T cell-derived lymphokines that are used as antigen in diagnostic kits and are also used therapeutically.

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Attempts to develop human hybridoma technology have not been very successful. To adapt the murine system for making human antibodies, recombinant DNA methodologies have been developed to “humanize” murine antibodies. The methods usually use murine V-region sequences coupled to human C-region sequences. There are many variations on this theme and the methodologies are also applicable to engineering receptors (for cytokines, etc.) into cell lines in which they are not normally expressed. HIGH THROUGHPUT IMMUNOSEQUENCING Recent advances in sequencing technology and computing algorithms have allowed for the development of methods to rapidly screen and characterize polyclonal immune responses to antigen. Collectively, these methods are referred to as high-throughput Ig sequencing (Ig-Seq) technologies, and they take advantage of the unique manner in which Ig genes recombine to specifically amplify heavy and light chain variable sequences isolated from class-switched B cells (or TCR from memory T cells). Genomic DNA isolated from a population of B cells is amplified using primers complementary to rearranged VDJ. Alternatively cDNA can be amplified using a primer pool complementary to leader peptides or framework regions of V gene segment combined and specific CH-specific primers for heavy or light chains (5’ RACE amplification can also be used). The samples are then sequenced, and various bioinformatics approaches are used to generate the output. Results from these analyses can be applied to many experimental and clinical questions to understand the generation of the antibody response and its role in human health and disease.    MICROARRAYS TO ASSESS GENE EXPRESSION Levels of expression of thousands of genes can be measured simultaneously using a technology called gene chips or microarrays. Briefly, thousands of short cDNA representing genes from all parts of the genome are attached to a slide. Samples of mRNA from cells in culture are used and reverse transcribed into cDNA and by labeling this cDNA from different sources (i.e. normal cells and tumor cells) with different fluorochromes, the differential expression of distinct sets of genes can be measured. By scanning with a laser, different spots can have different colors depending on the success of binding by the two different cDNA’s. This methodology has great potential in fields such as clinical diagnosis of lymphoid tumors.

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SUMMARY

1. In addition to functioning in vivo, antibodies are used in numerous diagnostic formats in the clinical laboratory.

2. The primary binding reaction of antibody with antigen follows the rules of the Law of Mass Action and an Association Constant (antibody affinity) can be accurately measured while functional avidity is defined as the affinity enhancement due to multivalency.

3. Secondary Ag-Ab reactions include the agglutination assay used in blood banking. A prozone in agglutination assays is due to a huge excess of antibody molecules. Zeta potential is an electrical repulsion of like-charged particles. Coombs tests utilize anti-Ig reagents.

4. Precipitation reactions between antibodies and soluble antigens occur regularly in vivo. The degree of precipitation depends on valency of antigen, ratio of antibody to antigen and the classes/subclasses of antibodies that predominate. Usually IgG is the only effective antibody class mediating precipitation.

5. Several precipitation reactions in gel media are widely used for different purposes. The Ouchterlony double diffusion assay is a qualitative assay for measuring antigen presence and comparing antigens. Immunoelectrophoresis is a qualitative method for measuring the numbers of components in mixtures and Radial immunodiffusion is a quantitative method.

6. Nephelometry is a widely used method for measuring Ig concentrations. 7. Radioimmunoassays and ELISA (Enzyme Linked ImmunoSorbent Assay)

are the two most widely used immunoassays used in US clinical laboratories although radioimmunoassays are slowly being phased out in favor of ELISA

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STUDY QUESTIONS - ANTIGEN-ANTIBODY REACTIONS

Study questions for Antigen-Antibody Interactions 1. What is the classic example where a Coomb’s type agglutination assay would be more sensitive than the direct agglutination method. 2. Describe the steps in setting up a quantitative precipitation reaction. What does the

experiment tell you? 3. Make a list of all the immunoassays in this chapter and categorize them as a)

Quantitative or as b) Qualitative. 4. Describe a situation where you would order an Ig class quantitation measurement

done on a patient’s serum. What instrument would the lab use to do this? 5. Describe the steps in developing an enzyme linked immunosorbent assay. How

would you make it quantitative? 6. Write the two equations that together define antigen-antibody Affinity. Answers to study questions may be found posted on Blackboard, and at: Study Question Answers

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IMMUNE EFFECTOR MECHANISMS I: ANTIBODY-MEDIATED REACTIONS Steven J. Norris, Ph.D.

Recommended Reading: Actor, 2012, Chapters 7 and 10. Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/genetic-basis-of-ab-structure/

I. INTRODUCTION The immune system cannot be understood in isolation from infectious diseases. All living

organisms exist in a hostile environment and are continually used as hosts and energy sources by other organisms. Since there are many different foreign invaders which can infect humans, we must have many different ways to defend ourselves. To provide the versatility required, the two major effector arms of specific immunity: antibody (humoral) and cellular, employ an incredible variety of accessory mechanisms. These lectures will introduce you to some of these mechanisms.

In defense against infections, antibody is generally operative against extracellular bacteria or bacterial products, whereas cell mediated immunity (CMI) primarily operates against intracellular viral and bacterial infections, as well as fungal infections. The killing effects of immune reactions are extremely efficient and, when specifically directed to a given infection, are able to eliminate large number of organisms in a short period of time.

The immune response is a double-edged sword. In most cases, the immune system is protective, providing life-saving defenses against infectious diseases and tumors. However, it can also be destructive, causing immunopathology, defined as tissue damage resulting from the immune response. These destructive responses result in some of the adverse effects of infections, in allergies or hypersensitivity reactions (antibody- or T cell-mediated reactions to environmental or administered antigens), and in distinct autoimmune disorders (antibody- or T-cell mediated reactions to self-antigens). The seven immune mechanisms listed below are active in both immunoprotective and immunopathologic reactions.

II. SEVEN IMMUNE MECHANISMS

Until the 1960s, immune reactions where not classified according to mechanism, but were presented as a bewildering list of lesions with peculiar names. The first working classification of Type I to Type IV immune mechanisms as introduced be Gell and Coombs was a major advance in understanding immunopathologic reactions; seven mechanisms are presented in this handout. The terms Type I - Type IV reactions, although out of date, are still used in some textbooks. TABLE 1: Classification of Immune Mechanisms

Handout Gell and General Properties Coombs (1963) Antibody-Mediated Inactivation or Activation -- Toxin, virus inactivation Cytotoxic or Cytolytic Type II Opsonization, ADCC, C’-mediated lysis Immune Complex Type III Ag-Ab complex formation in tissue Atopic or Anaphylactic Type I IgE mediated allergic reactions Cell-Mediated T-cell Cytotoxic (TCTL) -- Lysis of virus-infected cells; contact hypersensitivity Delayed Hypersensitivity (TDTH) Type IV CD4+ T cell-mediated activation of macrophages Either Granulomatous Reactions -- Chronic reaction to poorly degradable antigens

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These immune mechanisms are similar in many ways to antibody- or cell-mediated reactions observed in vitro. Primary reactions consist of the formation of Ag-Ab complexes or Ag-TCR reactions, secondary reactions the effects of this interaction that can be demonstrated in vitro, and tertiary reactions the corresponding in vivo manifestations (see figure).

Factors affecting the induction of different forms of immunity

• Type of infectious agent or antigen.

• Route of infection/exposure.

• Activation of Th1 vs. Th2 cells.

• Location/cell type involved in antigen presentation.

• Cytokines expressed by antigen presenting cells and T cells.

• Genetic factors.

• Non-genetic factors. (e.g. age and nutritional status)

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ANTIBODY-MEDIATED IMMUNE MECHANISMS 1. INACTIVATION (NEUTRALIZATION) REACTIONS

A. Definition - binding of antibody to an epitope (toxin, virus, cell receptor, etc.) resulting in inactivation (loss of function), neutralization (loss of infectivity), or abnormal activation.

B. Mechanisms 1. Binding of antibodies to a protein can stearically inhibit its binding to substrate, or

alter its conformation, resulting in loss of activity. 2. Antibody binding to viral receptor proteins can interfere with binding to cells, alter

viral structure, or mediate Ab- or C’-mediated opsonization and clearance 3. In some cases, antibodies against hormone or neurotransmitter receptors can either

block or activate the receptor.

C. Medical Aspects - examples 1. Protective

a. Immunization of individuals with diphtheria toxoid or tetanus toxoid results in expression of antibodies. These preformed antibodies do not prevent colonization by C. diphtheriae or C. tetani, but bind to the toxins and prevent them from interacting with the corresponding host cell receptors, thus preventing disease.

b. Infection or immunization with viruses (including polio, influenza, measles, mumps or rubella) results in expression of antibodies that bind to viral receptors and prevent infection upon subsequent exposures.

2. Immunopathologic

a. Myasthenia gravis - autoimmune antibodies bind to acetylcholine receptors at the neuromuscular junction, causing their internalization and downregulation. The synaptic folds also become decreased or ‘simplified’, reducing interaction with the neurotransmitter and inhibiting skeletal muscle contraction. (Aristotle Onassis had this disease.)

b. Graves disease - antibodies against the TSH receptor bind to thyroid cells and result in activation and abnormally high production of thyroxines. (George and Barbara Bush and dog Millie)

c. Pernicious anemia - antibodies against intrinsic factor interfere with its binding of vitamin B12 in the GI tract, resulting in B12 deficiency and anemia.

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2. CYTOTOXIC REACTIONS

A. Definition - reaction of antibodies with cell surface antigens may result in destruction of cells by opsonization, complement activation, or Antibody-Dependent Cellular Cytotoxicity (ADCC). Also called Type II hypersensitivity.

B. Mechanisms 1. Complement activation may lyse

bacteria directly through formation of the membrane attack complex (MAC). A single IgM molecule or 2 or more IgG molecules complexed to surface antigens are sufficient to activate the classical pathway.

2. Phagocytosis of infectious agents by macrophages or neutrophils can be enhanced through antibody binding (interaction with Fc receptors) or fixation of C3b (interaction with complement receptors).

3. ADCC results from IgG-mediated binding of null lymphocytes (and in some cases macrophages) to target cells via Fc receptors, and direct killing of the target cell through cytolytic mechanisms (see below).

4. In parasitic infections, IgE-mediated binding of eosinophils to helminths results in eosinophil degranulation and damage to the worm tegument (surface).

C. Medical Aspects (Examples)

1. Protective a. Many bacteria (particularly Gram

positive bacteria) are susceptible to C’-mediated killing and/or opsonization. This is particularly true of pyogenic bacteria (such as Staph and Strep) that result in massive accumulations of neutrophils (see Immune Complex reactions below).

b. Ab and C’-mediated MAC formation and opsonization are active against some protozoal infections, including Plasmodium and Trypanosoma.

Clinical Vignette – Inactivation Reactions (Geha and Notarangelo, “Case Studies in Immunology”, 6th edition) Case 42 Myasthenia Gravis – binding of anti-AchR antibodies results in skeletal muscle weakness

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c. ADCC may be active against virally-infected cells, tumor cells, protozoa, and helminths.

2. Immunopathologic

a. Transfusion reactions - ABO mismatches result in rapid lysis of transfused cells due to anti-A or anti-B isohemagglutinins, naturally occurring IgM antibodies that bind to the transfused erythrocytes and activate complement.

b. Rh reactions - birth of an Rh+ infant to a previously sensitized Rh- mother may result in binding of maternal anti-Rh antibodies to the infant’s erythrocytes, causing opsonization and phagocytosis hemolytic disease of the newborn.

c. Hemolytic anemia - autoantibodies can cause erythrocyte lysis, anemia. d. Goodpasture’s syndrome - autoantibodies to basement membrane components and

complement are bound in an even, ribbon-like pattern to glomeruli and other tissues. (Contrast with lumpy-bumpy appearance of immune complex disease; see below).

3. IMMUNE COMPLEX REACTIONS

A. Definition - formation of soluble or insoluble Ag-Ab complexes that can be deposited in tissue, leading to attraction of PMNs, inflammatory changes, and tissue damage. Also called Type III hypersensitivity.

B. Mechanisms 1. As increasing concentrations of

antigen-specific antibodies (particularly IgM and IgG) are expressed, any remaining antigen will form Ag-Ab complexes or so-called immune complexes.

2. The size of immune complexes formed in vivo will depend on the degree of cross-linking as it relates to antigen excess, equivalence, and antibody excess, similar to quantitative precipitation and agar double diffusion assays in vitro (see figure).

3. Depending on their size, immune complexes can fix complement, resulting in binding of C3b and release of the anaphylotoxins C3a and C5a. These cause local mast cell degranulation and attraction of neutrophils, leading to inflammation.

Clinical Vignettes – Cytotoxic Reactions (Geha and Notarangelo, “Case Studies in Immunology”, 6th ed., 2012) Case 46 Hemolytic Disease of Newborn– maternal anti-Rh antibodies cause hemolysis in Rh+ newborn

(Cynthia Waymarsh) Case 41 Autoimmune Hemolytic Anemia – patient Gwendolyn Fairfax develops hemolytic autoantibodies

following a mycoplasma infection

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1. Large immune complexes are typically phagocytosed and destroyed by phagocytic cells (such as resident macrophages of the reticuloendothelial system). Smaller complexes can become lodged in the walls of venules, in joints, and in glomeruli. Deposition of immune complexes causes complement activation, attraction of neutrophils, and release of lysosomal contents (“frustrated phagocytosis”), resulting in vasculitis, reactive arthritis, and glomerulonephritis.

2. The uneven distribution of immune complexes, complement components, and lysosomal contents results in the formation of lumpy-bumpy membrane deposits detectable by binding the anti-Ig or anti-C3 antibodies.

3. Injection of an antigen in a previously immunized individual can result in an Arthus reaction due to deposition of Ag-Ab complexes, complement activation, and resulting erythema, edema, and attraction of neutrophils. An Arthus reaction typically takes 2 to 6 hours to develop.

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C. MEDICAL ASPECTS (EXAMPLES) 1. Protective

In pyogenic infections (e.g. Staphylococcus aureus), immune complexes attract neutrophils which marginate on the endothelial cells and enter the tissue. A predominance of neutrophils constitutes an acute inflammatory response (occurs within a few days). Bacteria are killed through phagocytosis and release of lysosomal contents. Accumulation of dead bacteria, neutrophils and other cells killed by bacterial toxins or lysosomal contents, and fibrin accumulate, forming pus. This reaction may wall off the infection.

1. Immunopathologic a) Serum sickness - In the early

1900’s, serum from horses immunized with rabiesvirus or other agents was used for passive immunization. Administration of horse serum elicited antibodies against horse serum proteins in the patient, so that subsequent injections yielded immune complexes. These could cause severe muscle and joint pain and fever, as well as glomerulonephritis. Use of hyperimmune human serum antibodies for passive immunization has virtually eliminated this problem.

b) Systemic lupus erythematosus (SLE) and related autoimmune diseases (e.g. Sjogren’s syndrome and scleroderma) are caused by antibodies against DNA and other normal cell components. The accumulation of immune complexes results in skin rashes, glomerulonephritis, and pericarditis.

c) Rheumatic fever - infection with Streptococcus pyogenes can result in formation of antibodies cross reactive with heart antigens (cytotoxic reaction) and circulating immune complexes (immune complex disease). These cause heart and kidney damage and vasculitis in other tissues.

4. ANAPHYLACTIC OR ATOPIC REACTIONS

A. Definition - IgE-mediated activation of mast cells and other cells types and its effects. Also called allergic reactions, immediate type hypersensitivity and Type I hypersensitivity. The term anaphylactic (literally “away from protection”) arose from the recognition that immunization and subsequent challenge with some antigens lead to adverse reactions rather than protective effects (prophylaxis).

Clinical Vignettes – Immune Complex Reactions (Geha and Notarangelo, “Case Studies in Immunology”, 6th ed. 2012)

Case 37 Systemic Lupus Erythematosus – Nicole Chawner, age 16, butterfly rash after sun exposure. Immune complexes due to antibodies against DNA and other nuclear components cause tissue damage

Case 52 Drug-Induced Serum Sickness – Gregory Barnes, antibodies against penicillin cause vasculitis, hemorrhage

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B. Mechanisms 1. Requires the production of antigen-specific IgE, also called reagin or reaginic

antibody. Isotype switching to IgE during formation of memory cells requires Th2 expression of IL-4. IL-6 further enhances the production of IgE. An individual having significant levels of IgE against a certain antigen is said to be sensitized. Individuals vary greatly in levels of IgE production; those expressing high levels are called atopic patients.

2. Very little IgE is found in the circulation. Rather, most is bound to the surface of mast cells present in tissue around blood vessels, or basophils found in the circulation or tissue. IgE binds specifically to the FcR1 receptor, and can persist for weeks to months on the surface of mast cells.

3. Crosslinking of antigen-specific bound IgE by antigen causes a decrease in cyclic AMP levels and mast cell activation, resulting in rapid degranulation and de novo synthesis of arachidonic acid, which is subsequently converted to leukotrienes, prostaglandins, and thromboxanes.

4. Within seconds to minutes, the preformed contents of mast cell granules act locally to produce a typical wheal and flare reaction (in cutaneous exposures) or hayfever symptoms (in respiratory tract exposures).

Histamine - binds to tissue histamine receptors H1 (induces smooth muscle contraction, endothelial cell separation and leakiness vascular permeability) and H2 (mucus secretion, vasodilation).

Eosinophil chemotactic factor (ECF-A) - attracts eosinophils (present in late-phase or chronic anaphylactic reactions)

Neutrophil-chemotactic factors (NCF) - attract neutrophils (late-phase) Heparin - anti-coagulant, not directly involved in anaphylaxis Wheal and flare - local erythema (due to vasodilation), edema (due to increased

vascular permeability Hayfever - increased mucus secretion, mucosal swelling Prausnitz-Kustner reaction - passive cutaneous anaphylaxis, caused by

experimental injection of IgE and antigen into skin. 5. In severe cases, systemic effects can cause shock (vascular collapse, loss of blood

pressure) and/or airway obstruction (laryngeal edema, bronchoconstriction and mucus production resulting in suffocation).

6. Leukotrienes (formerly known as Slow-Reactive Substance A) cause long-term smooth muscle contraction which is not alleviated by antihistamines. Cause some

H2 RECEPTORS - DILATION

VASCULAR = SHOCK

H1 RECEPTORS - CONSTRICTION

LUNG = ASTHMA

GI = DIARRHEA

GU = URINATION

VASCULAR ENDO = EDEMA

AND ALLERGIC REACTIONS

SMOOTH MUSCLE DILATION

(INCREASED BLOOD FLOW)

ENDOTHELIAL CONTRACTION

(INCREASED VASCULAR

INFLAMMATORY EFFECTSHISTAMINE RECEPTORS

+

ALLERGEN

MAST

CELL

DEGRANULATION

IgECROSS LINKING

PERMEABILITY)

LEUKOTRIENES

PROSTAGLANDINS

NEUTROPHIL

EOSINOPHIL

INFILTRATE

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manifestations of asthma. Prostaglandins also promote bronchoconstriction, vasodilation, and chemotaxis of granulocytes.

7. Eosinophils attracted to the area also have bound IgE which can be crosslinked to cause release of granule contents:

Major Basic Protein - damages parasites, may provide some protection in parasitic diseases. Also causes damage to host epithelium cells, contributes to asthma.

Eosinophil Cationic Protein - also toxic to helminths, neurotoxin Platelet Activating Factor - yet another bronchoconstrictor.

8. Long-acting cells and substances contribute to late-phase reactions, including asthma.

9. Anaphylactic reactions can be reduced by a) avoidance of allergens; b) drugs such as cromolyn sodium (inhibits mast cell degranulation),

corticosteroids (block arachidonic acid metabolism, inflammation); antihistamines (block binding of histamine to receptors); and epinephrine (reverses bronchoconstriction, decreases vascular permeability);

c) hyposensitization - long-term injection of antigen to stimulate production of blocking IgG to reduce allergy symptoms; and

d) desensitization - short-term injection of small quantities of antigen to deplete IgE, desensitize mast cells (e.g. desensitization with penicillin prior to administration of therapeutic doses).

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C. Medical Aspects (Examples)

1. Protective a) Helminth (worm) infections. IgE-mediated responses are thought to aid in

the expulsion or killing of parasitic worms. In the GI tract, increased mucus secretion, intestinal mobility, and release of inflammatory products may result in dislodgement of intestinal worms such as Ascaris lumbridicoides. In addition, release of MBP and other products by eosinophils and mast cells damage schistosomes and trichinella parasites. Other parasites that also cause chronic inflammation against nematode associated antigens (Wuchereria bancrofti / Brugia malayi) may cause lymphatic obstruction and elephantiasis.

2. Immunopathologic a) Hay fever - allergic reactions to pollen and other allergens, causing

increased nasal secretions, watery eyes. b) Asthma - a more severe respiratory reaction causing bronchoconstriction,

increased mucus secretion. May be life-threatening. c) Cutaneous anaphylaxis - insect bites or exposure of skin to other allergens

may cause a rapid anaphylactic reaction. Distinct from contact hypersensitivity (see next lecture).

d) Food allergies - IgE-mediated reactions to seafood, nuts and other foods may cause severe anaphylactic reactions.

e) Systemic anaphylaxis - hypersensitive individuals may develop vascular shock and respiratory failure as the result of exposure to an allergen (e.g. bee stings). Can be reversed by rapid administration of epinephrine.

Clinical Vignettes – Anaphylactic Reactions (Geha and Notarangelo, “Case Studies in Immunology”, 6th Ed., 2012) Case 50 Allergic Asthma –14 yo Frank Morgan rhinitis and persistent wheezing Case 49 Acute Systemic Anaphylaxis – toddler John Mason has a near-fatal allergic reaction after repeated

exposure to cookies containing peanut butter

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SUMMARY -- ANTIBODY-MEDIATED REACTIONS

1. The immune response is a double-edged sword, in that it can be both protective and destructive. The immune mechanisms involved in both protective and destructive immune reactions are the same.

2. Immune mechanisms can be subdivided into antibody-mediated and cell-mediated reactions. The antibody-mediated reactions include inactivation or activation, cytotoxic or cytolytic, immune complex, and atopic or anaphylactic reactions. The cell-mediated reactions include T-cell cytotoxicity and delayed-type hypersensitivity. Granulomatous reactions can be caused by either humoral or cellular responses, but typically result from chronic reactions to poorly degradable antigens.

3. The type of response that occurs is dependent on several factors, including the type of agent or antigen, the route of infection or antigen exposure, the relative activation of Th1 or Th2 subpopulations, the cell type involved in antigen presentation, host genetic factors (such as HLA type), and other factors such as age and nutritional status. Cytokines produced by Th1 and Th2 cells play a central role in what type of responses occur.

4. Responses to a given infectious agent or antigen are rarely, if ever, of a single type. Rather, there is a mixture of several responses, some of which may be protective and others destructive.

5. Inactivation (or neutralization) reactions are caused by direct inactivation of toxins or neutralization of viruses by the binding of antibody. Binding of antibodies to host receptors can cause abnormal blocking (as in myasthenia gravis or pernicious anemia) or activation (as in Graves disease).

6. Cytotoxic reactions result in cell damage or lysis due to antibody binding and complement activation. Cell lysis through formation of the complement membrane attack complex or opsonization by antibody or C3b derivatives are possible outcomes. Cytotoxic reactions are particularly effective against many bacterial and protozoal infections, and antibody-dependent cellular cytotoxicity can kill infected host cells or tumors. Immunopathologic effects include transfusion reactions, Rh reactions, hemolytic anemia, and Goodpasture's syndrome.

7. Immune complex reactions result from formation of antigen-antibody complexes that can lead to complement activation, attraction of PMNs, inflammatory changes and tissue damage. The size and location of the complex formation determines the pattern of disease. Although immune complex reactions can aid in the attraction of PMNs to a region of infection, we typically think of them as being destructive, as in glomerulonephritis, serum sickness, and rheumatic fever.

8. Anaphylactic or atopic reactions occur through IgE-mediated activation of mast cells and other cell types. Crosslinking of surface-bound IgE results in release of preformed granule contents (such as histamine and eosinophil and neutrophil chemotactic factors) as well as the de novo synthesis of arachidonic acid metabolites including leukotrienes and prostaglandins. Anaphylactic reactions may participate in protection against helminth infections, but are also wide-spread causes of hayfever, asthma, and other allergic reactions.

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IMMUNE EFFECTOR MECHANISMS II: CELL-MEDIATED REACTIONS

Steven J. Norris, Ph.D. Recommended Reading: Actor, 2012, Chapters 7 and 10. Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/genetic-basis-of-ab-structure/ Cell-mediated immunity (CMI) is defined as immune reactions in which T cells play a central role as effector cells (as opposed to regulatory cells). CMI includes T-cell cytotoxicity and delayed type hypersensitivity (DTH). Granulomatous responses usually result from DTH reactions to poorly degradable antigens, although antibody responses can also be involved. 5. T-CELL CYTOTOXICITY

A. Definition - T mediated cellular cytotoxicity involving direct contact between the effector cell (CTL) and a target cell, resulting in target cell lysis or apoptosis.

B. Mechanisms 1. In general, T-cell

cytotoxicity involves CD8+ T cells. However CD4+ cytotoxic T cells also exist.

2. As in other effector mechanisms, naïve CD8+ cells must be activated by expo-sure to Ag-MHC I complexes and interleukins (e.g. IL-2) produced by helper T cells and must undergo proliferation and differentiation before becoming active Cytotoxic T Lymphocytes (CTLs).

3. The Ag-specific TCR of the cytotoxic T cell binds to the Ag-MHC-I complex on the surface of a target cell. In addition, a protein called Fas on the target cell binds to Fas ligand on the CTL. As in T-cell activation, other accessory proteins also form bridges between the cytotoxic cell and the target cell.

4. Binding of the TCR activates the release of granules containing perforin and granzymes by the CTL. The target cell is in close contact with the CTL, so most of the granule contents bind to the target cell. (Note: CTLs have mechanisms protecting themselves from self-destruction.)

5. Perforin forms a pore in the target cell, very similar to the pore formed by C9 in the complement pathway. If a sufficient number of pores are formed, the target cell can undergo rapid lysis.

6. Cytokines released by the CTL (including IFN-and TNF-may have cytotoxic effects on the target cell.

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7. Target cells can also undergo apoptosis or programmed cell death. In this case, killing is activated by two signals: the binding of Fas to the Fas ligand, and the leakage of granzymes into the target cell.

8. These two signals result the activation of two endogenous proteases in the target cell: JUN kinase and Caspase 8. These two enzymes act through a series of cytoplasmic and nuclear signals to start the irreversible process of apoptosis or cell death. Steps include nuclear condensation and fragmentation of nuclear DNA by endogenous Dnases. The process of cell death is complete in 1-2 days.

9. Once the target cell is ‘programmed’ to die, the CTL can detach and go on to kill many other target cells.

10. Null lymphocytes also generate lysis and apoptosis by similar mechanisms during natural killer (NK) activity and antibody-dependent cellular cytotoxicity (ADCC). However, T-cell receptor binding is obviously not involved in these activities. Apoptosis is also important in the elimination of self-reactive lymphocytes and the remodeling of tissues during development.

11. The protein Bcl-2 can block apoptosis by preventing the activation of caspases. It may be involved in the resistance of certain tumors to killing.

A. Medical Aspects (Examples)

1. Protective a) Viral infections - T cell-mediated cytotoxicity appears to be the principal

means of eliminating virally infected cells, although delayed type hypersensitivity must also play a role (see below). By killing cells expressing viral antigens on their surface, the host reduces virus production but may also destroy essential cells (e.g. neurons).

b) Cancer - CTL along with DTH and NK activities are also thought to be important in eliminating malignant cells before they proliferate and become tumors. This process is called immune surveillance. Tumor cells often express so-called tumor-specific transplantation antigens or TSTAs. In virally-induced tumors, the TSTAs are often the same from one patient to the next, whereas chemical- or radiation-induced tumors usually express unique TSTAs. This complicates experimental strategies for specific immunotherapy, in which the subjects are vaccinated with TSTAs or given TSTA-specific T cells or antibodies to aid in tumor elimination.

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c) Intracellular pathogens. Although less important than DTH, T cell cytotoxicity is also active in destroying intracellular pathogens. Most notably, CTL can destroy Plasmodium-infected hepatocytes during malaria. Also, this mechanism can lyse infected macrophages in tuberculosis, so that activated macrophages can then kill the released bacteria.

2. Immunopathologic

a) Autoimmune diseases. Although it is often difficult to separate out T cell

cytotoxicity and DTH, CTL almost certainly play a role in some autoimmune diseases. An example is insulin-dependent diabetes mellitus, in which the cells in the islets of Langerhans are destroyed by autoreactive immune responses. Cytolytic T cells specific for cells can be found at the scene in IDDM experimental models. Also, CTL are thought to be responsible for thyroid cell killing in Hashimoto’s thyroiditis (see figure). Reactive lymphocytes also surround target cells and separate them from neighboring cells and basement membranes, similar to what is seen in cell cultures. This ‘disorientation’ also favors target cell death.

b) Contact dermatitis. Again, both CTL and TDTH are involved in contact dermatitis (described in more detail below).

c) Viral exanthems. The eruptive lesions and fever characteristic of many viral infections are partially due to the host immune response. Tissue damage due to cytotoxic T cell responses may cause permanent loss of function.

d) Graft rejection. Cytotoxic T cell (and DTH) responses are involved in acute graft rejection in transplant patients.

SPECIFIC T-CTL

TISSUE CULTURE

TARGET CELLS

DYING CELLS

A. TISSUE CULTURE MONOLAYER

DYING FOLLICULAR

CELLS

BASEMEMT MEMBRANE OF THYROID GLAND

T-CTL TO THYROID

FOLLICULAR CELLS

THYROID

FOLLICULAR

CELLS

B. AUTOIMMUNE THYROIDITIS

T-CTL

T-C

TL

T-C

TL

T-CTL

T-CTL

SPOROZOITES

CLASS I MHC

IFN-gIL-1

ENDOGENOUS ANTIGEN PROCESSING AND T-CTL IMMUNITY

MALARIA

ENDOGENOUSPROCESSING

INFECTION OF HEPATOCYTES

INDUCTIONEXPRESSION

CIRCUMSPORATE

ANTIGEN

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Chromium release assay - measure of cytotoxic activity. Used to screen potential donor-recipient pairs in transplant patients. 1. Incubate virus-infected cell culture and

normal cell culture with 51Cr to radiolabel cells.

2. Wash to remove excess radioactivity. 3. Incubate cell cultures with lymphocytes

from virus-infected subject. 4. CTL activity will result in cell lysis and

release of radioactivity into culture medium.

5. Determine radioactivity in supernatant, compare to control. Results are typically expressed as “percent specific killing”:

% killing = cpm releasedexp - cpm releasedcontrol total cpm 6. What would be the percent specific killing

in this example? 7. Another control would be to perform the

same experiment with lymphocytes from an uninfected individual. What results would you expect?

6. DELAYED TYPE HYPERSENSITIVITY (DTH)

A. Definition - an in vivo reaction involving activation of macrophages by cytokines produced by lymphocytes (TDTH). Also called Type IV Hypersensitivity.

B. Mechanisms 1. Naïve T cells are stimulated by specific interaction of their TCR with Ag-

MHC II complexes on the surface of antigen presenting cells. They must undergo activation, proliferation and differentiation, as in other immune responses.

2. Upon restimulation with antigen (typically in the ‘target’ tissue such as skin, lung, or transplanted organs), the resulting memory TDTH cells (which have Th1 characteristics) express large quantities of cytokines including IL-2, macrophage chemotactic factor (MCF), IFN- and tumor necrosis factor (TNF-.

Virus-infected cell monolayer

lysis, releaseof Cr

25 cpm

viral Ag-MHC complex

No lysis, little release of Cr

MHC

450 cpm

50 cpm

475 cpm

Normal cell monolayer

Chromium Release Assay

51

51

51

Clinical Vignette –T-Cell Cytotoxicity (Geha and Notarangelo, “Case Studies in Immunology”, 6th Ed., 2012) Case 45 “Acute Infectious Mononucleosis” – 15 yo Emma Bovary had a severely sore throat,

lymphadenopathy, and 2 weeks of fever, but eventually improves with supportive therapy.

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3. IL-2 activates additional T cells, MCF attracts macrophages to the area, and IFN- activates macrophages, increasing their motility, phagocytic activity, and ability to kill intracellular bacteria (e.g. by oxidative mechanisms). TNF- can be cytotoxic.

4. Even in a sensitized individual, it takes 1-2 days for a sufficient number of T cells and macrophages to accumulate to cause a visible reaction (e.g.

erythema and induration [hardening] in a tuberculin skin test). That is why the reaction is called delayed type hypersensitivity. In contrast, anaphylactic reactions take minutes and immune complex reactions are maximal within ~6 hours after exposure of sensitized individuals.

5. TDTH cells have little or no direct effect on pathogens or tissues. Their main activity is the recruitment and activation of macrophages. These guys do the dirty work of phagocytosing and killing pathogens or damaging tissue (in contact hypersensitivity, transplants, autoimmune reactions, etc.). Nonactivated macrophages are relatively quiescent; for example, they are incapable of killing M. tuberculosis and actually serve as hosts for its intracellular growth.

6. The DTH activity of a patient can be tested by using antigens to which everyone is exposed, such as Candida albicans extracts. Patients who give negative skin test reactions to such antigens are considered to be anergic, i.e. deficient in cellular responses.

7. DTH reactions can be inhibited by corticosteroids or blocked by cyclosporin and other immunosuppressive agents. These agents are commonly used to control autoimmune diseases and transplant rejection.

8. Recent studies have shown that basophils may play a role in certain types of DTH reactions.

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a) Destruction of intracellular bacteria and other pathogens. DTH is the principal protection against mycobacterial infections and most parasitic and fungal infections. The importance of DTH is underscored in AIDS patients, who extremely susceptible to these organisms.

b) Cancer - as mentioned above, DTH most likely plays a role in the immune surveillance for malignant cells. Unusual tumors occur at high frequency in patients with decreased CD4+ cell function (e.g. Kaposi’s sarcoma, lymphomas in AIDS patients).

2. Immunopathologic a) Contact hypersensitivity - skin reactivity to certain

environmental agents, including poison oak/ivy, nickel, rubber products (including latex exam gloves!), PABA in suntan lotions, adhesives, and many other compounds. Typically the sensitizing agent is a hapten that binds to tissue proteins to form a hapten-carrier conjugate. These are processed and presented by Langerhans cells that are present in the skin and may migrate to lymph nodes. TDTH cells are sensitized and will react to subsequent exposures to the antigen. When exposed to irritants, keratinocytes often express MHC Class II proteins and cytokines, enhancing the hypersensitivity response.

b) Autoimmune diseases - DTH reactions are involved in many autoimmune diseases, including multiple sclerosis, insulin dependent diabetes mellitus, Hashimoto’s thyroiditis, and rheumatoid arthritis. None of these appear to be ‘pure’ DTH responses, but rather involve a mixture of different effector mechanisms.

c) Transplant rejection - DTH is active in acute allograft rejection, along with CTL reactions. In this type of reaction, activated macrophages cause tissue damage by release of lysosomal contents and oxygen radicals (rather than phagocytosis). Reactive T cells apparently recognize the allograft MHC proteins as “altered self”, and therefore are able to respond despite MHC restriction. An unusually high proportion of T cells (up to 10%) respond during allograft rejection.

Clinical Vignette – DTH Reactions (Geha and Notarangelo, “Case Studies in Immunology”, 6th Ed., 2012) Case 51 Atopic Dermatitis – Tom Joad, 2 yo male with severe eczema Case 53 Contact Hypersensitivity to Poison Ivy – 7 yo Paul Stein develops itchy eruptions after a hiking trip

which responded to corticosteroids; the lesions ‘rebounded’ after the corticosteroids were stopped. Case 48 Lepromatous Leprosy – Ursula Iguaran has leprosy, and develops disseminated lesions with large

numbers of M. leprae due to a Th1-Th2 imbalance and a resulting poor DTH response.

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Blast transformation assays - in vitro measures of T cell reactivity. 1. Peripheral blood lymphocytes are incubated in the

presence of: a) Mitogens - agents that cause nonspecific

proliferation of certain populations of lymphocytes: Measure of the overall activity of that cell population.

Concanavalin A (ConA) and phytohemagglutinin (PHA) - plant proteins that cause proliferation of T cells

Lipopolysaccharide (LPS) - causes proliferation of B cells

b) Antigens - provides information on the reactivity of the individual to specific antigens. Example:

Mixed leukocyte culture - inactivated recipient cells mixed with donor lymphocytes. Shows whether CD4+ cells of recipient react to Class II MHC of donor.

2. If reactive, lymphocytes begin to proliferate. 3H-

thymidine is added, and the amount of radioactivity incorporated into DNA determined as a quantitative measure of proliferation.

3. High levels of incorporation relative to controls indicate a response. Why are responses to mitogens typically much higher than responses to specific antigens?

7. GRANULOMATOUS REACTIONS

A. Definition - space-occupying lesion consisting of a predominantly mononuclear infiltrate (lymphocytes and macrophages) at the site of deposition of a poorly degradable antigen.

B. Mechanisms 1. Usually caused by DTH reactions, but sometimes brought about by nonspecific

reactions (e.g. silicosis) or antibody-mediated reactions. The archtypical example is the granuloma characteristic of tuberculosis.

2. CD4+ lymphocytes and macrophages accumulate at the site of the antigen in a typical DTH response. If the antigen (such as M. tuberculosis) continues to replicate or is not easily degraded, it will persist and cause continued accumulation of cells. The resulting granuloma can be up to several cm in diameter, and contains epithelioid cells (enlarged macrophages expressing TNF) and multinucleate giant cells (formed by the fusion of macrophages). In large granulomas, the center can become necrotic, forming a cavity. The granuloma can also displace normal tissue and cause fibrosis, decreasing tissue function (e.g. in the lung).

3. In inactive TB, granulomas containing viable M. tuberculosis can persist for decades without affecting health. However, breakdown of the granuloma or changes in the immune status of the individual may allow mycobacteria to grow out, resulting in active disease.

4. Persistence of immune complexes can also cause granuloma formation.

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C. Medical aspects 1. Mycobacterial infections - as described above, granulomas are important in

tuberculosis and leprosy. They can be detected in chest Xrays and are indicative of past or present active TB.

2. Parasitic infections - attempts to destroy or wall off parasites (such as worms) can result in granulomas. In extreme cases (e.g. Roundworm Wuchereria bancrofti), these can occlude lymphatic vessels and cause elephantiasis.

3. Sarcoidosis - disease of unknown etiology that causes granulomas in multiple sites, including the lungs and skin.

4. Crohn’s disease - inflammatory disease of the bowel, in which granulomatous reactions can cause stricture (obstruction) and fistula formation. Etiology unknown.

T-DTH

C1->C3b

OPSONIZATION

C3a, C5a, C5-7

CHEMOTAXIS

LYMPHOKINES

ACTIVATED

MACROPHAGES

IgG ANTIBODY

SENSITIZED

CELLS

+

INSOLUBLE ANTIGEN

GRANULOMA - SPACE OCCUPYING MASS

TUBERCULOSIS

LEPROSY

PARASITIC INFECTIONS

SARCOIDOSIS

GRANULOMATOSES

CLINICAL CONDITIONS

GRANULOMATOUS REACTIONS

MACROPHAGE

Clinical Vignette – Granulomatous Disease, Geha and Notarangelo, “Case Studies in Immunology”, 6th Ed. Case 26 Chronic Granulomatous Disease – Randy

Johnson develops granulomas and is unable to ward off Aspergillus and other opportunistic pathogens due to inability of his phagocytes to produce H2O2 and superoxide anion.

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SUMMARY -- CELL-MEDIATED REACTIONS

1. Cell-mediated reactions come about when T cells play a central role as effector cells. Another term is cell-mediated immunity or CMI. These reactions include T-cell cytotoxicity and delayed type hypersensitivity.

2. T-cell cytotoxicity occurs when an activated cytotoxic T cell (usually CD8+) binds directly to a target cell via a specific interaction of the TCR with Ag-MHC complexes on the target cell surface. Killing of the target cell occurs through two mechanisms. Release of granules containing perforins and granzymes result in formation of a pore in the target cell membrane, causing rapid lysis. A second major mechanism involves apoptosis, where programmed cell death is activated through a complex cascade involving Fas-Fas ligand interaction, activation of Jun kinase, Caspase 8, and other target cell signal transduction proteins, nucleus fragmentation, organelle destruction, and DNA cleavage. Cell death occurs over a 1-2 day period. T-cell cytotoxicity is protective against many viral infections, tumors, and intracellular pathogens, but is also involved in autoimmune diseases, contact dermatitis, viral rashes, and graft rejection. It can be quantitated through cell lysis assays, including the chromium release assay.

3. Delayed-type hypersensitivity (DTH) (also called Type IV hypersensitivity) is the activation of macrophages by cytokines produced by lymphocytes, typically Th1 cells. When Th1 cells are activated by exposure to antigen, they produce macrophage chemotactic factor, interferon-gamma, and tumor necrosis factor which attract and activate macrophages. These activated macrophages are much more effective in destroying intracellular pathogens and tumor cells. DTH is protective against many intracellular bacteria and protozoa, including mycobacteria and Pneumocystis carinii. Adverse effects include participation in contact hypersensitivity, autoimmune diseases, and transplant rejection. DTH responses can be measured indirectly by blast transformation assays or more directly by quantitation of cytokine production.

4. Granulomatous reactions are collections of lymphocytes and enlarged macrophages resulting from a chronic response to an antigen that is difficult to destroy. Persistent M. tuberculosis infection is an example of a disease process leading to granuloma formation. CD4+ Th1 cells attract macrophages to the area, but they continue to collect due to failure to eliminate the antigen. Granulomatous reactions are prominent in mycobacterial infections, some parasitic infections, sarcoidosis, and Crohn's disease.

178

IMMUNOLOGY OF HIV INFECTION Steven J. Norris, Ph.D.

Required Reading: Geha and Notarangelo, 6th edition (2012). Case Studies in Immunology. Garland Publishing, New York, NY. Case 10: AIDS. Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/immunology-of-aids/ I. HUMAN IMMUNODEFICIENCY VIRUS

A. AIDS Related Retroviruses 1. Human Immunodeficiency Virus-I (HIV-1) is the type most commonly

associated with HIV infection and AIDS in the United States and Europe. HIV-2, which shares ~50% nucleotide identity to HIV-1, is associated with a small number of cases in the U.S., but is prevalent in regions of Africa.

2. HIV-1 and HIV-2 are members of the retrovirus family, a group of viruses that have an RNA genome but form a DNA intermediate that is incorporated into the genome of the host cell. There are oncogenic (tumor causing) and cytolytic (cell-killing) subfamilies of retroviruses. HIV is part of a group called lentiviruses, slow-acting cytolytic retroviruses (lento means slow in music). An example of an oncogenic retrovirus is Human T Lymphocyte Virus (HTLV), which is associated with T cell lymphomas.

Virus Disease Lentiviruses Human immunodeficiency virus Cause of human AIDS Simian immunodeficiency virus AIDS in monkeys Visna/maedi virus Neurologic and lung disease in sheep

Equine infectious anemia virus Horse anemia . Caprine arthritis/encephalitis virus Goat encephalitis

B. The HIV Genome and Structure

1. The HIV genome consists of a 9,000 bp segment of single-stranded RNA. It encodes a series of gene products that are cleaved by the HIV protease to form important structural and nonstructural proteins (see figure).

2. Proteins important in the immune response to HIV include: a) The envelope (env) glycoproteins gp120 and gp41, and their

precursor gp160 b) The group antigen (gag) proteins p24 (major core protein) and

p17 (protein that forms a scaffold during virion assembly) c) The pol proteins p66 and p51 (form reverse transcriptase),

protease, and p32 (endonuclease) d) Regulatory proteins including tat (transactivator), rev (regulator of expression), vif

(virion infectivity factor), and nef (negative factor). These regulate HIV virus gene expression and assembly.

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C. HIV Replication and Gene Expression (see figure)

1. Binding and internalization. gp120 on the surface of the virion binds with high affinity to CD4 on the surface of CD4+ T cells and some other cells types. Gp120 is removed, exposing gp41 underneath, which then promotes fusion between the cell membrane and viral membrane. As a result, the viral core is released into the cytoplasm of the cell. Alternatively, antibody or C3b bound to the surface of the virion can bind to Fc or complement receptors on macrophages and other cells, resulting in internalization (antibody dependent enhancement). Lastly, other “co-receptors” such as chemokine receptors (e.g. CXCR4 and CCR5) and the glycolipid galactosyl ceramide can interact with HIV, promoting infection of other cell types, albeit at much lower efficiency than CD4 binding. A 32-bp deletion in the CCR5 gene (ccr532) that eliminates CCR5 expression has been linked to resistance to HIV infection.

2. Reverse transcription and incorporation. The virion RNA is replicated by virus-associated reverse transcriptase, resulting in a double-stranded DNA copy of the viral genome. This DNA becomes circularized and then incorporated into host chromosome.

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3. Transcription and translation of viral genes. Host transcription factors along with viral factors such as tat activate transcription of the viral genes, resulting in protein expression. Large precursor proteins are cleaved into the final protein products by HIV protease. This step is blocked by drugs called protease inhibitors, thus inhibiting viral replication.

4. Assembly and budding. The viral core, including two copies of the RNA genome, assemble and bud through the cell membrane to form an infectious virion.

5. Latent infection vs. virus production. Resting CD4+ T cells typically exhibit latent infection, that is they contain HIV DNA but do not actively produce virus. Cell activation as indicated by expression of HLA-DR and other Class II MHC proteins is required for high level virus production. Enhanced viral production is linked to expression and activation of nuclear factor kappa B (NF-B) and other host cell transcription factors. During primary HIV or late symptomatic infection, 1 out of 10 peripheral blood CD4+ T cells may be latently infected, whereas only 1 out of 300 to 400 are actively producing virus. Tissue macrophages are an important reservoir of infection, in that they can become infected and produce low levels of virus without being killed. Many other cell types, including epithelial cells, can be infected. Macrophages and other cells can be latently infected for long periods and then express virus when activated by exposure to cytokines, viruses or other infectious agents, and other factors.

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II. CLINICAL COURSE OF HIV INFECTION HIV infection and AIDS are not equivalent. HIV infection means, quite literally, infection with HIV-1 or HIV-2. HIV+ patients can lead normal, healthy, productive lives. Unfortunately, HIV infection almost inevitably progresses to the profound immunodeficiency and opportunistic infections of Acquired Immunodeficiency Disease Syndrome (AIDS). This process usually takes 8 to 12 years for sexually transmitted infection, fewer years for blood transmission, and <1 year for congenital infection. The rapidity of progression is related to the viral dose and, in the case of congenital infection, the immature immune system of the host.

A. Primary infection 1. Nearly all cases of HIV infection arise from sexual contact, transfer of

infected blood (by used needles, transfusion, etc.), or maternal/fetal transmission.

2. Although blood, semen, and other body fluids often contain HIV virions, most sexual transmission is thought to be due to transfer of infected cells. In infected individuals, the percentile of infected cells in peripheral blood mononuclear cells is ~0.001-1%, and in semen is 0.01-5%. Virion and infected cell levels are highest during primary infection and late stages (AIDS).

3. Sexual transmission occurs through mucus membranes and is apparently enhanced by tissue disruption or ulcerative diseases (such as syphilis and chancroid).

4. One to three weeks after exposure, the patient may develop primary infection symptoms, consisting of headache, retro-orbital pain, muscle aches, low- to high-grade fever, and lymphadenopathy (swollen lymph nodes). A characteristic maculopapular erythematous rash (red spots or raised bumps) may appear on the trunk and spread to the extremities. These symptoms last for a few weeks.

5. HIV virions are found in the blood, cerebrospinal fluid, and seminal fluid in high numbers at this stage. The blood viral load in these patients averages 5 x 106 (see table). Anti-HIV antibodies may be detected in some patients within 6 to 14 days after the development of symptoms. Over 90% of HIV-infected patients have seroconverted (have detectable anti-HIV antibodies) within 3 months of infection. A small percentage may remain seronegative after 6 months.

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Viral Load at Different Stages of HIV Infection. Viral load is the total number of virions per ml, as determined by a quantitative competitive polymerase chain reaction (QC-PCR) technique. It is estimated that the number of infectious virions is about 60,000-fold less than the total number of virions determined by QC-PCR and other means. The noninfectious virions may have defective RNA or be missing other required components. Clinical Stage Ave. Viremia (Virions/ml blood) Primary (acute) infection 5 x 106 Asymptomatic 8 x 104 Early symptomatic 35 x 104 AIDS 2.5 x 106 .

B. Asymptomatic Infection (Category A)

1. After primary infection, patients enter an asymptomatic phase in which they appear healthy. Although the patients have high viral loads, they have normal CD4+ lymphocyte levels and immune responses.

2. Originally, it was thought that virus production was low during the asymptomatic stage of HIV infection. However, recent studies have shown that up to 109 virions are produced per day, and that the average half-life of infected CD4+ cells is 2 days. Therefore asymptomatic infection actually is a steady state involving massive turnover of both new virions and newly produced CD4+ T cells.

C. Early symptomatic infection (Category B)

1. At this stage (previously called AIDS-related complex or ARC), the years of viral infection and cell destruction begin to take their toll. CD4+ cell numbers begin to decline, and other changes (e.g. chronic lymphadenopathy) occur. CD4+ cell counts in peripheral blood decline to 200-499 per l (between normal levels and those characteristic of AIDS)

2. CD4+ cell function, as measured by lymphocyte proliferation assays and other tests, declines before decreases in cell numbers are evident.

3. May be accompanied by fever, chronic diarrhea, oral or persistent vulvovaginal candidiasis, and other manifestations.

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Late symptomatic infection (AIDS) (Category C) 1. Acquired immunodeficiency disease syndrome (AIDS) is defined as

HIV infection together with the occurrence of unusual infections, tumors, or other manifestations (e.g. HIV-related encephalopathy) or blood CD4+ cell levels of <200/l (1993 AIDS Surveillance Case Definition, Centers for Disease Control & Prevention).

2. Unusual infections include a long list, but most typically involve Pneumocystis carinii pneumonia, chronic or disseminated fungal infections, M. tuberculosis, M. avium complex, or M. kansasii pulmonary or extrapulmonary infections, herpesvirus or CMV infections, or intestinal parasites (e.g. Cryptosporidium).

3. Tumors include Kaposi’s sarcoma, a variety of lymphomas, and invasive cervical cancer.

4. Other conditions are HIV-related encephalopathy (apparently caused by CNS infection by HIV), progressive multifocal leukoencephalopathy, and generalized wasting.

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III. HIV INFECTION AND THE IMMUNE RESPONSE The interaction between HIV and the immune system is, to say the least, complex. Most of the manifestations of AIDS are directly related to decreased CD4+ T cell numbers and function, which increases susceptibility to intracellular pathogens, viruses, fungi, and certain tumors. CD4+ T cells play a central role as helper cells and mediators of delayed type hypersensitivity. Other patients with depressed CD4+ T cell activity (e.g. renal transplant patients on immunosuppressive therapy) have a similar pattern of increased susceptibility (although usually not as severe). As described below, virtually every arm of the immune response is involved in combating HIV infection, but is also compromised by the effects of CD4+ cell deficiency. Because the virus genomic sequence becomes incorporated into the host cell DNA and can establish long-term latent infection, the virus eventually overwhelms the immune response, leading to the breakout of rampant HIV infection, opportunistic infections, and tumor growth that characterizes AIDS. Highly Active Anti-RetroViral Therapy (HAART) prevents this progression by reducing the number of infectious virions to extremely low levels; however, the viral load will again increase if HAART is discontinued. A. Antibody responses

1. Antibodies against a variety of HIV proteins are expressed following primary infection. These can be detected in serum by an HIV ELISA test or Western blot assay (see below). The combination of two positive ELISA tests and one positive Western blot assay is considered to be diagnostic of HIV infection. Negative tests obtained within 6 months of potential HIV exposure should be repeated later, because the patient may be infected but not have expressed sufficient antibodies for a positive result.

ELISA TEST FOR WESTERN BLOT ANALYSIS ANTI-HIV ANTIBODIES

1. Add test serum to well coated coated with HIV proteins

2. Incubate and wash 3. Add goat anti-human IgG

horseradish peroxidase conjugate 4. Incubate and wash 5. Add substrate for

horseradish peroxidase 6. Color change indicates

presence of anti-HIV antibodies in test serum (positive result)

NOTE: DESCRIPTION OF HIV ELISA AND WESTERN BLOT PROCEDURES: Geha and Notarangelo, “CASE STUDIES IN IMMUNOLOGY”, 2012, CASE 10.

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2. Anti-HIV antibodies may have neutralizing activity, i.e. be able to inactivate

the virus. Such protective antibodies are generally directed against the surface proteins gp120 and gp41. These antibodies may also mediate antibody-dependent cellular cytotoxicity (ADCC) of infected cells.

3. Anti-gp120 antibodies may also increase the efficiency of infection in vitro (so-called Antibody Dependent Enhancement or ADE). Antibody and complement components on the surface of HIV can bind to Fc receptors and complement receptors on macrophages, increasing the efficiency of binding and internalization. Neutralization vs. enhancement may depend on the particular epitopes recognized by the antibodies. As with many other in vitro findings, the clinical significance of ADE is not known.

4. Immune complexes of virions, antibodies, and complement have been shown to be infectious, indicating that immune complex formation may also increase the efficiency of macrophage infection.

5. During late symptomatic infection, antibody levels (both total and anti-HIV) decline due to the lack of functional T helper cells, further exacerbating HIV and opportunistic infections.

6. Vaccination protocols must take into account the possibility that the resulting antibody responses may enhance the efficiency of viral infection.

B. CD4+ T Cell responses - help and DTH

1. CD4+ T cells are the principal target of HIV infection, and as such are the most profoundly affected cell type. CD4+ T cell levels are measured as the total number of cells per l blood using a flow cytometer (also called a fluorescent activated cell sorter; see pages 103-104 in book).

2. Even during the so-called asymptomatic phase, there is extensive HIV-mediated killing of CD4+ T cells, so that the HIV-infected patient must produce many-fold more cells to maintain normal levels (>500/l).

3. After several years, the ability of the host to replace the CD4+ lymphocytes declines, resulting in reduction of CD4+ counts into the 200-499 cells/l range.

4. When CD4+ cell counts fall below 200 cells/l, the patient becomes deficient in DTH and helper activities, and thus much more susceptible to opportunistic infections, tumors, and rampant HIV infection. This threshold corresponds to the onset of AIDS symptoms.

5. Some CD4+ T cells have cytotoxic activity and may be involved in combating HIV infection.

6. Functional activities of Th1 cells such as IL-2 and IFN-production decline long before cell numbers decrease. Conversely, IL-4 and IL-10 expressed by Th2 cells increase late in infection. This shift in cytokine expression may play an important immunoregulatory role during HIV infection.

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C. CD8+ T cell activities – cytotoxicity and antiviral activity 1. CD8+ lymphocytes from HIV+ patients can kill HIV-infected cells. This

CTL activity is high during the asymptomatic phase, and decreases with progression of the disease.

2. CTLs may also be involved in the destruction of uninfected CD4+ cells (see figure below). Soluble gp120 produced by HIV-infected cells can bind to CD4 molecules on uninfected cells and result in expression of Fas. Fas ligand on CTLs will bind to Fas and initiate apoptosis of the target cell. (see Cell-Mediated Reactions lecture for review). Uninfected CD4+ cells could be destroyed by this mechanism.

3. In addition, CD8+ cells express a cell antiviral factor (CAF) that inhibits HIV replication in infected cells without killing the cells. When the factor is removed, viral replication resumes. Recently, CAF activity was found to be associated with expression of -defensins by CD8+ T cells. -defensins are peptides have anti-bacterial activity and are usually produced by neutrophils. The mechanism by which defensins inhibit HIV-1 replication is unknown currently. Defensins are expressed in higher quantities by long-term nonprogressors (LTNPs), i.e. HIV-infected patients who remain healthy.

IV. PROSPECTS FOR IMMUNOTHERAPY AND VACCINATION At present, therapy for HIV infection involves treatment with nucleoside analogs such as zidovudine (AZT) to inhibit viral reverse transcriptase and protease inhibitors, which block the cleavage of protein precursors by HIV protease. In addition, antimicrobial compounds can be administered prophylactically (as a preventative measure); an example is the use of aerosolized pentamidine to prevent Pneumocystis carinii pneumonia. Ideally, it would be best to prevent HIV infection through vaccination or other means. Alternatively, immunotherapeutic measures could be used to augment the immune response of HIV-infected individuals.

A. Immunotherapy 1. Immune reconstitution. The idea behind this therapy is akin to a

bone marrow transplant – replace the infected cells with those of an HIV-negative donor. However, human donor lymphocytes would

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rapidly become infected. Transplant of baboon bone marrow cells into an HIV+ patient was attempted, with the rationale that baboons are resistant to HIV infection. However, the baboon cells failed to “take” (survive) in the patient.

2. Passive immunotherapy. Injection of “cocktails” human anti-HIV monoclonal antibodies into HIV+ patients is being tried as a means of decreasing viral loads. However, it is unclear how these cocktails are in any way better than the patient’s own antibodies, particularly given the degree of antigenic heterogeneity in HIV (see below).

B. Vaccination 1. Given that vaccines against Feline Leukemia Virus, a retrovirus, are successful

in preventing transmission of FLV in cats, it is possible that an effective vaccine against HIV could be developed. It is also possible (although unlikely) that vaccination of infected individuals could enhance viral clearance.

2. Stage II clinical trials have shown that a recombinant form of gp160 or gp120 (VaxGen) is safe and induces antibodies against HIV-1. Thus far, the antibodies neutralize strains expressing the same gp160, but not other primary HIV isolates (due to sequence variation). This vaccine will is being tested for its ability to prevent maternal/fetal transmission of HIV.

3. Recombinant Live-Vector Vaccines consist of a carrier virus containing HIV genes. Ones that are being tested extensively are a recombinant vaccinia-HIV gp160 (a modified smallpox vaccine) and a canarypox-HIV vaccine. The canarypox form has improved safety, because it does not replicate in human cells. “Prime boost” strategies with a boost of recombinant gp120 or gp160 have been more effective. Several other live vectors are being developed.

4. Virus-like particles (VLPs) consist of the protein capsid without the nucleic acid, and thus are non-infectious. A p17/p24 VLP of the HIV core is being tested, and a p55 (gag) form is also being produced.

5. Vaccines using peptides corresponding to important epitopes on gp120 (e.g. the V3 loop and the CD4 binding region) could potentially be used. Branched and lipidated forms (to increase immunogenicity) are being tried.

6. DNA vaccines are naked DNA encoding viral proteins that is taken up and expressed by the vacinee’s own cells. These show promise, particularly in combination with boosts with protein vaccines.

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7. Attenuated viruses represent another approach. Monkeys vaccinated with a Simian Immunodeficiency Virus variant lacking the nef gene were resistant to infection with “wild-type” SIV. Intentional infection of humans with an attenuated form of a virus that incorporates into host DNA and mutates rapidly has hefty ethical implications.

8. Immunization of chimpanzees with formalin-inactivated HIV has not provided convincing evidence of protection.

C. Problems with vaccination - multiple

1. Intracellular location/latency - latently infected cells that have HIV DNA incorporated into their genome can be activated at a later time, after the effects of vaccination have waned.

2. Antigenic modulation - RNA viruses have a high mutation rate, resulting in rapid changes in antigenic structure. In the human population there are many different antigenic variants of HIV, called clades. In addition, a patient infected with one genotype of HIV later has several different genotypes, some of which differ in the structure of important antigens (e.g. gp120). Immunization with a single ‘serotype’ of HIV may not be effective in preventing infection or enhancing immune clearance.

3. Inappropropriate or ineffective immune responses. As mentioned earlier, antibodies against HIV may actually enhance the infection of macrophages. T-CTLs activated by vaccination may destroy uninfected T cells by the mechanism involving gp120 binding described above. Furthermore, activation of infected macrophages and T cells through immunotherapeutic means may increase virus production.

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D. Animal models

Animal models of HIV infection provide a means of studying HIV pathogenesis and immunity. However, none of these closely resemble the human disease. One of the most effective models is the SCID mouse- human lymphoid cell chimera. In this case, a mouse strain with severe combined immunodeficiency can be injected with human lymphoid cells. The human cells persist, and can be infected with HIV upon subsequent injection of the virus. This system permits the study of human cell infection in a surrogate model. Animal Virus Result Used to study: Chimpanzee HIV-1 Latent Infection Vaccine efficacy Macaques SIV AIDS-like wasting

disease Vaccine efficacy, therapy, pathogenesis

Rabbits HIV-1 Defective infection Latent infection Mice- HIV genome transgenic

-- AIDS-like illness Pathogenesis

Mice - tat transgenic -- Kaposi’s sarcoma Carcinogenesis SCID-human lymphoid cell chimera

HIV-1 AIDS-like disease Pathogenesis, therapy

Sheep Visna Chronic neurodegeneration

Lentivirus pathogenesis

Goats Caprine arthritis, encephalitis Lentivirus pathogenesis Horses EIAV hemolytic anemia Lentivirus pathogenesis

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III. SUMMARY 1. HIV-1 and HIV-2 are lentiviruses that cause CD4+ T lymphocyte depletion and

immunodeficiency in humans. 2. Infection of CD4+ T cells, macrophages, and other cell types can lead to virus

production and cytolysis or long-term latent infection. 3. HIV infection progresses through primary infection, asymptomatic infection,

early symptomatic infection, and late symptomatic infection (AIDS). 4. Depletion of CD4+ cells leads to profound defects in DTH and T helper activity,

resulting in susceptibility to rampant HIV infection, opportunistic infections, and tumors.

5. Antibody and cytotoxic lymphocyte activities combat virus production, but eventually are overwhelmed by the virus’ ability to inactivate and destroy CD4+ cells, to change its antigenic structure, and to alternate between latent and active infection.

6. In their current forms, immunotherapy and vaccination have not demonstrated the ability to prevent or combat HIV infection. However, intensive research for the development of an HIV vaccine is ongoing.

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INFECTION AND IMMUNITY Jeffrey K. Actor, Ph.D.

713-500-5344

Objectives: Understand the course of response and major immune defense mechanisms to infectious agents. Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York, NY. 6th edition, 2009. Chapter 20. Geha and Notarangelo. Case Studies in Immunology. Garland Publishing, New York, NY. 6th edition, 2012. Case 48: Lepromatous Leprosy. Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/infection-and-immunity/ The course of response against typical acute infections can be subdivided into distinct stages. Initially, the level of infectious agent is low, beginning with breach of a mechanical barrier (e.g. skin, mucosal surface). Once inside the host, the pathogen encounters a microenvironment for suitable replication. The agent replicates, releasing antigens that trigger innate immune function, generally characterized as non-specific. This innate function assists in limiting expansion of the organism. After 4 or 5 days, effector cells and molecules of the adaptive and specific immune response enable control and eventual clearance of the infectious agent. Once the agent is cleared, the host is left with residual effector cells and antibodies, as well as immunological memory to provide lasting protection against reinfection. The response to initial infection is therefore subdivided into 3 phases. The first is an early innate and non-specific response. Preformed effector cells and molecules recognize microorganisms within the first 4 hours of infection. Although this may be enough to clear the organism, typically more help is necessary. The second phase lasts from 4 hours to 4 days. Again, this is primarily a non-specific encounter with the organism. This phase is characterized by recruitment of effector cells (professional phagocytes, NK cells) to the site of infection, and specific activation of these effectors. The final phase is one where one where adaptive immunity occurs. Antigen specific cells (B and T lymphocytes) undergo clonal expansion to become specific effectors. Some of these effector cells remain even after clearance of the organism, and are able to provide a much more rapid and specific memory response if the organism is re-encountered. A wide variety of pathogenic microorganisms exist. They may be globally classified into groups: Bacterial, Mycobacterial, Viral, Protozoal, Worms, and Fungal. The major immune defense mechanisms are summarized in the following chart: Type of Infection Major Immune Defense Mechanisms

Bacterial Antibody (Immune complex and cytotoxicity) Mycobacterial DTH and granulomatous reactions Viral Antibody (Neutralization), TCTL and DTH Protozoal DTH and antibody Worms Antibody (Atopic, ADCC) and granulomatous reactions Fungal DTH and granulomatous reactions

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The host defense is based upon availability of resources to combat a localized pathogen. Virtually all pathogens have an extracellular phase where they are vulnerable to antibody-mediated effector mechanisms. An extracellular agent may reside on epithelial cell surfaces, where antibodies (IgA) and non-specific inflammatory cells may be sufficient for combating infection. If the agent resides within interstitial spaces, in blood or in lymph, then protection may also include complement components, macrophage phagocytosis and neutralization responses. Intracellular agents require a different response to be effective. For cytoplasmic agents, T lymphocytes and NK cells, as well as T-cell dependent macrophage activation, are usually necessary to kill the organism.

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Pathogens can damage host tissue by direct and indirect mechanisms. Organisms may directly damage tissue by release of exotoxins that act on the surface of host cells, or via released endotoxins that trigger local production of damaging cytokines. Pathogens may also directly destroy the cells they infect. Adaptive mechanisms can cause disease; formation of antibody:antigen complexes can lead to the release of proteins and factors that both mediate control of disease as well as cause tissue damage. Some of the representative infectious agents and the common names of associated diseases are listed in the figure.

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BACTERIAL INFECTIONS

Bacterial infections begin with a breach of a mechanical barrier. Release of bacterial factors upon replication initiates a cascade of events. Initially, infection may be resisted by antibody-mediated immune mechanisms, including neutralization of bacterial toxins. With the help of complement factors direct cytotoxic lysis can occur. Release of C3a and C5a in the complement cascade will cause vasodilation and vasopermeability resulting in an influx of professional phagocytes and acute polymorphonuclear infiltration (Arthus reaction). Opsonization of bacteria leads to increased phagocytosis and acute anaphylactic vascular events permitting

exudation of inflammatory cells and fluids. Phagocytosis may also occur via specific surface receptors for ligands such as mannose or sialic acids. During the chronic stage of the infection cell mediated immunity is activated. TDTH-cells that react with bacterial antigens may infiltrate the site of infection, become activated and release lymphokines that attract and activate macrophages. The activated macrophages phagocytose and degrade necrotic bacteria and tissue, preparing the lesion for healing. The role of complement in response to bacterial infection must be stressed. There are three major biological components of the complement system. They are (1) activation of phagocytes including macrophages and neutrophils, (2) direct cytolysis of target cells, and (3) opsonization of microorganisms and immune complexes for cells expressing complement receptors. The polymorphonuclear cells, especially neutrophils, are an excellent example of the first line of innate defense. Puss is composed of dead and dying polymorphonuclear and host cells, local fluids and exudates, and dead and dying bacteria. Important factors released by macrophages in response to bacterial antigens include cytokines that exert both local and systemic. Locally, IL-1, TNF-, and IL-8 cause inflammation and activate vascular endothelial cells to increase permeability and allow more immune cells to enter infected area. TNF- will also destroy local tissue to limit growth of bacteria. In addition, IL-6 can stimulate an increase in B cell maturation and antibody production, and IL-12 will lead to activation of NK cells and priming of T cells towards a T Helper 1 (TH1) response. Systemically, IL-1, IL-1, TNF-, IL-6 and IL-8 all contribute to elevated body temperature (fever) and production of acute-phase protein production.

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MYCOBACTERIAL INFECTIONS

Mycobacterial infections such as tuberculosis and leprosy are extremely complex. The Mycobacteria have evolved to inhibit normal macrophage killing mechanisms (e.g. phagosome-lysosome fusion) and survive within the “disarmed’ professional phagocyte. These organisms are resisted mainly by TDTH initiated cellular mechanisms, including granulomatous hypersensitive responses, but only after the infection have become established. T cells are mainly responsible for control and containment of the infection. They recognize mycobacterial antigens expressed on the surface of infected cells and release factors that recruit additional immune effectors. A local environment is established to contain infection. Healing of the infected center may occur, with limited necrosis of the infected tissue. However, if the infection

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persists, an active caseous granuloma results. Here, an infected nidus is comprised of infected and active macrophages, ringed by T cells. A host mediated destructive response occurs inside a contained area. The infection itself is surrounded by activate epithelioid cells, with the presence of Giant cells (activated syncytial multinucleated cells,). At one time it was thought that the tissue lesions of the disease tuberculosis required the effect of delayed hypersensitivity. The term hypersensitivity was coined because animals with cellular immune reactivity to tubercle bacilli developed greater tissue lesions after re-inoculation of bacilli than did animals injected for the first time. The granulomatous lesions seen in tuberculosis do depend upon immune mechanisms for their formation. However these lesions are not really the cause of the disease but an unfortunate effect of the protective mechanisms; the granulomatous inflammatory reaction to the infective mycobacterium results in destruction of normal tissue. In the lung, for instance, extensive damage done by the formation of large granulomas in response to a tuberculosis infection can result in respiratory failure. The granulomatous immune response produces the lesion, but the mycobacterium causes the disease. VIRAL INFECTIONS

Immune resistance to viral infections is mainly mediated by cell-mediated-immunity, but humoral (antibody) responses also play a role by preventing virus from attaching to cell receptors. The antiviral response id dictated by availability to be seen by the immune system. Viruses live within the host's cells and can spread from cell to cell. To be effective in attacking intracellular organisms, an immune mechanism must have the capacity to react with cells in solid tissue. This is a property of cell-mediated reactions, in particular TCTL, but not of antibody mediated reactions. Antibodies do play a role during the extracellular life cycle of the virus. Antibodies can bind to virus forming complexes to inactivate virions, and allow them to be cleared effectively

by profession phagocytes. Humoral antibody can prevent the entry of virus particles into cells by interfering with the ability of the virus to attach to a host cell, and secretory IgA can prevent the establishment of viral infections of mucous membranes. However, once the virus is within cells, it

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is not susceptible to the effects of antibody. Natural Killer cells are an early component of the host response to viral infection. NK cells will non-specifically recognize and kill virally infected targets, although the mechanism of recognition still remains unclear. Infected cells have a limited mechanism to down regulate viral replication by the production of IFN- and IFN-. Non-specific interferon responses are not sufficient to eliminate the virus. NK cells release IFN- (physically different from the other interferons) and IL-12, molecules which both activate macrophages and help to prime T cells for an effective anti-viral TH1 response. Non-specific response can not totally eliminate virus.

Many cells infected with a virus will, at some stage of the infection, express viral antigens on the cell surface in combination with Class I molecules. It is at this stage that specifically sensitized CD8+ TCTL cells can recognize and destroy the virus-infected cells through the release of factors (granzymes, perforins, and/or interferons) that either kill the infected host or limit viral replication. Adverse effects of this reaction occur if the cell expressing the viral antigens is important functionally, as is the case for certain viral infections of the central nervous system. If the virus infects macrophages, TDTH-cells can activate the macrophages to kill their intracellular viruses through the activation of the infected macrophages by lymphokines. Lymphokine activated macrophages produce a variety of enzymes and

cytokines that can inactivate viruses. Patients with deficiencies in antibody production alone usually do not have serious viral infections but develop life threatening bacterial infections. Patients with defects in cell-mediated-immunity develop serious virus infections. HELMINTH (WORM) INFECTIONS Host response to helminth infections are generally more complex because the pathogen is larger, and not able to be engulfed by phagocytes. They also typically undergo life cycle changes as they adapt for life in the host. Worms are located in the intestinal tract and/or tissues. Tapeworms, which exist in only the intestinal lumen, promote no protective immunologic response. On the other hand, worms with larval forms that invade tissue do stimulate an immune response. The tissue reaction to Ascaris and Trichinella consists of an intense infiltrate of polymorphonuclear leukocytes, with a predominance of eosinophils. Therefore, a variety of antigens that are life cycle stage dependent are displayed in changing tissue environments. Numerous cells play a role, depending on the location of the organism. Antigens on surface of organisms, or antigens released into the local environment, may stimulate T cells and macrophages to interact with B cells to secrete specific antibodies. One T cell factor (type II) is also instrumental in stimulation of eosinophils (IL-5). The eosinophils act by associating with specific antibody to kill worms by antibody dependent cell cytotoxic mechanisms (ADCC), or by releasing enzymes from granules to exert a controlling effect on mast cells.

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Antigen reacting with IgE antibody bound to intestinal mast cells stimulates release of inflammatory mediators, such as histamine, proteases, leukotrienes, prostaglandins and serotonin. These agents cause an increase in the vascular permeability of the mucosa, exposing worms to serum immune components, stimulate increased mucous production and increase peristalsis. These activities are associated with expulsion of parasitic worms from the gastrointestinal tract through formation of a physical barrier to adherence and interactions with the mucosal surface. Eosinophils contain granules containing basic proteins which are toxic to worms. Eosinophils may be directed to attack helminths by cytophilic antibodies that attach to the eosinophil through the Fc region and to the helminth by specific Fab binding (ADCC). Anaphylactic antibodies (IgE) are also frequently associated with helminth infections, and intradermal injection of worm extracts elicits and wheal-and-flare reaction. Children infested with Ascaris lumbricoides have attacks of urticaria, asthma, and other anaphylactic or atopic types of reactions presumably associated with dissemination of Ascaris antigens. FUNGAL INFECTIONS A great deal is not known concerning immune response to fungal agents. Cellular immunity appears to be the most important immunologic factor in resistance to fungal infections, although humoral antibody certainly may play a role. The importance of cellular reactions is indicated by the intense mononuclear infiltrate and granulomatous reactions that occur in tissues infected with fungi and by the fact that fungal infections are frequently associated with depressed immune reactivity of the delayed type (opportunistic infections). Chronic mucocutaneous candidiasis refers to persistent or recurrent infection by Candida albicans of mucous membranes, nails, and skin. Patients with this disease generally have a form of immune deviation, i.e., a depression of cellular immune reactions, with high levels of humoral antibody; similar to lepromatous leprosy. Fungi appear to be resistant to the effects of antibody, so that CMI is needed for effective resistance. LEPROSY -- IMMUNE DEVIATION The protective function of granulomatous reactivity is exemplified by the spectrum of leprosy. The clinical manifestations of leprosy are determined by the immune response of the patient. The high resistance of tuberculoid leprosy is associated with delayed hypersensitivity and the

formation of granulomas, whereas the low resistance characteristic of lepromatous leprosy is associated with the accumulation of "foamy" macrophages and the presence high levels of humoral antibodies. Immune deviation or split tolerance is defined as the dominance of one immune response mechanism over another for a specific antigen and has

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FORMS OF LEPROSY

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been implicated in the tendency for certain individuals to develop IgE (allergy) antibodies rather than IgG antibodies. In addition, for reasons that are unclear, but may be genetically determined, some individuals tend to make strong cellular immune responses, but weak antibody response to certain antigens, whereas other individuals will have the opposite response. The course of leprosy depends upon the immune reaction of the patient. Leprosy is classified into three major overlapping groups: tuberculoid, borderline and lepromatous. In tuberculoid leprosy there are prominent well-formed granulomatous lesions, many lymphocytes and few if any organisms. Delayed hypersensitivity skin tests are intact and there is predominant hyperplasia of the diffuse cortex (T-cell zone) of the lymph nodes. The level of antibodies is low. In lepromatous leprosy granulomas are not formed, there are few or no lymphocytes and lesions consist of large macrophages filled with viable organisms. Delayed hypersensitivity skin tests are depressed and there is marked follicular hyperplasia in the lymph nodes with little or no diffuse cortex. The levels of antibodies are high and vascular lesions due to immune complexes are seen (erythema nodosum leprosum). Borderline leprosy has intermediate findings. The prognosis in tuberculoid leprosy is good and the response to chemotherapy is excellent. In borderline leprosy a good response to therapy is associated with a conversion to the tuberculoid form. The prognosis in lepromatous leprosy and the response to chemotherapy is poor. The example of the forms of leprosy illustrates the role of cellular immunity (delayed hypersensitivity) in controlling the infection, and the lack of protective response provided by humoral antibodies. This concept is also considered valid for immunity to Candida albicans (chronic mucocutaneous candidiasis). Depressed cellular immunity is associated with chronic mucocutaneous candidiasis, a condition in which the infected individual is unable to clear Candida infections. A diagram illustrating the relationship of the degree of cellular and humoral immune response to the stages of leprosy is shown. The overlapping triangles indicate the relative strength of delayed hypersensitivity and antibody production. The cross-hatched triangle indicates delayed hypersensitivity; the open triangle, antibody production. High levels of delayed hypersensitivity (DTH) are associated with cure of tuberculoid leprosy; weak DTH is associated with progressive disease; balanced DTH and antibody production with borderline leprosy and slowly progressive disease. The cytokine patterns in the two polar forms of the disease are different. Typically T Helper 2 (TH2) cytokines (IL-4, IL-5 and IL-10) dominate in the lepromatous form, while cytokines produced by TH1 cells (IFN-, TNF and IL-2) predominate in tuberculoid leprosy. IFN-g would be expected to activate macrophages to kill intracellular pathogens and control organism expansion; high IL-4 may explain hypergammaglobulinemia in lepromatous patients. EVASION OF IMMUNE DEFENSE In the ongoing evolution of host-parasite relationships between humans and their infections, infectious organisms have developed "ingenious" ways to avoid immune defense mechanisms. Organisms may locate in niches not accessible to immune effector mechanisms (protective niche) or hide themselves by acquiring host molecules (masking). They may change their surface antigens (antigenic modulation), hide within cells, produce factors which inhibit the immune response (immunosuppression), or fool the immune system into responding with an ineffective effector mechanism (immune deviation). The ultimate endpoint of co-evolution of the human

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host and its infectious organisms results in an eventual mutual co-existence with most environmental organisms. No better evidence of this is the loss of this coexistence when the immune mechanisms do not function properly. Then organisms which do not normally cause disease become virulent. The lesson of AIDS demonstrates that new infectious organisms will become dominant when introduced into a previously unexposed population. In a fully evolved, mature relationship host and infectious agent initially co-exist without detrimental affects. Thus the ultimate evolution of the host parasite relationship is not "cure" of an infection by complete elimination of the parasite, but least mutual co-existence without deleterious effects of the parasite on the host. In fact, in many human infections, the infectious agent is never fully destroyed and the disease enters a latent state that can be reactivated under different conditions.

Bacteria have evolved to evade different aspects of the phagocyte-mediated killing. For example, they may (1) secrete toxins to inhibit chemotaxis, (2) contain outer capsules that block attachment, (3) block intracellular fusion with lysosomal compartments, and (4) escape from the phagosome to multiply in the cytoplasm. Viral entities also subvert immune responses usually through the presence of virally encoded proteins. Some of these proteins block effector functions of antibody binding, block complement mediated pathways, and inhibit activation of infected cells. The Herpes virus produces a factor that inhibit inflammatory responses by blocking effects of cytokines through receptor mimicking, and another that blocks proper antigen presentation and processing. Finally, Epstein-Barr virus encodes a cytokine homolog of IL-10 which leads to immuno-suppression of the host by activating TH2 rather than TH1 responses.

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Summary: Infection and Immunity The response to initial infection may be divided into 3 phases. The first is an early innate and non-specific response, where preformed effector cells and molecules recognize microorganisms. The second phase is again primarily a non-specific encounter with the organism, characterized by recruitment of professional phagocytes and NK cells to the site of infection. The final phase involves antigen specific cells (B and T lymphocytes) effectors which undergo clonal expansion; these cells provide memory responses in case of reinfection. The host defense is based upon availability of resources to combat a localized pathogen. Virtually all pathogens have an extracellular phase where they are vulnerable to antibody-mediated effector mechanisms and complement components, macrophage phagocytosis and neutralization responses. Intracellular agents usually require T lymphocytes (helper and cytotoxic) and NK cells, as well as T-cell dependent macrophage activation, to kill the organism. Pathogens can damage host tissue by direct and indirect mechanisms. The main immune mechanisms against pathogens are as follows: Bacterial, Antibody (Immune complex and cytotoxicity); Mycobacterial, DTH and granulomatous reactions; Viral, Antibody (Neutralization), TCTL and DTH; Protozoal, DTH and antibody; Worms, Antibody (Atopic, ADCC) and granulomatous reactions; Fungal, DTH and granulomatous reactions. Virtually all classes of infectious agents have devised ways to avoid host defenses. These mechanisms include: non-accessibility in protective niches, antigenic modulation of surface molecules, and release of factors to either suppress the immune response, or cause immune deviation and ineffective response to the agent.

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Clinical Vignette - The case of Ursula Iguaran (Case 48 in Geha and Notarangelo): Ursula Iguaran, a native of Columbia, developed hypopigmented lesions on her hands and arms when she was 16, with progressive lesions developing through the next two years. Blood tests revealed normal white blood counts. Dermatological evaluation revealed numerous Virchow's cells (foamy macrophages) and few lymphocytes within the lesions. Histological analysis of a forearm biopsy revealed clumps of acid-fact bacilli. She was diagnosed as having Mycobacterium leprae. Ursula was aggressively treated with a multiple drug regime (dapsone, clofazamine and rifampin). Her skin lesions gradually flattened and improved. The immune response in patients with Lepromatous Leprosy is skewed towards the production of T helper 2 cytokines. On this basis, would Ursula be more susceptible to certain types of infections? Which ones and why?

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IMMUNE REGULATION: TOLERANCE

Dat Q. Tran, MD | Assistant Professor Division of Pediatric Research Center Division of Allergy | Immunology | Rheumatology Department of Pediatrics MSE R428 713-500-5422 Dat.Q.Tran@uth.tmc.edu Learning Objectives

1. Understand mechanisms of central and peripheral tolerance 2. Understand clinical manifestations when tolerance is perturbed or disrupted 3. Appreciate potential targets for therapeutic intervention and development of

curative treatments

Keywords immune tolerance, immunosuppression, apoptosis

Required Reading: Coico and Sunshine, 2009. Chapter 12.

Geha and Notarangelo. Case Studies in Immunology. Garland Publishing, New York, NY. 6th edition, 2012. Case 40 Multiple Sclerosis; Case 50 Allergic Asthma. Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/immune-regulation-tolerance/ See also notes from Spring 2013: compliments of Dr. Shen-An Hwang, Ph.D. Overview: control of immune response. The major goal of the adaptive immune response is directed towards recognition of specific non-self antigens. Specificity allows recognition of foreign (non-self) antigens. The intensity and duration of the response dictates sufficient protection against invading pathogens with prompt and specific downregulation when foreign antigen is no longer present. Recognition of self-antigens does occur, with immunological tolerance defined as specific unresponsiveness to an antigen while allowing the rest of the host response to remain intact and capable of response. Autoimmunity - loss of tolerance Central Tolerance - B cells Balance of genes, immune regulation and environment Immune Regulation - B cells Cellular strategies used to regulate self-reactive receptors during differentiation B cell activation - anergy Role of CD40

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Peripheral tolerance - B cells Maturation of B cells Autoantibody effector function

Example: Autoimmune Hemolytic Anemia Example: Idiopathic Thrombocytopenia Purpura (ITP)

Mechanism of autoantibody production Antinuclear antibodies

Autoimmune disorders: Myasthenia gravis – antagonist; targets and symptoms Graves disease – agonist; target and symptoms

Central - Peripheral tolerance Primary and Secondary lymphoid tissues Central tolerance - t cells Positive vs Negative selection Diseases resulting from defect in central tolerance Apeced (aps1) - autoimmune polyendocrinopathy candidiasis ectodermal dystrophy Immune Regulation - T cells Priveleged sites, Regulatory cells, Anergy, Death signals T cell activation – anergy Two signal theory Peripheral tolerance – costimulation Normal response vs Clonal anergy Inhibitory signals Inhibitory cytokines; mediation of immune suppression Peripheral tolerance – Activation Induced Cell Death (ACID) Fas and Fas Ligand Apoptosis Diseases resulting from defect in apoptosis

Alps - autoimmune lymphoproliferative syndrome

Inhibitory signals Inhibitor receptor engagement vs lack of engagement

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Peripheral tolerance - dendritic cells Peripheral immunity – regulation T cell subsets Peripheral tolerance – barriers Sequestration and privileged sites Fetal materal tolerance – Role of Tregs Foxp3; TGF-beta Oral tolerance - mucosal immunity Antigen dose Diseases with early onset and multiple autoimmune conditions Foxp3+ regulatory T cells (Tregs) Immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome Scurfy mouse model Tregs

Development Suppressive mechanisms Relationship to autoimmunity diseases

Diseases associated with dysregulation in tolerance

Example: Type 1 diabetes Example: Multiple sclerosis Example: Cancer - anti-tumor immunity

Summary

Immune response must be regulated to allow sufficient response to protect host with excessive or inappropriate responses that may create disease

Immunological tolerance is specific unresponsiveness to an antigen while allowing the rest of the host response to remain intact and capable of response

Tolerance can be at the level of B or T lymphocytes or both and can be accomplished by a variety of mechanisms including apoptosis, anergy, and

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suppressor T cell activity

Many factors influence the nature, intensity and duration of an immune response including age, neuroendocrine hormone levels, HLA allotypes, antigen dose, antigen access, and cytokine milieu.

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AUTOIMMUNITY AND AUTOIMMUNE DISEASES

Sandeep K. Agarwal, M.D., Ph.D. Medicine-Immunology, Allergy & Rheumatology, BCM

713-798-3390 skagarwa@bcm.edu Objectives

1. Define and discuss autoimmunity. 2. Use autoimmune diseases to illustrate mechanisms of autoimmunity. 3. Provide you with clinical correlations and applications of the basic principles of

immunology. Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York, NY. 6th edition, 2009. Chapter 12 (p190-204). R. S. Geha and Notarangelo, L. Case Studies in Immunology: A Clinical Companion. (6th Ed) Garland Publishing, New York, 2012. Chapter 36. Rheumatoid Arthritis; Chapter 40. Multiple Sclerosis; Chapter 41. Autoimmune Hemolytic Anemia. Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-

lectures/autoimmunity/ INTRODUCTION: The regulation of immune function and overall immuno-homeostasis is under control of multiple factors that include genetic and environmental components. HLA allotypes, antigen dose, and existing cytokine milieu can all influence responses to both pathogenic agents and self antigens. It is highly recommended to review the reading materials PRIOR to lecture, as the lecture will primarily concentrate on clinical manifestations of autoimmune disorders.

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Autoimmune Lecture Outline Autoimmunity

• Specific adaptive immune response mounted against a self-antigen – Loss of Self-tolerance to self-antigens – Loss of central and peripheral tolerance • Loss of central tolerance likely occurs all the time

• May have a physiological role to clear defective or denatured molecules through the RE system

• Normally kept in check by mechanisms of peripheral tolerance • May be triggered by infections or aging • May or may not cause disease

Autoimmune Disease

• Termed “horror autoxicus” by Paul Ehrlich • Tissue response and damage triggered by autoimmunity • Results from the dysregulation of immune processes and pathways that

are involved in normal immunity Architecture of an Autoimmune Response

• Innate and Adaptive components Autoimmune Disease and Clinical Phenotypes: AUTOIMMUNE DISEASE CLINCAL PHENOTYPE

Systemic Lupus Erythematosus Rash; inflammation of joints and serosal linings; glomerulonephritis; hemolytic anemia, systemic symptoms

Rheumatoid Arthritis Inflammation of synovium of diarthroidal joints, systemic inflammation

Scleroderma Inflammation, dermal fibrosis, internal organ fibrosis, vasculopathy

Ankylosing Spondylitis Inflammation of spine, joints, and tendon insertions; uveitis

Multiple Sclerosis Demyelination, optic neuritis, neurological deficits

Myasthenia Gravis Skeletal muscle weakness, diplopia, dysarthria, dysphagia

Hashimoto’s Thyroiditis Hypothyroidism

Graves Disease Hyperthyroidism, opthalmopathy

Celiac Disease Diarrhea and malabsoprtion

Autoimmune hemolytic anemia Anemia through lysis of red blood cells

Type I diabetes Failure of insulin production and glycemic control

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Genetic Susceptibility to Autoimmune Diseases Simple Genetic Traits Associated with Autoimmune Diseases Autoimmune Diseases and Concordance in Twins Common diseases: Multiple SNPs

• Common diseases are believed to result from a combination of susceptibility alleles at multiple loci, environmental factors and stochastic events

• Non-Mendelian Inheritance Patterns • Single nucleotide polymorphisms (SNPs)

– Individual bases that exist as either of two alleles in the population Major Histocompatibility Complex – Association with Autoimmune Diseases Class I MHC Associations Ankylosing Spondylitis HLA-B27 Grave’s Disease HLA-B8 Class II MHC Associations Rheumatoid Arthritis HLA-DR4 Sjogren’s Syndrome HLA-DR3 Systemic Lupus Erythematosus

HLA-DR3, DR2

Type I Diabetes HLA-DR3 Celiac Disease HLA-DR3 Myasthenia Gravis HLA-DR3 Multiple Sclerosis HLA-DR2 HLA-B27and Autoimmune Disease HLA-DR4 and Rheumatoid Arthritis Single Nucleotide Polymorphisms in Autoimmune Diseases Mechanisms of Autoimmune Disease

• Previous attempts to classify them as T-cell and B-cell mediated are outdated • Involve Innate and Adaptive Components • Classified based on the effector mechanisms that appear to be most responsible

for organ damage: – Autoantibodies – T-cells

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Autoantibodies • Antibodies against to self-antigens • Can be found in normal, healthy individuals • Important effectors in autoimmune disease

Autoimmune Hemolytic Anemia

• Autoantibodies against RBC antigens – Warm autoantibodies

• IgG, react with Rh antigen on RBC at 37degC • Result in opsonization of RBCs and macrophage phagocytosis

– Cold autoantibodies (cold agglutinins) • IgM, react with I or i antigen on RBC when <37degC • Activate complement and result in complement mediated lysis

– Drug induced antibodies • Penicillin acts as a hapten, binds to RBC and form antibodies

against RBCs Myasthenia Gravis

• Target antigen is alpha chain of the nicotinic acetylcholine receptor in the neuromuscular junction

• Autoantibodies act as antagonist • Symptoms of muscle weakness, diplopia, dysarthria, dysphagia • May be associated with a thymoma • Can be transmitted to fetus through placental transmission of autoantibodies

Graves Disease

• Symptoms of hyperthyroidism – Heat intolerance, Increased metabolism, weight loss – Palpitations, increased HR, Hair loss, Fatigue – Nervousness, Opthalmopathy

• Autoantibodies against thyrotropin stimulating hormone receptor (TSH-receptor)

• Autoantibodies act as an agonist • Symptoms of hyperthyroidism • Maternal antibodies can be transmitted to fetus through the placenta resulting

transient neonatal hyperthyroidism Systemic Lupus Erythematosus

• Autoimmune disease characterized by – systemic autoimmunity – multi-organ involvement – production of autoantibodies against nuclear components – immune complexes

• Autoantibodies and immune complexes deposit in tissues including skin, joints, blood vessels, kidneys, etc.

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Antinuclear Antibodies: Presence in multiple autoimmune diseases SLE: ANA Associations and Immunofluorescence Viral Triggering of Autoantibody Production TLRs and Autoimmunity Scleroderma Associated Autoantibodies Multiple sclerosis

• A T-cell mediated autoimmune disease of the central nervous system characterized by

– Demyelination in brain and spinal cord – inflammation and dissemination of lesions in space and time

• Symptoms: visual defects, weakness, sensory deficits, diplopia, ataxia, cognitive deficits, bowel/bladder incontinence

Pathology of MS • An immune-mediated disease in genetically susceptible individuals • Demyelination leads to slower nerve conduction • Axonal injury and destruction are associated with permanent neurological

dysfunction • Lesions occur in optic nerves, periventricular white matter, cerebral cortex,

brain stem, cerebellum, and spinal cord Possible Mechanism of Demyelination and Axonal Loss in MS

• Activation of autoreactive CD4+ T cells in peripheral immune system against myelin proteins

• Migration of autoreactive Th1 cells into CNS • In situ reactivation by myelin autoantigens • Activation of macrophages, B cells • Secretion of proinflammatory cytokines, antibodies • Inflammation, demyelination, axonal transection, and degeneration

Other Autoimmune Diseases

• Hashimoto’s Thyroiditis – Autoantibodies and autoreactive T-cells to thyroglobulin and thyroid

microsomal antigens – Th1 cells also play a role – Destruction of thyroid gland leading to hypothyroidism – Symptoms of hypothyroidism: fatigue, goiter, dry skin, brittle hair and

nails, cold intolerance, weight gain, depression

• Rheumatoid Arthritis – Antibodies to citrullinated peptides (anti-CCP antibodies) – Antibodies to Fc portion of IgG (rheumatoid factor)

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– Immune complex formation and T-cell infiltration in synovium – Leads to activation of innate immune system components through Fc

receptors – Synovial inflammation, destruction of cartilage and bone erosions

• Type I Diabetes Mellitus

– Autoreactive CD8+ T-cells to pancreatic islet cells – Destruction of islet cells and failure of insulin production – Autoantibodies to insulin and islet cell antigens (GAD) are also present,

might be a result and not causative Targeted Therapeutics

• As our understanding of the pathogenesis increases, targeted therapeutic approaches are becoming available

– TNF-alpha inhibitors for the treatment of rheumatoid arthritis, ankylosing spondylitis, psoriasis, inflammatory bowel disaese

– CTLA-4 Ig for the treatment of rheumatoid arthritis – antiCD20 antibody (targeting B-cell) for the treatment of rheumatoid

arthritis – Beta interferon for the treatment of multiple sclerosis – Anti-type I interferons for the treatment of systemic lupus

erythematosus (in development) – Many others in development

CONCLUSION: “…. The mechanisms underlying all autoimmune diseases are not fully elucidated; however, genetic polymorphisms of MHC class II genes (alleles of HLA-DR and/or HLA-DQ) are associated with increased susceptibility to autoimmune diseases. Possible mechanisms for a loss of tolerance leading to autoimmune reactions include (1) a lack of Fas-Fas ligand–mediated deletion of autoreactive T cells in the thymus during development, (2) loss of T-regulatory or T-cell suppressor function, (3) cross-reactivity between exogenous and self-antigens (molecular mimicry), (4) excessive B-cell function due to polyclonal activation by exogenous factors (of viral or bacterial origin), (5) abnormal expression of MHC class II molecules by cells that normally do not express these surface molecules, and (6) release of sequestered self-antigens from privileged sites, thus priming for responses not previously seen by the immune system. Autoimmune diseases can be classified as organ specific or systemic in nature …. Three major types of autoimmune reaction mechanisms are recognized as causing different autoimmune disorders ... Two of these mechanisms involve autoantibodies directed against self-antigens; for both, classical complement pathway activation exacerbates local damage and inflammatory response. In the first case, autoantibodies may be directed against a specific self-component,

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such as a surface molecule or receptor. Examples include antibodies against the acetylcholine receptor producing myasthenia gravis, and antithyroid- stimulating hormone receptor antibodies producing Graves’ disease. Autoantibodies may also bind with antigens present in the blood, forming antigen-antibody (immune) complexes that later deposit in organs, thus inciting an inflammatory response. An example is seen in lupus glomerulonephritis in which complexes of anti-DNA antibodies and free DNA accumulate in the kidney. The third mechanism is that of autoreactive T cells that recognize targeted self-antigens on organs, leading to direct damage to tissue. In many cases, autoreactive T cells coexist with autoantibody responses, leading to exacerbation of disease and organ damage. In the case of multiple sclerosis, T cells reactive to myelin basic protein destroy the protective layer surrounding axons, thereby eliminating effective transfer of signals through nerves." [adapted from Actor, J.K. Elsevier’s Integrated Immunology and Microbiology, Mosby/Elsevier, Philadelphia, 2007.] SUMMARY (included materials from required reading):

1. Autoimmunity represents a failure of effective tolerance to self-antigens.

2. Genetic and environmental factors play a role in the etiology of disease.

3. Mechanisms of disease include autoantibodies that are directed against specific self-components, deposition of circulating antibody-antigen complexes, and deleterious responses by autoreactive T cells.

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CLINICAL CORRELATIONS Faculty Taught Class Correlations

Clinical Cases will be presented by faculty. Cases are taken from the Geha and Notarangelo. Case Studies in Immunology. Garland Publishing, New York, NY. 6th edition, 2012.* Clin. Corr. Date Time First Case Second Case Class 2/12 11:00-11:50 AM 36. Rheumatoid Arthritis

37. Systemic Lupus

Erythematosus Clinical Correlation Cases will be presented by faculty. This is NOT an extra credit assignment, but rather a clinical correlate that is part of the Immunology curriculum. Review the study questions at the end of each chapter for the cases presented. There is no formal requirement to complete the questions below. No extra credit points will be given for this Faculty taught Clinical Correlate. Rather, it is recommended that you be prepared to answer the following during class discussion:

Self Study Questions: 1. Define the deficiency/hyperreactivity involved in these cases, if one can be identified.

(Hyporeactivity) (Hyperreactivity) Immunodeficiency Health Immunopathology /\

2. How does this immune disorder directly or indirectly involve or impact each of the following (answer all):

Innate immune system activities B cell activities T cell activities

3. Describe the underlying mechanism(s) (e.g. at the organ, cellular or molecular level) in these cases (brief paragraph). 4. Give a short, succinct summary of the immunologic principle illustrated by these cases.

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PRIMARY IMMUNODEFICIENCIES WILLIAM T. SHEARER, M.D., PH.D.

Objectives

Define immunodeficiency and note its frequency and inheritance patterns.

Understand the genetic basis for primary immunodeficiency.

Describe deficiencies in B and T lymphocytes.

Describe deficiencies in NK and phagocytic cells and complement.

Learn how to diagnose immunodeficiencies.

Consider treatment options for patients with congenital immunodeficiencies.

I. Key Words

SCID, Severe Combined Immunodeficiency

XLA, X-linked Agammaglobulinemia

CVID, Common Variable Immunodeficiency

Hyper IgM, Immunodeficiency with Elevated IgM

Wiskott-Aldrich Syndrome

DiGeorge Syndrome, Cellular Immunodeficiency with Hypoparathyrodism

CGD, Chronic Granulomatous Disease

LAD, Leukocyte Adhesion Deficiency

II. Definitions The immunodeficiency disorders are a diverse group of illnesses that, as a

result of one or more abnormalities of the immune system, predispose a person to infection. The abnormalities of the immune system can involve absence or malfunction of blood cells (lymphocytes, granulocytes, monocytes) or soluble molecules (antibodies, complement components) and can result from an inherited genetic trait (primary) or from an unrelated illness or treatment (secondary).

III. Required Reading Assignments

A. Chinen J, Kline MW, and Shearer, WT. Infections of the Compromised Host. Chapter 67: Primary Immunodeficiencies. in Feigin and Cherry’s Textbook of Pediatric Infectious Diseases. 7th Edition. Elsevier. 2014. [posted on Blackboard]

B. Coico R, Sunshine G. Chapter 17: Immunodeficiency disorders and neoplasias of the lymphoid system. In Immunology: a short course, 6th Edition. New York: Wiley-Liss, 2009.

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Additional Reading (recommended – not required) – Posted to Blackboard C. [recommended reading] Shearer WT, Fischer A. Editorial. The last 80 years in primary

immunodeficiency: How far have we come, how far need we go? J Allergy Clin Immunol 2006;117:748-752.

D. [recommended reading] Notarangelo L, Casanova JL, Conley ME, Chapel H, Fischer A, Puck J, Roifman C, Seger R, Geha RS; International Union of Immunological Societies Primary Immunodeficiency Diseases Classification Committee. Primary immunodeficiency diseases: an update from the International Union of Immunological Societies Primary Immunodeficiency Diseases Classification Committee Meeting in Budapest, 2005. J Allergy Clin Immunol 2006;117:883-896.

E. [recommended reading] Orange JS, Hossny EM, Weiler CR, Ballow M, Berger M, Bonilla FA, Buckley R, Chinen J, El-Gamal Y, Mazer BD, Nelson RP Jr, Patel DD, Secord E, Sorensen RU, Wasserman RL, Cunningham-Rundles C; Primary Immunodeficiency Committee of the American Academy of Allergy, Asthma and Immunology. Use of intravenous immunoglobulin in human disease: A review of evidence by members of the Primary Immunodeficiency Committee of the American Academy of Allergy, Asthma and Immunology. J Allergy Clin Immunol 2006;117:S525-S553.

F. [recommended reading] Puck JM, Malech HL. Gene therapy for immune disorders: good news tempered by bad news. J Allergy Clin Immunol 2006;117:865-869.

WEB RESOURCE: HTTPS://MED.UTH.EDU/PATHOLOGY/COURSES/IMMUNOLOGY/LINKS-FOR-LECTURES/DISORDERS-OF-THE-IMMUNE-RESPONSE/ IV. General Considerations

There are greater than 120 types of primary immunodeficiency that have

been characterized and their incidence in the general population is 1:10,000 (aside from the extremely common selective IgA deficiency, 1:500). The most serious forms of immunodeficiency, such as severe combined immunodeficiency and X-linked agammaglobulinemia, occur in 1:100,000 individuals. At least 58% of cases are diagnosed in children (less than 5 yr) and 83% of these patients are male. Autosomal recessive, X-linked recessive, and familial inheritance patterns are observed.

V. Etiology

It is likely that genetic abnormalities underlie all primary states of

immunodeficiency. The simplest concept to remember is the scheme of the developing immune system. T and B lymphocytes pass through unique development stages as they mature and differentiate from the primordial stem cells in the bone marrow. Probably due to an underlying genetic lesion causing absent or altered enzymes, lymphocyte maturation may stop at a certain stage, deemed an “arrest point." Lymphocyte maturation arrest points may lead to clinical immunodeficiency states; for example, in patients with X-linked agammaglobulinemia (X-LA, B lymphocyte maturation is interrupted between the pre-B and B cell stage. By virtue of their lack of mature B cells, patients with X-LA have no circulation of antibody-producing plasma cells, and are very susceptible to infection with multiple organisms. Patients with common variable immunodeficiency possess B cells which are present but malfunctional, due to intrinsic developmental block between B cells and plasma cells, failure to secrete immunoglobulin, or lack of effective T helper cell function. Patients with X-linked severe immunodeficiency lack maturation of stem cells into mature T cells.

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VI. Clinical Features The principal manifestation of immunodeficiency is an increased susceptibility

to infection as documented by: a) increased frequency of infection, b) increased severity of infection, c) prolonged duration of infection, d) development of an unexpected complication or unusual manifestation, e) infection with organisms of low pathogenicity. Certain types of cancer, e.g., Epstein-Barr virus-induced B-cell lymphomas, appear in patients with certain immunodeficiencies. Autoimmune conditions also appear in patients with immunodeficiencies.

VII. Antibody (B Cell) Disorders

A. X-Linked (Bruton's) Agammaglobulinemia (X-LA) Patients with X-LA suffer from recurrent serious infections that begin in

early childhood, and are found to lack all classes of immunoglobulin and circulating B cells. Plasma cells do not develop and antibody responses are absent. Pre-B cells are present in the bone marrow of patients indicating a failure of differentiation into mature B cells.

Recurrent bacterial and pyogenic (pus producing) infections begin between 6 and 9 months due to wane of maternal IgG acquired transplacentally. Since the half-life of IgG is 23 days, in about 5-6 months the infant's serum IgG is virtually nil. Infections include pneumonia, sinusitis, otitis, pyoderma, osteomyelitis, and meningitis. Sepsis may result from infection with organisms such as pneumococci and streptococci, which have an exterior polysaccharide capsule normally coated by host IgG (opsonin) and digested by phagocytic cell (contain Fc receptor for IgG).

The molecular basis of X-linked agammaglobulinemia has been shown to be due to the lack of a cytoplasmic tyrosine kinase, which prevents B cell maturation, and production of immunoglobulins. There are also autosomal recessive forms of agammaglobulinemia that involved mutations in the mu heavy chain of immunoglobulins.

B. Selective IgA Deficiency

This is the most common form of immunodeficiency, which occurs in 1/500 individuals and has significant associations with several other diseases usually of an autoimmune nature. There is a strong familial association--possibly autosomal dominant inheritance with incomplete penetrance, although recessive patterns have been seen. Patients with IgA deficiency may develop CVID or have relatives with CVID (see below.). There are low or normal numbers of mature B cells that fail to secrete IgA due to an unknown genetic lesion (the alpha heavy chain gene is intact). Since IgA coats mucosal surfaces, it is not unreasonable that gastrointestinal symptoms of infection and malabsorption should be prominent in the IgA-deficient population. C. Common Variable Immunodeficiency (CVID)

This name represents a heterogenous group of patients that sometime after infancy, usually from 15-35 years of age, develop recurrent bacterial infections, decreased immunoglobulin levels and impaired antibody responses. Cellular immunity is usually normal or minimally defective except when patients become debilitated. Approximately 20% of cases of CVID are now known to be

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due to an autosomal dominant defective gene called TACI (transmembrane activator and calcium-modulator and cyclophilin interaction) located on B cells that normally binds to B cell activating factor of the TNF family (BAFF) and a proliferation-inducing ligand (APRIL) which regulates isotype switching. Autosommal recessive inheritance patterns have been noted in a subset of CVID patients who lack the normal inducible co-stimulator (ICOS), leading to inability to make specific antibody.

Prominent clinical problems are those of bronchiectasis (persisting infections of the periphery of the lung tissue) and intestinal giardiasis (parasite) leading to extreme debilitation and premature demise. Immunization with an antigen leads only to production of low levels of IgM antibody without the normal switch to IgG upon repeated immunization. There is also a high incidence of associated autoimmune disease and malignancies and a familial tendency for these diseases. Both CVID and IgA deficiency may map to a susceptibility gene on the 6th chromosome in the Class II MHC region.

D. Hyper IgM (HIM) Disorders

This immunodeficiency is usually X-linked and appears in males (HIM-1); but females are also affected, indicating an autosomal inheritance as well (i.e., defective activation-induced deaminase [AID]) or a defect in uracil DNA glycosylase (UNG), both termed HIM-2. Another autosomal recessive defect (abnormal CD40 molecule on B cells) has been discovered (HIM-3). In all types, the serum IgG and IgA are usually totally absent or markedly reduced; IgM concentration is normal to very high. Patients may have high titers of some IgM antibodies but often no or very low specific antibody formation. The percentage of peripheral blood lymphocytes with surface Ig is normal. There are usually normal T cell mitogen responses but some patients develop T cell deficiency with time. Patients often have associated neutropenia and/or autoimmune phenomena and infections are common. A high incidence of malignancies is also seen in these patients. In males, the abnormal gene in the X-linked type of HIM-1 has been mapped to Xq26,27. The normal gene product is the ligand (CD40L present on T cells) that binds to CD40 on B cells, leading to cell activation. Thus, the B cells appear to be intrinsically normal.

E. Wiskott-Aldrich Syndrome

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder with variable clinical phenotypes that correlate with the type of mutations in the WAS protein (WASP) gene. WASP, a key regulator of actin polymerization in hematopoietic cells, has 5 well-defined domains that are involved in signaling, cell locomotion, and immune synapse formation. WASP facilitates the nuclear translocation of nuclear factor kappaB and was shown to play an important role in lymphoid development and in the maturation and function of myeloid monocytic cells. Mutations of are located throughout the gene and either inhibit or dysregulate normal WASP function. Analysis of a large patient population demonstrates a phenotype-genotype correlation: classic WAS occurs when WASP is absent, X-linked thrombocytopenia when mutated WASP is expressed, and X-linked neutropenia when missense mutations occur in the Cdc42-binding site. The progress made in dissecting the function of WASP has

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provided new diagnostic possibilities and has propelled our therapeutic strategies from conservative symptomatic treatment to curative hematopoietic stem cell transplantation and toward gene therapy.

VIII. Cellular Disorders

A. T-Cell Deficiency: DiGeorge Syndrome (DGS) DiGeorge first described (1965) the association of infection, absent

thymus, and congenital hypoparathyroidism. There are other associated congenital abnormalities: cardiovascular defects, abnormal facies, urinary tract abnormalities, and orthopedic abnormalities. There is failure of the 3rd and 4th pharyngeal pouches to develop at about 10 weeks of embryonic life. Originally, DGS was believed to occur on a sporadic basis but familial associations have been noted. Children with the syndrome come to the attention of the physician because of seizures on the first day of life due to low calcium in the blood. If the children survive the neonatal period (1 mo.) increased susceptibility to opportunistic infection occurs with fungi, such as Candida albicans and Pneumocystis carinii. About 95% of DGS patients have chromosomal deletions (22q11 [also have velocardiofacial syndrome] or 10q13). A chest x-ray is frequently helpful in making the diagnosis, since the thymic shadow is absent or reduced in size. An animal model of DiGeorge syndrome lacks the Tbx1 gene, but this abnormal gene is not universally found in DGS.

It is now becoming clear that, in addition to the complete DGS, where there is a total lack of T-cell immunity, there exist partial syndromes where T-cell numbers and T-cell responses to mitogens and antigens may be completely absent or intermediate in value. Infants usually have normal serum immunoglobulins and normal circulating B lymphocytes but do not mount a specific antibody response to antigens because of lack of T cell help. B. T- and B-Cell Deficiency

1. Severe Combined Immunodeficiency (SCID) There are many types of combined (B- and T-cell) immunodeficiency

which result in recurrent life-threatening infections, severe diarrhea, and failure-to-thrive. The classical form of SCID is X-linked, but there are autosomal recessive forms, as well as sporadic forms. The usual lymphocyte analysis reveals an absence of lymphocytes bearing mature T-cell antigens (CD3, CD4, CD8) and inability of lymphocytes to respond to mitogenic and antigenic stimulation. Frequently, there are small numbers of B cells with mature antigens (CD19, CD20), but most infants with SCID do not make serum immunoglobulins. The genetic and molecular defect in X-linked SCID is a mutation in the gene that codes for the gamma chain of the IL-2 receptor. Without a normal IL-2 receptor, normal T-cell maturation and proliferation cannot take place. Consequently, all normal T-cell functions are absent. The profound nature of the SCID defect is due to the fact that the defective gamma chain renders not only the IL-2 receptor dysfunctional but also the IL-4, IL-7, IL-15, and IL-21 receptor.

There are several other forms of SCID involving genetic lesions (all inherited in autosomal fashion) in the: 1) T-cell receptor complex, CD3; 2) kinase

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enzyme, JAK-3; 3) interleukin receptor, IL-7R; 4) IL-2R; 5) T-cell receptor recombinase enzymes, RAG1/RAG2; 6) maturity marker, CD45; 7) major histocompatibility complexes, MHC-1 due to TAP-1, -2 (transporter associated with antigen processing) deficiency; 8) MHC-2; 9) kinase enzyme, ZAP-70 (absence of CD8+ T cells); 10) unknown gene termed ARTEMIS; 11) nucleic acid enzyme adenosine deaminase, ADA; and 12) nucleoside phosphorylase, NP.

These defects can also be classified according to T-cell phenotypes, e.g., X-linked SCID is T cell (-) B cell (+) NK cell (-); ADA deficiency is T cell (-) B cell (-) NK cell (-); RAG1, RAG2 deficiency is T cell (-) B cell (-) NK cell (+); and IL-7 deficiency is T cell (-) B cell (+) NK cell (+).

2. Non-SCID forms of T- and B-Cell Deficiency These conditions, although eventually fatal, permit up to several years of

life, although the quality is poor because of recurrent infections and malignancy. These conditions are: 1) Wiskott-Aldrich syndrome (WAS), 2) Ataxia-telangiectasia (AT), 3) Nijmegen breakage syndrome (NBS), 4) X-linked lymphoproliferative disorder (X-LP), 5) NFkB essential modifier (NEMO), 6) warts, hypogammaglobulinemia, infections, myelokathexis (WHIM), 7) absence of caspase 8, and 8) hyper IgM due to X-linked (HIM-1), and an autosomal recessive gene defect in the gene coding for CD40 on B cells (HIM-3). C. Other Cell-Derived Combined Immunodeficiency Defects in non-B/T cells can cause serious immunodeficiencies as severe as SCID. For example, defects in the IFN-R or IL-12R on antigen-presenting monocytes/macrophages cause repetitive serious infections, most commonly with atypical mycobacteria. Natural killer (NK) cell deficiency leads to chronic viral infections and malignancy because of lack of immunosurveillance. Although these other cells are not part of the adaptive immune system, their impact upon B- and T-cell function is so profound that the result of their dysfunction is tantamount to severe B- and T-cell deficiency. In the case of IFN-R/IL-12R deficiency, there is so much infection with mycobacteria that B- and T-cell resistance prove inadequate. In the case of NK-cell deficiency, the innate function of viral clearance is missing, and again B- and T-cell resistance is overwhelmed.

IX. Phagocytic Immunodeficiency

A. Chronic Granulomatous Disease

The engulfment process in phagocytic cells is associated with increased anaerobic glycolysis and ATP consumption. There is a burst of respiratory oxidative activity, increased oxygen consumption, and a shift to glucose metabolism via the hexose monophosphate shunt. As glucose is utilized by the leukocytes, reduced pyridine nucleotides (NADH and NADPH) accumulate, and cytochrome oxidase activities increase with the final production of H2O2, which (after subsequent metabolism) is toxic to bacteria (O2

-, superoxide). CGD leukocytes, however, demonstrate no increased O2 uptake, no shift to the hexose monophosphate shunt and no H2O2 production. There are several genetic defects, the principal defect being the abnormal gp91 in the membrane portion of the cytochrome B558 which is inherited in X-linked fashion (chromosome

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XP21.1). The white blood cell count (WBC) is high in an attempt to compensate for lack of bacterial killing.

Laboratory tests used to diagnose CGD are the nitroblue tetrazolium dye test (NBT), dihydro rhodamine test (DHR), and the chemiluminescence assay. All tests measure H2O2 and subsequent superoxide production by the granulocyte. Gamma interferon has been shown to augment the effectiveness of antimicrobial treatments in CGD, but bone marrow transplantation offers the only permanent therapy.

B. Leukocyte Adhesion Deficiency

Leukocyte adhesion deficiency-1 (LAD-1) is a rare autosomal recessive disorder characterized by recurrent bacterial and fungal infections and impaired wound healing in spite of leukocytosis. Classically there is a history of delayed separation of the umbilical stump in affected individuals. In these patients, most adhesion-dependent functions of leukocytes are abnormal. The molecular basis of the defect is absent or deficient expression of the β2 integrins, or the CD11CD18 family of glycoproteins (chromosome 21q22.3), which includes leukocyte function-associated antigen-1 (LFA-1 or CD11aCD18), Mac-1 (CD11bCD18), and p150,95 (CD11cCD18). These proteins participate in the adhesion of leukocytes to other cells and in the phagocytosis of complement-coated particles. In all patients studied so far, the defect has been mapped to the 95 kD β chain (CD18). The gene encoding this chain may be mutated, producing an aberrant transcript, or its transcription may be reduced.

Leukocyte adhesion deficiency-2 (LAD-2) is another disorder described in a very small number of patients that is clinically indistinguishable from LAD-1 but is not due to integrin defects. In contrast, LAD-2 results from an absence of sialyl-Lewis X, the carbohydrate ligand on neutrophils that is required for binding to E-selectin and perhaps P-selectin on cytokine-activated endothelium. It is likely that LAD-2 patients have mutations in genes encoding enzymes involved in fucose metabolism.

Like other genetic defects affecting leukocytes, leukocyte adhesion deficiencies are candidates for bone marrow transplantation and ultimately specific gene therapy.

X. Disorders of the Toll-Like Receptor and Complement Systems A. Toll-Like Receptors Toll-like receptors (TLRs) are a series of 10 receptors in humans that participate in detecting pathogens. TLRs recognize a small number of pathogen-associated molecular patterns (PAMPs). PAMPs represent molecules (LPS, lipoproteins, flagellin, unmethylated DNA, double-stranded and single-stranded RNA) that promote for the survival of a pathogen. Activation of TLRs leads to recruitment of neutrophils and macrophages to sites of infection and augmentation of antimicrobial activity. On engagement of TLRs, dendritic cells (DCs) undergo maturation and migrate to draining lymph nodes, where they present antigen to T cells. PAMPs binding to all known Toll-like receptors cause the production of inflammatory cytokines, including TNF-α. IRAK-4 (interleukin receptor-associated kinase-4) is a critical effector in signaling by TLRs and the IL-1 receptor, which share homology in their intracellular domain. Patients with IRAK-4 deficiency are susceptible to invasive bacterial infections and to viral infections.

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B. Complement The complement system is a complex of approximately 31 innate host defense proteins that acts in three activation patterns (classical, alternate, and lectin-binding) to augment adaptive host defense mechanisms and to exert a bacteriolytic effect of its own. Genetic deficiencies or mutations of any one of these proteins leads to impaired host defense.

The prototype complement component deficiency disease is that of C3 deficiency, a very rare disorder but one that allows us to assess the crucial importance of the total complement pathway in host-defense mechanisms. Since C3 is the pivotal complement component through which both classical and alternative pathways act, its absence does not permit complete activation of complement. Because of the lack of C3 chemotactic factors, C3a and C5a are not released and phagocytic cells are not drawn to the focus of infection. Moreover C3b serves as an opsonin of bacteria and by virtue of a chemical affinity of C3b for a receptor on phagocytic cells (polymorphonuclear leukocytes and monocytes) the engulfment of a C3b-coated bacterium by a phagocytic cell is facilitated. Persons with complement component deficiencies of C1, C2, or C4 can still activate the complement cascade via the alternative pathway and persons with complement component deficiencies of C5, C6, C7, C8, or C9 can still generate chemotactic factors and have opsonin (C3b) function.

Pyogenic infection (streptococcal, staphylococcal) and autoimmune diseases are associated with early complement component deficiencies (i.e., C1, C2, C4) and meningococcal and gonococcal infections are associated with late complement component deficiencies (i.e., C5-C9). XI. Treatment of Immunodeficiency Diseases A. Antibody (B Cell) Disorders 1. Replace IgG deficiencies with intravenous immunoglobulin 2. General supportive care very important 3. Treat complications with appropriate medications, e.g., autoimmune

disease with immunomodulators. B. Cellular Disorders (T and B cells, monocyte/macrophages) 1. Bone marrow stem cell transplantation 2. Intravenous IgG 3. Gene therapy where possible 4. General supportive care 5. Cytokine therapy in selected defects, e.g., IL-2 for IL-2R

deficiency C. Phagocyte Deficiency 1. IFN- for CGD 2. General supportive care 3. Bone marrow stem cell transplants (patients bone marrow must be

ablated with chemotherapy)

D. Complement Deficiency 1. General supportive care

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XII. Diagnosis of Immunodeficiency Diseases

A. Medical History 1. Age of patient at onset of symptoms 2. History of live microbial immunizations 3. Severity of illness 4. Family history

B. Physical Examination 1. Tonsils present 2. Palpate lymph nodes 3. Organomegaly 4. Growth - measurements

C. Laboratory Investigation 1. Antibody function

a. Immunoglobulin levels b. Specific antibody responses

- Isohemagglutinins (anti A, anti B) - Anti-diphtheria and tetanus antigen antibodies - Anti X 174 antibody

2. B and T Lymphocyte, NK cell, and monocyte/macrophage function a. T cell

- Delayed hypersensitivity skin testing (e.g. SK-SD, monilia antigens) - T cell subsets (flow cytometry) - Mononuclear cell phenotypes (subsets-monoclonal antibodies) - PHA reactivity and antigen reactivity - Specific antigen stimulation in vitro - T cell excision circles (TREC) - V TCR spectratyping

b. B cell (B cell subsets by flow cytometry) - Memory (CD27+) B cells c. Nucleic acid enzyme assay

- Adenosine deaminase - Nucleotide phosphorylase

d. NK cell surface markers and functional assay e. Monocyte/macrophage receptor assays

3. White blood cell function a. WBC b. NBT, DHR tests c. CD11a,b,c CD18 assay by flow cytometry d. Chemotaxis and opsonization assays

4. Complement function a. Total hemolytic complement b. C3 c. C4

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5. Molecular and genetic studies XIII. Summary (Key Concepts)

A. Inherited gene defects are causes of primary immunodeficiency. B. Lack of immune effector function produces infection. C. Defective function occurs in B, T, NK, PMN, or M cells. D. Diagnostic tests augment medical history and physical exam. E. Treatment attempts to replace what is missing.

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MODIFICATION OF THE IMMUNE RESPONSE: IMMUNOPROPHYLAXIS AND IMMUNOTHERAPY

[Special thanks to: Semyon A. Risin, MD PhD]

OBJECTIVES:

1. To understand the application of the major immunological principles and concepts to modification of immune response, immunoprophylaxis and immunotherapy of human diseases.

2. To define the current approaches and future strategies to immunoprophylaxis and immunotherapy of immune-mediated and non-immune-mediated diseases

KEYWORDS: Immunoprohylaxis, vaccine, active and passive

immunization, immunotherapy READING: Coico and Sunshine, 2009. Chapter 20 WEB RESOURCE: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/immunoprophylaxis-vaccines/

I. INTRODUCTION Considering the number of saved lives, the active process of deliberate exposure of individuals to infectious agents or their antigenic components - vaccination or immunoprophylaxis - has been one of the most important medical advances in the history of mankind. Historically it was based on early recognition of resistance to secondary exposure to infectious challenges of individuals recovered from infectious diseases. Reports of immunization go back to the ancient Chinese (10th century) when they used crusts from smallpox lesions (variolation) to provide an attenuated episode of smallpox with subsequent protection. Jenner, in 1798, introduced the use of vaccinia (cow) virus as a cross reactive agent for protection against smallpox (from which the terms vaccine and vaccination are derived). In 1860-65, Pasteur introduced the concept of attenuated vs. killed vaccine to prevent anthrax. These were examples of early active immunizations. Later the concept and technology of passive immunization –use of sera of convalescent individuals or sera from hyperimmunized animals for immediate protection- was introduced. It was particularly successful in treating toxic complications of diphtheria and tetanus. Since then the theory and practice of immunization and the vaccine-manufacturing technology developed significantly. It incorporated the major advances of molecular biology and biotechnology, including DNA-

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recombinant technology, gene transfer, use of bacterial and viral vectors, modification of protein structure, “humanization” of animal-derived antibodies, use of hybridoma technology for manufacturing of monoclonal antibodies, etc. In addition, based on understanding the complexity of the immune system, new approaches to modification of the immune response targeting such key elements as affector cells, cell interaction and cytokines were introduced. These achievements also revolutionized the field of immunodiagnostics (ELISA, immunostaining, Flow-cytometry) as well as triggered the use of monoclonal antibodies for blocking biologically active molecules playing a key role in non-immune pathology (herceptin, antiplatelet antibodies etc.), and thus creating the basis for immunotherapy of non-immune diseases.

Smallpox vaccination based on cross-reactivity between cowpox and smallpox viruses

II. IMMUNIZATION BASICS

A. Types of Immunizations 1. Active – exposure to antigen with the host generating

protective immunity. Objective: provide long lasting immunity against future exposures

2. Passive – administration of humoral and/or cellular factors that provide immunity for the host. Objective: provide temporary immediate protection against an imminent or ongoing exposure

3. “Heard” immunity in preventing spread of infection

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B. Examples of Active and Passive Immunization Type of immunity How acquired Active

Natural (unintended) Infection

Artificial (deliberate) Vaccination

Passive Natural Transfer of antibody

from mother to infant in placental circulation or though breast-feeding (colostrums)

Artificial Passive antibody therapy (serum therapy, immune human globulin, monoclonal antibodies)

III. Active Immunization

1. Current US Recommendations for active immunization

1a. Schedule for Active Immunization of Children and Adults Age Vaccine Birth Hepatitis B (Hep B) 1–2 months Hep B 2 months Diphtheria and tetanus toxoids and acellular

pertussis (DTP), Haemophilus influenzae type b (Hib), inactivated polio (IPV)

4 months DTP, Hib, IPV, rotavirus (Rv) 6 months Hep B, DTP, Hib, IPV, Rv 12–15 months Oral poliovirus vaccine (OPV), measles,

mumps, rubella (MMR), varicella vaccine for susceptible children

4–6 years DTP, OPV, MMR 11–12 years Hep B, MMR, varicella 25–64 years Measles, rubella >65 years Influenza, pneumococcal disease

Adapted from JAMA, Vol 281:601–603, with permission.

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1b. Vaccines for Specific Populations a. BCG – vaccine against TB in endemic populations

(Obscures PPD skin testing – not used in US)

b. PneumoVax – susceptible populations i. Cancer ii. Elderly iii. Immunocompromised, postsplenectomy

c. Meningococcus vaccine – military recruits and institutionalized subjects d. Travel vaccines – typhoid, anthrax, yellow fever, plague, etc.

Depends upon endemic area for travel

2. BASIC MECHANISMS OF PROTECTION

2A. Primary vs. Secondary Immune Response

1. Rapidity of response is critical in light of incubation period of infection 2. If incubation period short (i.e. 3-4 days), there may not be enough time for anamnestic response to develop before disease ensues

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2B. Age and Timing of Immunizations Fetus a. IgM appears at 6 months gestation b. decreases to 10% adult level at birth c. IgG appears at 6-8 weeks gestation, maternal origin d. majority of IgG at birth is of maternal origin

(transplacental)

Infants

a. generally do not respond well to polysaccharide antigens at less than 2 yrs of age

b. antigenicity improves when conjugated to protein or toxoid

2C. Mixed or multiple antigen vaccines a. routine vaccines often group by antigen types

toxoids polysaccharides virus coats

b. concern about relative competition between vaccine responses

c. with 1012 lymphocyte repertoire, competition not a practical issue

2D. Route of vaccine administration a. parenteral vs. oral or respiratory b. deltoid muscle vs. gluteus (e.g. Hepatitis B) c. systemic vs. mucosal immunity

- Oral administration should stimulate mucosal immunity, parenteral often does not (e.g. Sabine vs. Salk) - Mucosal immunity stops infection, systemic -> stops illness

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2E. Hazards

a. Live vaccines in immunocompromised individuals and pregnant women

b. Reversion to wild type c. Arthralgias/myalgias d. Hypersensitivity reactions

1. Arthus phenomenon 2. arthritis and arthralgia 3. anaphylaxis

3. Vaccine Production Methods a. Recombinant DNA – makes antigen-specific oligopeptide b. Conjugated polysaccharides – add protein to involve T

cells c. Synthetic peptides – largely covered by rDNA-induced

peptides – must be big enough to induce T and B cell memory

d. Specific receptor blockade –stops pathogen entry (i.e. virus)

e. Antiidiotype vaccines f. Gene constructs – virus vector or naked DNA g. Bacterium – carrier : bacterium acts as adjuvant h. Toxoids – inactivated toxins which may produce better

immunity than natural infection due to relative amounts of antigen exposure

IV. PASSIVE IMMUNIZATION

A. Natural Placental Antibody Transfer 1. majority of IgG in neonate’s plasma is passive from mom 2. protection wanes by 6mo as infant makes own immunoglobulin 3. specific immunization of mother antenatal can protect

neonate (i.e. tetanus neonatorum)

Colostrum protection 1. contains enzymes, cells, antibodies 2. B cells migrate to breast from intestine (enteromammary) 3. antigen-specific T cells also transmitted but role is unclear

B. Artificial Passive Antibodies – Specific vs. Nonspecific

specific antigen raised in animal sera (e.g. horse) result was serum sickness with repeated exposure

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now use hyperimmune Ig purified from human donors although peak levels may be lower, end result is an

extended duration of circulating protective antibody use of humanized monoclonal antibodies

C. Monoclonal vs polyclonal antibody

monoclonal highly specific for single epitope can make very large amounts in biologically active form polyclonal represents activity against larger number of

antigens must be purified from serum of human donors if single

antigen specificity more common to use IVIG

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D. Intravenous Immunoglobulin (IVIG) purified from pooled sera of thousands of donors advantage of multiple specificites dilutes out any adverse influences (drug, infections, etc.) IgG1 is major component – 25 fold higher concentration

than plasma Comparison of immunoglobulin contents of Human Immune Serum Preparations

Immunoglobulin (mg/100 ml)

Source IgG IgA IgM Whole serum 1,200 180 200 Immune serum globulin 16,500 100–500 25–200 Intravenous immunoglobulin 3000–5,000 trace trace Placental immune serum globulin 16,500 200–700 150–400

E. Uses for Immune serum globulins Hyperimmune globulins

Rhogam – prevent Rh immunization CMV-IGIV – prevent CMV in bone marrow

transplants Rabies Ig – prevent clinical rabies VZIG – leukemia patients exposed to VZV

IVIG Humoral (IgG immunodeficiency) producing

chronic infection Idiopathic thrombocytopenia purpura

Precautions IM – aggregates may cause anaphylactoid reaction aseptic meningitis noninfectious hepatitis anaphylactoid in selective IgA deficiency

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V. IMMUNOTHERAPY Use of immunological approaches for treatment of immune-based and non-immune-based human diseases

A. Immune-based diseases

1. Mechanisms

Deficiency Dysregulation Dysfunction

2. Clinical Manifestations

Infectious Hypersensitivity Cancers Others

3. Potential Roles for Cytokine Therapy in Immune Diseases

Disease Mechanisms Diagnosis Prognosis Monitoring response to therapy

B. Non-immune-based diseases (examples of immunoprophylactic and immunotherapeutic approaches)

1. Cardio-vascular (antiplatelet Ab abciximab) 2. Tumors

a. nonspecific stimulation of innate immunity by BCG b. use of ex vivo propagated tumor infiltrating lymphocytes (TIL) in melanoma c. use of dendritic cells loaded ex vivo with multiple tumor epitopes d. new antitumor vaccines (melanoma, prostate cancer, HPV vaccine for cervical cancer, H. pylori vaccine for gastric and gastro-esophageal cancer etc.) e. use of bcr-abl vaccine for CML Herceptin for breast cancer, rituximab for B-cell malignancies, alemtuzumab for CLL)

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C. Clinical Examples with Potential for Using Cytokine Therapies (immune-

based and non-immune-based diseases) 1. Metabolic Diseases

Osteoporosis – IL-6 Diabetes mellitus – TH1

2. CNS diseases

Multiple sclerosis – TH1 ALS –TH1 Alzheimer’s – TH2 (?)

3. Infectious Diseases

Opportunistic infection – T cell deficit HIV disease – CD4 T cell deficit

4. Inflammatory bowel disease

Crohn’s TH1 Ulcerative colitis TH2 Rheumatoid arthritis – blocking inflammatory cytokines

5. Sepsis syndrome/ARDS –TNF, IL-1, IL-6

6. Hypersensitivity Diseases

Allergic/asthmatic diseases – TH2 Autoimmune/inflammatory diseases – TH1 and TH2

D. Rationale for immunotherapy of bronchial asthma

a. Asthma is a classic example of TH2 disease.

b. IL-4 serves not only as a signal for isotype switches to IgE but is, along with IL-3 and GM-CSF, a mast cell growth factor

c. IL-4 can upregulate expression of VCAM-1/VLA-4, which is an adhesion molecule pair that facilitates eosinophil-specific inflammation.

d. IL-4 appears to be involved in goblet cell hypertrophy and hyperplasia which result in increased mucus production, a hallmark of asthma inflammation.

e. IL-4 may also be involved in airway remodeling. Additionally, IL-5 induces eosinophil differentiation from myeloid precursors in the bone marrow

f. IL-5 in conjunction with eotaxin serves as an important chemotactic factor for eosinophils,

g. IL-5 inhibits apoptosis thus prolonging survival of eosinophils in the periphery

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h. IL-5 activates eosinophils to release cytotoxic products such as major basic protein, eosinophilic cationic protein and others.

Thus, when considering the therapeutic utility of various new biotechnology molecules, a fundamental approach would attempt to regulate the milieu that activates mast cells and eosinophils and recruits them to airways. If mast cell numbers and/or activities can be regulated, asthma activity can likewise be affected. Thus, many efforts are underway to regulate activity and/or production of TH2 cytokines IL-4 and IL-5 as well as allergen-specific IgE. In 2003 FDA approved a humanized monoclonal antibody against IgE – Xolair (omalizumab) - for clinical use. SUMMARY

Immunization can be by either exposing the host to an antigen preparation that induces a protective immune response (active) or by supplying the immune products (i.e. antibody or effector cells ) from another immune host (passive)

Immunizations occur after a primary exposure that creates a sensitization

and a secondary “booster” challenge that provides an accelerated, heightened response capable of protecting the host against subsequent infection and disease

There are multiple vaccine preparation methods, each with their own

advantages and disadvantages

Passive immunization can be natural - from maternal source (placental transfer or colostrums) or artificial – from an exogenous source of immunoglobulins or immune cells. The exogenous antibody preparation can be for either a specific antigen source (antiserum) or for more general immunoglobulin replacement (i.e. intravenous Immunoglobulin). Antibody preparations may be monoclonal or polyclonal.

Immunotherapy is used primarily as either a modulator of the immune

response based upon the notion of TH1/TH2-based immunological diseases that can be treated by altering the underlying imbalance (such as increasing one helper population over another), or as a cancer treatment modality to block the expression of biologically important molecules and suppress cancer cell proliferation.

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IMMUNOLOGY OF CANCER Jeffrey K. Actor, Ph.D.

MSB 2.214, 713-500-5344 [Special thanks to Priya Weerasinghe, M.D., Ph.D.]

Objectives

(1) Discuss Tumor Antigens. (2) Review effector mechanisms to combat tumors and tumor development. (3) Discuss role of antibodies in diagnostics and in immunoprophylaxis and

immunotherapy. Reading: Coico and Sunshine (2009), Chapter 19.

Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/cancer-immunology/

Cell-Mediated Responses to Tumor Cells Many concepts discussed to date also apply to protection against tumor cell development. Please refresh these concepts by visiting Chapter 19 of the Coico and Sunshine text, beginning on page 303.

Introduction What are tumor antigens/tumor specific transplantation antigens (TSTAs)? Some tumor antigens consist of molecules that are unique to the tumor cell but not to the normal cell. Some tumor antigens are qualitatively not different from those found on normal cells but are over expressed on the tumor cell. Examples include the HER in some breast and ovarian cancers- over expression of the HER-2/neu-1 oncogene, and the ras oncogene on some human prostate cancer cells.

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Carcinogen-induced tumor antigens Carcinogens can induce mutations in normal genes that were silent. These mutations can give rise to an array of different gene products. There is very little or no cross reactivity in these tumor antigens. This lack of cross reactivity is due to the random mutations induced by the chemical or physical carcinogen. Example: When methylcholanthrene is applied repeatedly to genetically identical animals, tumors will develop. But tumor antigens will be different and there will be no cross reactivity. Same is true for physical carcinogens such as UV light or x ray. Categories of tumor antigens

Category Type of Antigen Name of Antigen

Types of Cancer

Normal cellular gene products

Embryonic Oncofetal antigens

MAGE-1 MAGE-2 CEA AFP

Several Several Lung, pancreas, breast, colon, stomach Liver, melanoma, carcinoma of bladder, lung, testis

Differentiation Normal intracellular enzymes Oncoprotein Carbohydrate

Prostate specific antigen, CT antigen tyrosinase HER-2/neu Lewis

Prostate Melanoma Breast, ovary Lymphoma

Clonal amplification

Ig isotype Specific antibody of B cell clone

Lymphoma

Mutant cellular gene products

Point mutations

Oncogene product Suppressor gene product CDK

Mutant RAS protein Mutant P53 Mutant CDK-4

Several Several Melanoma

Viral gene products

Transforming viral gene

Nuclear protein E6 and E7 proteins of HPV

Cervical

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Activation of cellular proto-oncogenes in human cancer

Proto-oncogene

Activation mechanism

Chromosomal change Associte cancer

C-myc Genetic rearrangement

Translocation: 8-14. 8-2 or 8-22

Burkitt’s lymphoma

C-abl Genetic rearrangement

Translocation 9-22 CML

C-H-ras Point mutation Bladder carcinoma

C-K-ras Point mutation Lung and colon carcinoma

Effector mechanisms in tumor immunity

Effector Mechanism Comment

B cells and antibodies (ADCC, CDC) Role in immunity– poorly understood

T cells (cytolysis, apoptosis) Virally- and chemically–induced tumors

NK cells (cytolysis, apoptosis, ADCC) Tumor cells not expressing MHC class 1 alleles- rejected by NK cells

LAK cells (cytolysis, apoptosis) Anti tumor response- to adoptive transfer to LAK cells

Macrophages and neutrophils Activated– by using bacterial products

Cytokines (apoptosis, recruitment of inflammatory cells)

Using adoptively transferred tumor cells- eg: GM-CSF

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Limitations of effectiveness of immune responses against tumors

Tumor Related Mechanisms of Escape Related Mechanisms of Escape

Failure of tumor to provide a suitable antigenic target or an effective immune response;

- lack of tumor antigen - lack of MHC class 1 - deficient antigen processing - antigen modulation - antigenic masking of tumor - resistance of tumor to tumoricidal pathways - lack of co-stimulatory signals - production of inhibitory cytokines - shedding of tumor antigens

Failure of host to antigenic tumor cells: - immuno-supression or immuno- deficiency - deficiency in inducing apoptosis and cell death signaling - infections or old age - deficiency in tumor antigen presentation by host APC - failure of host effector cells to reach the tumor (eg: stromal barrier) - failure of host to kill variant tumor cells - T reg hindrance to tumor immunity

Immuno-diagnosis

1. Immunohistochemistry-

Antibodies to specific antigens detected by amplified signals. Applications: Diagnosis on surgical specimens. to identify the original cancer to classify the type of cancer to predict the aggressiveness of the tumor Tumor immunoprophylaxis Cervical cancer vaccine:

-Gardasil (by Merck Pharmaceuticals) – Prevents human papilloma virus (HPV )16, 18, 6, 11.

-Cervarix: (by GlaxoSmithKline)- Prevents HPV 16 and 18

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Tumor Immunotherapy (1) Stimulate the immune system, reject and destroy tumors.

- BCG immunotherapy for early stage bladder cancer. - Imiquimod: topical therapeutic to supplement local production of IFN-γ

(2) Monoclonal antibodies to target and destroy tumors. Examples: Antibody Immunotherapy targeted towards cancers

Name Trade name Used to treat Target Year approved

Rituximab Rituxan Non-Hodgkin’s lymphoma

CD20 1997

Trastuzumab Herceptin Breast cancer Erb b2 1998

Gemtuzumab ozogamicin

Mylotarg Acute myelogenous leukemia (AML)

CD33 2000

Alemtuzumab Campath Chronic lymphocytic leukemia (CLL)

CD52 2001

Ibritumomab tiuxetan

Zevalin Non-Hodgkin’s lymphoma

CD20 2002

Panitumumab Vectibix Colorectal cancer EGFR 2006

Cetuximab Erbitux Colorectal cancer, Head and neck cancers

EGFR 2004

Bevacizumab Avastin Colorectal cancer VEGF 2004

See also diagram in Appendix for additional mechanisms to regulate immune responses to tumors.

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Summary: 1. Tumor cells differ from normal counterparts by indefinite proliferation, changes in

growth regulation. 2. Normal cells can be transformed in vitro by chemical and physical carcinogens, or

by transforming viruses. 3. Tumor cells express TSTAs and TATAs. 4. Some tumor antigens are recognized by CTL cells: They are TSTAs, antigens that

are over expressed in various tumors, antigens that are normally expressed in certain stage of differentiation and antigens from mutated proteins.

5. Proto-oncogenes encode proteins that control normal cellular growth. Key step in induction of human cancer is conversion of proto-oncogenes to oncogenes. This conversion may result from mutation, translocation or amplification of an oncogene.

6. Immune responses to tumors include: CTL mediated cell lysis, NK cell killing, ADCC and macrophage mediated cell killing. There are several cytotoxic factors such as TNF-∞ and TNF-β.

7. Some tumors cells utilize immune response evading mechanisms. 8. Cancer immune-therapy includes monoclonal antibodies, antibodies coupled with

toxins, chemotherapeutic agents or radioactive elements. 9. There are new strategies for cancer immune therapy: identification of specific

tumor antigens, effective presentation of tumor antigens, generation of activated CTLs and T helper cells.

Spring Semester, 2015

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Team Based Learning Exercise

The Immunology course will have one Team Based Learning exercise where students will be required to address a clinically based scenario and provide answers to related questions. Students will be assigned specific reading prior to the session, which will assist in mastering of the material so as to allow participation in the group activities. Materials will include new material in Immunology, as well as materials already mastered in other courses. The format will be similar to the Clinical Applications course. The Team Based Learning Exercise is mandatory. The Team Based Learning Exercise encompasses a graded set of exercises related to multiple integrated aspects of a clinical scenario. The exercise is worth a maximum of 10 points towards your overall Immunology grade. The session will utilize a clinical scenario to present a problem. Students are divided into teams; utilizing the groups already in place for the Clinical Applications course. Problem questions arising from the clinical scenario are crafted to foster discussion within the teams; each team is required to come to a consensus as to the solution to the problem. Written justification may be required for the team solution, to be prepared and handed in for grading at the end of the session. Team Based Learning Exercise: Immunology

February 26th

8:00-9:50 a.m.

Persons missing the session must provide written notice explaining circumstances for not attending. Written approval must be obtained from the Office of Educational/Student Affairs prior to consideration for any makeup session or alternate assignment.

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TRANSPLANT IMMUNOLOGY

Keri C. Smith, PhD

MSB 2.218 713-500-7235 Keri.C.Smith@uth.tmc.edu

Objectives: (1) Discuss the immunobiology of transplantation. (2) Appreciate the importance of innate and adaptive functions in graft recognition. (4) Define molecular aspects of hyperacute, acute and chronic rejection. (5) Recognize clinical consequences of transplantation.

Keywords: Histoincompatibility, Allorecognition, Rejection, GVHD, Tolerance

Reading: Coico and Sunshine. Immunology: A Short Course. John Wiley & Sons, Inc, New York, NY. 6th edition, 2009. Chapter 18; Geha and Notarangelo. Case Studies in Immunology. Garland Publishing, New York, NY. 6th edition, 2012. Case 11. Graft-Versus-Host Disease. Kidney Graft Complications (Blackboard file, case #46).

Web Resource: https://med.uth.edu/pathology/courses/immunology/links-for-lectures/transplantation/

The response to a transplant, or “non-self”, may involve nearly every facet of the immune system that you have learned about thus far in this course. In this section, it is important to cross-reference chapters in the Coico, as well as syllabus sections, to review the cellular and molecular mechanisms at work in various forms of rejection.

Terms used in transplantation

Autologous = “self”

Syngeneic = genetically identical (same MHC). An autologous graft, such as a skin graft from one area of the body to another as sometimes performed to treat burns, is a syngeneic graft. Also, grafts from one individual to another who share the same MHC (as in the case of identical twins) are also syngeneic. Syngeneic grafts (also called isografts) are histocompatible, that is, the donor tissue does not induce an immune response in the recipient.

Allogeneic = genetically different (different MHC). Thus, a graft from one genetically distinct individual to another is called an allograft. It is histoincompatible and induces an immune response in the recipient. Another histoincompatible transplant is a xenograft, which is a graft between a donor and a recipient from a different species.

The majority of transplant rejection is due to differences in MHC expression between donor and recipient. If you need a refresher on class I and class II MHC expression, please review the “Role of the MHC in the Immune Response” syllabus chapter as well as Chapter 8 in Coico.

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The Adaptive Immune Response and Graft Rejection

Lab studies, particularly studies of transplants between inbred strains of mice, provided insight into the role of immune memory in allograft rejection. As shown below, an allogeneic (MHC-mismatched) skin graft will be rejected within 14-21 days (mechanisms of acute rejection are described below). If the mouse that rejected the first skin graft receives another graft from the same donor, the second set rejection will occur much faster, within 6-8 days. This tells us that the immune system “remembers” the graft and mounts a more effective response to the second transplant. If the mouse that rejects the first graft receives a graft from another MHC mismatched donor (unrelated to the first donor), the 2nd graft will be rejected within “1st set” kinetics, that is, the immune system is responding to it as a novel antigen and does not have memory for the 3rd party MHC. This result indicates that immune responses to transplant are specific to individual unique MHC. Thus, transplant rejection is indeed an adaptive immune response to the “foreign” tissue and rejection responses can proceed in a variety of manners depending on context (as discussed in the next sections)

Categories of Allograft Rejection

The rate and mechanism of rejection are dependent upon multiple factors, including previous exposure of recipient to donor antigen, lack of control of T cell response to the allograft, and inflammation and injury of the transplanted tissue. These mechanisms are defined as follows:

Hyperacute rejection occurs within hours following transplant. Graft loss is due to preformed antibodies directed against donor MHC or major blood group antigens. Mediated primarily by activation of the complement cascade, hyperacute rejection is characterized by: 1) recruitment and activation of neutrophils 2) disruption of vascular integrity leading to edema and hemorrhage, and 3) activation of coagulation that leads to fibrin deposition and thrombosis.

The pathobiology of hyperacute rejection most closely resembles Type III hypersensitivity, as it is mediated by pre-existing antibody forming complexes with cells on the donor tissue. You may want to review these mechanisms in Coico Chapter 15 (especially figure 15.2), and in Dr. Norris’ syllabus section regarding antibody-mediated reactions.

Thanks to the institution of the cross-matching test in 1969, hyperacute rejection is rarely observed in transplant today.

Acute rejection is cell-mediated and occurs within 10-21 days of transplant (depending on the transplanted tissue). T cells are absolutely required for this response, and the end result is an infiltration of lymphocytes and macrophages into the transplanted tissue and subsequent loss of

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tissue function. The specific mechanisms by which T cells mediate acute rejection are discussed below. Acute rejection is currently managed by corticosteroids, cyclosporine, and other drugs.

Chronic rejection occurs months or years following the transplant. It is caused by both antibody- and cell-mediated immunity. Multiple episodes of acute rejection, even if they were eventually controlled, probably contribute to the development of chronic rejection as the transplanted tissue remodels in response to injury from the immune attack. Mechanisms of chronic rejection vary depending on the transplanted tissue: in general, pathology is associated with fibrosis and thickening of arterioles in the transplant. There is no cure for chronic rejection; once the process begins it is impossible to stop.

Mechanisms of Alloantigen Recognition by T cells

Direct recognition occurs when recipient T cells bind to transplant MHC and are “tricked” into responding to the foreign MHC-peptide complex presented. This can be mediated by recipient CD8+ T cells binding to donor MHC class I molecules expressing self (transplant) peptides, or by recipient CD4+ T cells responding to graft-derived APC presenting MHC class II molecules.

Remember that T cell activation doesn’t happen by antigen-MHC recognition (Signal 1) alone! A co-stimulatory signal (Signal 2) must also be received. Since the transplanted tissue has undergone some serious trauma, the transplant cells quite capably supply these signals in the form of “danger signals” such as up-regulation of Toll-like receptors (TLRs). The transplant and recipient APC

respond to these signals by up-regulating co-stimulatory molecules and increasing expression of MHC class II on their cell membranes.

If you need to review your understanding of the “3 signal” model of the T cell response to antigen presentation in the context of MHC and co-stimulation see the “Adaptive Immune Response” section of your syllabus and Coico Chapter 10 (Fig 10.4)

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Indirect recognition is the presentation of donor peptides by recipient MHC. Recipient APC process allogeneic proteins and present the resulting peptides on self-MHC molecules to recipient T cells. Current research indicates that most of the peptides presented to recipient T cells are categorized as “minor histocompatibility antigens” (mH). The mH are encoded by genes outside of the MHC, and these polymorphic proteins are usually expressed on MHC as “self” antigens (see figure at right).

Tissue typing

As a rule, the fewer the MHC and mH mismatches, the less response to a transplant. Several tests are employed to attempt to make the best “match” between donor and recipient.

These tests are often referred to as tissue typing because they determine the HLA allele expression on donor and recipient.

Research has shown that some HLA mismatches are “better” than others in terms of transplant survival. These data, combined with our increased knowledge of HLA antigen sequencing, have been combined to form the basis for the commonly used panel reactive antibody test (PRA). The PRA score is expressed as a percentage between 0% and 99%. It represents the proportion of the population to which the person being tested will react via pre-existing antibodies.  

http://optn.transplant.hrsa.gov/converge/resources/allocationcalculators.asp?index=78

The “panel” in this case is a well-characterized panel of lymphocytes expressing known HLA subtypes. Peripheral blood cells from the recipient are incubated with these targets, and the amount of complement dependent cell cytotoxicity that occurs in response to recognition of the target cell is determined for each antigen. Recent advances in flow cytometry and multiplex technology have also been employed for highly sensitive detection of recipient cells that can bind to donor HLA.

Another “old school”, but very effective method to determine if a recipient will respond to donor HLA is to measure the mixed leukocyte response (MLR).

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If leukocytes from a donor and recipient are cultured together (for 72 hours or so), the CD4+ cells will respond to the foreign MHC and activate, secrete cytokines, and proliferate. CD8+

cells respond by CTL response against their targets. Since both the donor and recipient are responding to each other (this is known as a “2-way” MLR), this pretty much results in proliferation and mutually assured destruction of both populations. If we want to analyze the response of just the recipient, or just the donor leukocytes, we can treat one population to stop it from proliferating (using mitomycin C or irradiation), then only the untreated population response will be measured (this is a “one way” MLR as shown at left)

Hematopoetic stem cell transplantation (HCT)

In the case of inherited blood or immune disorders where the mutant gene is expressed in blood-forming cells in bone marrow, transplantation of healthy bone marrow can cure the disease. This type of treatment usually requires allogeneic HCT. HCT is also employed as a treatment for blood cancers.

Even though these are hematopoietic cells, they may still viewed as “foreign” by the recipient immune system. Thus, in order to make sure that the donor bone marrow or blood cells survive and populate the recipient bone marrow, the recipient must be treated with intense immunosuppressive therapy (usually chemotherapy and/or radiation). This induction therapy takes care of any malignancies and also “makes room” in the recipient bone marrow for the newly transplanted cells.

Since the recipient is highly immunosuppressed and the transplanted cell population includes all the progenitor cells necessary to generate a brand new immune system, a serious complication of HCT is graft vs. host disease (GVHD). The “new” immune cells recognize the mismatched host MHC, and can generate immune responses exacerbated by the inflammatory conditions which the host tissue very often displays due to underlying disease, or from damage caused by induction therapy. As the response can theoretically be directed against nearly every MHC expressing cell in the host’s body, the consequences of GVHD are systemic and quite often fatal.

See Case #11: Graft vs. Host Disease

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Immunosuppressive Therapies

Since pretty much every facet of the immune system can play a role in rejection of a transplant, multiple therapies are needed to control the anti-graft response.

The broadest acting agents are the corticosteroids, including prednisone, prednisolone, and methylprednisolone. Corticosteroids exert their anti-inflammatory effects by binding to intracellular steroid receptors. Most of the effects of the corticosteroids are linked to their down-regulation of the inflammatory response. They can downregulate the genes that code for inflammatory cytokines, inhibit leukocyte migration, and reduce the activity of and MHC expression by APC.

For a review of inflammation, see Dr. Actor’s Innate Immunity and Inflammation syllabus section and page 16-17 of Coico.

Since corticosteroids are so broad-acting, they have potent side effects (including edema, weight gain, and diabetes) and thus are usually used sparingly and in combination with more targeted immunotherapies.

As T cells are the major players in acute allograft rejection, agents which interfere with lymphocyte signaling and activation are very effective in the transplant setting. There are several agents with potent effects on T cells:

Calcineurin inhibitors, Cyclosporine and FK506 (tacrilomus) block TCR signal transduction (signal 1) and prevent secretion of IL-2, IL-4, and IFNγ.

mTOR inhibitors (rapamycin, temsirolimus, everolimus) bind to mammalian target of rapamycin (mTOR), which is crucial for the propagation of downstream signaling following binding to co-stimulatory molecules (signal 2) and cytokines such as IL-2 (signal 3).

Inhibitors of DNA synthesis including cyclophosphamide azathioprine (a purine analog), prevent activated T cells and B cells from rapidly proliferating.

Monoclonal antibodies are some of the newest immunosuppressives on the scene. OKT3 is a mouse anti-CD3 antibody that blocks TCR signaling (probably by inducing CD3 to be internalized from the cell surface). The humanized antibodies Daclizamab and Basiliximab bind to the IL-2 receptor (CD25) and prevent T cell proliferation in response to this cytokine. Rituximab is a B-cell depleting antibody that is sometimes used in cases where antibody response to transplant is evident.

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Tolerance

The ultimate goal of HLA matching efforts and immunosuppressive therapies is to induce tolerance to the graft. Unfortunately, true tolerance of the immune system to a graft is nearly impossible to achieve. However, there are records of remarkably long lasting grafts (the longest known allogeneic kidney transplant survived longer than 30 years!) Factors that probably keep the recipient immune system in check include the development of graft antigen-specific T regulatory cells, induction of anergy in graft-reactive CD8+ cells, the secretion of immunosuppressive cytokines such as TGFβ (this is a main player in the maintenance of the eye as an “immune privileged” site), and finally, the development of “microchimerism”, that is, the establishment of a very small percentage of donor cells in the bone marrow of the recipient.

“Transplant biology is not the story of foreignness repulsed. It is the story of transplant tissues impeded by the immune system in their struggle to return to functional homeostasis.”*

- Charlie Orosz, PhD (1945-2005)

*Introduction, Immunobiology of Organ Transplantation 1st edition, Kluwer Academic, New York. 2004.

249

MODERN IMMUNOTHERAPY TBA

Objectives: (1) Discuss the emergence of emergence of natural and engineered antibodies as a tool in scientific discovery, and, (2) understand their potential for utility in both diagnostic and therapeutic applications. Full Syllabus Chapter to be distributed via Blackboard prior to lecture presentation.

Time Line 1

Timeline of Immunology

Sources: Wikipedia, Timeline of Immunology; Immunology History IV, History of Immunology Time Line (Keratin.com); Stewart Sell and Scott L. Rodkey, A short history of Immunopathology.

Also see: http://aai.org/timeline/digital-timeline/

3000 B.C.E. – Fever (Mesopotamia) 2000 B.C.E. - Recognition of “adaptive” protection against disease (Egypt, China) 400 B.C.E. – Anatomic identification of organs (Hippocrates) 80 B.C.E. – Acquired resistance to poinsons (Mithridate Eupator, King of Pontus) 25 – Four cardinal signs of inflammation (Celsus) 1000 – “Snuff” variolation for smallpox (Sung Dynasty, China) 1590 – Bursa of birds described (Fabricius) 1690 – Lymphoid tissue identified in small intestine (Peyer) 1718 - Lady Mary Wortley Montagu, the wife of the British ambassador to Constantinople,

observed the positive effects of variolation on the native population and had the technique performed on her own children.

1798 - First demonstration of vaccination smallpox vaccination (Edward Jenner) 1837 - First description of the role of microbes in putrefaction and fermentation (Theodore

Schwann) 1838 - Confirmation of the role of yeast in fermentation of sugar to alcohol (Charles

Cagniard-Latour) 1840 - First "modern" proposal of the germ theory of disease (Jakob Henle) 1850 - Demonstration of the contagious nature of puerperal fever (childbed fever) (Ignaz

Semmelweis) 1855 – Tuberculous granulomas identified (Rokitansky) 1868 – Langhans Giant Cells identified (Langhans) 1857-1870 - Confirmation of the role of microbes in fermentation (Louis Pasteur) 1862 - phagocytosis (Ernst Haeckel) 1867 - First aseptic practice in surgery using carbolic acid (Joseph Lister) 1876 - First demonstration that microbes can cause disease-anthrax (Robert Koch) 1877 - Mast cells (Paul Ehrlich) 1878 - Confirmation and popularization of the germ theory of disease (Louis Pasteur) 1880 – Birth of Cellular Pathology (Virchow) 1880 - 1881 -Theory that bacterial virulence could be attenuated by culture in vitro and

used as vaccines. Proposed that live attenuated microbes produced immunity by depleting host of vital trace nutrients. Used to make chicken cholera and anthrax "vaccines" (Louis Pasteur)

1883 - 1905 - Cellular theory of immunity via phagocytosis by macrophages and microphages (polymorhonuclear leukocytes) (Elie Metchnikoff)

1885 - Introduction of concept of a "therapeutic vaccination". First report of a live "attenuated" vaccine for rabies (Louis Pasteur)

1887 – Anti-rattlesnake venom discovered (Sewall)

Time Line 2

1888 - Identification of bacterial toxins (diphtheria bacillus) (Pierre Roux and Alexandre Yersin)

1888 - Bactericidal action of blood (George Nuttall) 1890 - Demonstration of antibody activity against diphtheria and tetanus toxins. Beginning

of humoral theory of immunity. (Emil von Behring) and (Shibasaburo Kitasato). Attempt to cure tetanus with passive immunotherapy (Behring)

1891 - Demonstration of cutaneous (delayed type) hypersensitivity (Robert Koch) 1893 - Use of live bacteria and bacterial lysates to treat tumors-"Coley's Toxins" (William

B. Coley) 1894 - Bacteriolysis (Richard Pfeiffer) 1896 - An antibacterial, heat-labile serum component (complement) is described (Jules

Bordet) 1900 - Antibody formation theory “side chain theory” “horror autotoxicus” (Paul Ehrlich) 1901 - blood groups (Karl Landsteiner) 1901-8 Carl Jensen & Leo Loeb, Transplantable tumors 1902 - Immediate hypersensitivity anaphylaxis (Paul Portier) and (Charles Richet) 1902 Paul Portier & Charles Richet, Anaphylaxis 1903 - Intermediate hypersensitivity, the "Arthus reaction" (Maurice Arthus) 1903 - Opsonization (Almroth Wright & Stewart Douglas) 1905 - "Serum sickness" allergy (Clemens von Pirquet and (Bela Schick) 1905 – successful organ transplantation (Correl and Guthrie) 1906 – Clemens von Priquet, coined word “allergy” 1907 - Svante Arrhenius, coined the term immunochemistry 1910 - Emil von Dungern, & Ludwik Hirszfeld, Inheritance of ABO blood groups 1910 - Peyton Rous, Viral immunology theory 1911 - 2nd demonstration of filterable agent that caused tumors (Peyton Rous) 1914 - Clarence Little, Genetics theory of tumor transplantation 1915-20 - Leonell Strong & Clarence Little, Inbred mouse strains 1917 - hapten (Karl Landsteiner) 1921 - Cutaneous allergic reactions (Carl Prausnitz and Heinz Küstner) 1922 – Fleming found lysozyme and penicillin 1924 - Reticuloendothelial system (Aschoff) 1925 – Chemical mediators of inflammation (Lewis) 1926 - Lloyd Felton & GH Bailey, Isolation of pure antibody preparation 1935 Quantitative precipitin reaction (Heidelberger) 1936 - Peter Gorer, Identification of the H-2 antigen in mice 1938 – Gammaglobulin identified (Tiselius and Kabat) 1938 - Antigen-Antibody binding hypothesis (John Marrack) 1940 - Identification of the Rh antigens (Karl Landsteiner and Alexander Weiner) 1941 – Hemolytic disease of the newborn (Rh antigens) (Levine) 1941 - Albert Coons, Immunofluorescence technique 1942 - Anaphylaxis (Karl Landsteiner and Merill Chase) 1942 - Adjuvants (Jules Freund and Katherine McDermott) 1944 - hypothesis of allograft rejection (Peter Medawar) 1945 - Passive transfer of cell mediated immunity (Chase) 1946 - identification of mouse MHC (H2) by George Snell and Peter A. Gorer

Time Line 3

1947 – Twins do not demonstrate transplant rejection (Owen) 1948 - antibody production in plasma B cells (Astrid Fagraeus) 1949 - growth of polio virus in tissue culture, neutralization with immune sera, and

demonstration of attenuation of neurovirulence with repetitive passage (John Enders) and (Thomas Weller) and (Frederick Robbins)

1949 - immunological tolerance hypothesis (Macfarlane Burnet & Frank Fenner) 1950 - Richard Gershon and K Kondo, Discovery of suppressor T cells 1952 - Ogden and Bruton, discovery of agammagobulinemia (antibody immunodeficiency) 1951 - vaccine against yellow fever 1953 - Graft-versus-host disease (Morton Simonsen and WJ Dempster) 1953 - immunological tolerance hypothesis (Rupert Billingham, Leslie Brent, Peter Medwar, &

Milan Hasek) 1953 - James Riley & Geoffrey West, Discovery of histamine in mast cells 1955-1959 - Niels Jerne, David Talmage, Macfarlane Burnet, Clonal selection theory 1957 - Clonal selection theory (Frank Macfarlane Burnet) 1957 - Discovery of interferon (Alick Isaacs & JeanLindermann) 1957 Ernest Witebsky et al., Induction of autoimmunity in animals 1958-1962 - Discovery of human leukocyte antigens (Jean Dausset and Snell) 1959-1962 - Discovery of antibody structure (independently elucidated by Gerald Edelman

and Rodney Porter) 1959 - Discovery of lymphocyte circulation (James Gowans) 1960 - Discovery of lymphocyte "blastogenic transformation" and proliferation in response

to mitogenic lectins-phytohemagglutinin (PHA) (Peter Nowell) 1961-1962 Discovery of thymus involvement in cellular immunity (Jacques Miller) 1961- Demonstration that glucocorticoids inhibit PHA-induced lymphocyte proliferation

(Peter Nowell) 1962 – Classification of immune mechanisms (Gell and Coombs) 1963 - Development of the plaque assay for the enumeration of antibody-forming cells in

vitro (Niels Jerne) (Albert Nordin) 1963 - Jaques Oudin et al., antibody idiotypes 1964-1968 T and B cell cooperation in immune

response (Anthony Davis) 1964 – Mixed lymphocyte reaction (Bain, Vas, et al.) 1965 - Discovery of the first lymphocyte mitogenic activity, "blastogenic factor" (Shinpei

Kamakura) and (Louis Lowenstein) (J. Gordon) and (L.D. MacLean) 1965 - Discovery of "immune interferon" (gamma interferon) (E.F. Wheelock) 1965 - Secretory immunoglobulins (Thomas Tomasi et al.) 1966 - Identification of T and B cells (Claman) 1967 - Identification of IgE as the reaginic antibody (Kimishige Ishizaka) 1968 - Passenger leukocytes identified as significant immunogens in allograft rejection

(William L. Elkins and Ronald D. Guttmann) 1968 – Accessory cell role in immune response (Mosier) 1969 - The lymphocyte cytolysis Cr51 release assay (Theodore Brunner) and (Jean-Charles

Cerottini) 1969 – Immune response genes (Benacerraf and McDevitt) 1971 - Donald Bailey, Recombinant inbred mouse strains 1971 - Peter Perlmann and Eva Engvall at Stockholm University invented ELISA

Time Line 4

1972 - Structure of the antibody molecule 1974 – Network theory for antibody control on immune response (Niels K. Jerne) 1974 - T-cell restriction to major histocompatibility complex (Rolf Zinkernagel and (Peter

Doherty) 1975 - Generation of the first monoclonal antibodies (Georges Köhler) and (César Milstein) 1975 – Identification of natural killer cells (Kiessling, et al.) 1976 - Identification of somatic recombination of immunoglobulin genes (Susumu Tonegawa) 1979 - Generation of the first monoclonal T cells (Kendall A. Smith) 1980 – Immunoglobulin structure (Kabat) 1980-1983 - Discovery and characterization of the first interleukins, 1 and 2 IL-1 IL-2

(Kendall A. Smith) 1981 - Discovery of the IL-2 receptor IL2R (Kendall A. Smith) 1981 – Appearance of AIDS on a global scale 1983 - Discovery of the T cell antigen receptor TCR (Ellis Reinherz) (Philippa Marrack) and

(John Kappler) (James Allison) 1983 - Discovery of HIV (Luc Montagnier) 1984 - The first single cell analysis of lymphocyte proliferation (Doreen Cantrell) and

(Kendall A. Smith) 1984 - Robert Good, Failed treatment of severe combined immunodeficiency (SCID, David

the bubble boy) by bone marrow grafting 1984-1987 - Identification of genes for the T cell receptor (Leroy Hood, et al.; Hedrick

Davis, Mak) 1985 Tonegawa, Hood et al., Identification of immunoglobulin genes, somatic generation of

Ig variable regions 1985-onwards - Rapid identification of genes for immune cells, antibodies, cytokines and

other immunological structures 1987- Structure of MHC I defined (Wiley and Strominger) 1986 - Hepatitis B vaccine produced by genetic engineering 1986 - Th1 vs Th2 model of T helper cell function (Timothy Mosmann) 1988 - Discovery of biochemical initiators of T-cell activation: CD4- and CD8-p56lck

complexes (Christopher E. Rudd) 1989 – Catalytic antibody cleavage of peptide bonds (Sudhir Paul) 1990 - Yamamoto et al., Molecular differences between the genes for blood groups O and A

and between those for A and B 1990 - Gene therapy for SCID using cultured T cells 1991- Role of peptide for MHC Class II structure (Sadegh-Nasseri & Germain) 1992 – Hepatitis A vaccine developed 1993 - NIH team, Treatment of SCID using genetically altered umbilical cord cells 1994 - 'Danger' model of immunological tolerance (Polly Matzinger) 1995 - Regulatory T cells (Shimon Sakaguchi) 1996-1998 - Identification of Toll-like receptors 2001 - Discovery of FOXP3 - the gene directing regulatory T cell development 2005 - Development of human papillomavirus vaccine (Ian Frazer) 2011 – Nobel Prize awarded to Bruce A. Beutler, Jules A. Hoffmann, and Ralph M. Steinman

for landmark discoveries indicating TLRs are gatekeepers of innate immunity 2011 – Roles of innate lymphocytes in mucosa homeostasis identified.

Spring Semester, 2015

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK. Glossary1

Glossary

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for

Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK.

Also see on-line Glossary: Roitt: Essential Immunology (Wiley-

Blackwell, 12th ed)

Active Immunity: Present immunity acquired through the presence of protective antibodies and

memory T lymphocytes.

Accessory Molecule: Cell surface molecules participating in cellular interactions to modulate

strength and direction of specific immune response.

Acquired Immunodeficiency Syndrome (AIDs): An infectious disease caused by the human

immunodeficiency virus (HIV) characterized by loss of CD4+ T helper lymphocytes.

Acute Phase Proteins: Any of the non-antibody proteins found increased in serum during active

and immediate innate responses; includes complement factors, C-reactive protein and fibrinogen.

Acquired Immunity/Adaptive Immunity: Network of antigen specific specialized lymphocytes

that function to eliminate or prevent systemic infection. Responses take days or weeks to

develop, and results in immune readiness (memory) that may be sustained for long periods.

Adjuvant: Excipient added to an immunogen to direct immune response during vaccination.

Affinity: Binding strength of antibody for its cognate antigen.

Affinity Maturation: Process by which B lymphocytes mature response to increase specificity

of antibody for is cognate antigen.

Allele: Variants of a polymorphic gene at a genetic locus.

Allelic Exclusion: Expression of only one gene while the alternate copy (allele) remains silent.

Spring Semester, 2015

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK. Glossary2

Allergen: Antigen that elicits Type I hypersensitivity (allergic reactions).

Allergy: Misdirected hypersensitive immune reaction to normally harmless foreign substances.

An “allergen” is any antigen that elicits Type I hypersensitivity (allergic reactions).

Allogeneic: Genetically different from a similar species member.

Alternative Pathway: Complement activation pathway involving complement C3, Factor B, and

Factor D. Generates the alternative pathway C3 convertase (C3bBb) in the presence of a

stabilizing microbial activator.

Allograft: Tissue graft from a non-self donor of the same species.

Anaphylactic Shock: Systemic allergic reaction to circulating antigen, resulting from interaction

with IgE antibodies on connective tissue mast cells, flowed by release of inflammatory mediators

which confer “shock”.

Anaphylotoxin: Complement system enzymatic fragments (C3a, C4a, C5a) that mediate host

defense functions, including chemotaxis and activation of cells bearing fragment receptors.

Causes enhanced vascular permeability, and mast cell histamine release.

Anergy: Specific immunological tolerance where lymphocytes become functionally

nonresponsive.

Antibody: A two chain protein on the surface of B lymphocytes that can be secreted in large

amounts in response to an antigen. Five subclasses exist, each which uniquely function to confer

protection against infectious assault. See: Immunoglobulin.

Antibody Dependent Cell Cytotoxicity (ADCC): Cytolytic process directed towards an

Antibody-coated target cell via mechanisms whereby effector cells (mostly Natural Killer cells)

with Fc receptors recognize the constant region of target bound immunoglobulin.

Antigen: Foreign substance capable of eliciting an immune response. May be of protein,

carbohydrate, lipid, or nucleic acid in nature.

Spring Semester, 2015

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK. Glossary3

Antigen Presentation: Organized display of processed antigenic fragments bound to presenting

cell surface histocompatability molecules to allow targeted recognition by the T cell receptor.

Antigen Presenting Cell (APC): Specialized bone-marrow derived cell, bearing cell-surface

class II major histocompatibility complex molecules to function in antigen processing and

presentation to T cells.

Antigen Processing: Act of protein degradation into small peptide fragments that can interact

with MHC molecules for presentation to T cells.

Antigen Receptor: Specific antigen-binding receptor on T or B lymphocytes; comprised of

amino acids produced from genetic sequences with physical rearrangements and translocation of

V, D, and J gene subsets.

Antigen Receptor: Specific antigen-binding molecule on T or B lymphocytes; comprised of

amino acids produced from genetic sequences with physical rearrangements and translocation of

V, D, and J gene subsets.

Antigenic: Substance capable of recognition by an immunoglobulin or an antigen receptor. The

“antigenic determinant” is the site or epitope on a complex molecule recognized by an antigen

receptor (antibody or T cell receptor). The antigen-binding site “paratope” represents the

physical location on the receptor that contacts the molecules.

Antigenic Determinant: Site or epitope on a complex antigenic molecule or particle recognized

by an antigen receptor (antibody or T cell receptor). The antigen-binding site represents the

physical location on the antigen receptor that contacts the molecules.

Apoptosis: Process of programmed cell death.

Autograft: Tissue grafted from one person to that same individual, with complete match of

histocompatability molecules.

Autoimmune Disorder: Pathological condition where the body’s own immune system is

directed towards self antigens. Autoreactivity describes immune cells mounting a response

Spring Semester, 2015

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK. Glossary4

against self.

Autologous: From the same individual.

Autoreactive: Describes immune cells mounting a response against self antigens.

Avidity: Combined strength of antibody-antigen interaction, taking into account multiple

binding sites between molecules.

β2-microglobulin: A 12 kDa protein that interacts with MHC class I-encoded molecules.

B Cell/B Lymphocyte: Type of lymphoid cell produced in the bone marrow from lymphoid

progenitor stem cells that possesses specific antibody cell-surface antigen receptors; cell capable

to produce antibodies when activated.

Basophil: Polymorphonuclear granulocytic cell involved in allergic reactions during Type I

hypersensitivity.

Blood Group Antigens: Red blood cell surface molecules detectable with antibodies produced

by sensitization to environmental substances. Major blood group antigens include ABO and Rh

(Rhesus) markers used in routine blood screening to designate blood type.

CD3 Complex: Set of signal transduction molecules assisting in T cell activation once the

antigen receptor has been engaged.

CD4: Cell surface glycoprotein on helper T-cells that recognizes MHC class II molecules on

antigen-presenting cells.

CD8: Cell surface glycoprotein on cytotoxic T-cells that recognizes MHC class I molecules on

target cells.

Cell Mediated Immunity/Cellular Immunity: Adaptive immune responses initiated by antigen

specific T cells.

Chemokines: Family of related small polypeptide cytokines involved in directed migration and

Spring Semester, 2015

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK. Glossary5

activation of leukocytes. “Chemotaxis” is targeted movement in response to a chemical stimulus.

Class Switch: Change in production of antibody isotype due to maturation of the B lymphocyte

towards a particular antigen.

Classical Pathway: Complement activation pathway involving components C1, C2 and C4

following fixation of C1q, by antigen–antibody complexes; produces the classical pathway C3

convertase C4b2b.

Cluster of Differentiation (CD designation): Commonly used designation for specific cell

surface molecules. CD molecules are useful markers to discriminate different cell phenotypes,

and to classify cells according to functional activity.

Combinatorial Joining: Physical joining of nucleic acid sequences during development to

generate novel proteins involved in antigen binding receptors on B and T lymphocytes.

Complement: System of serum proteins involved in inflammation and immunity; mediates

activities which include activation of phagocytes, direct cytolysis of target cells, and coating

(opsonization) of microorganisms for uptake by cells expressing complement receptors.

Concanavalin A (con A): Mitogenic lectin derived from the jack bean that stimulates T

lymphocytes to undergo mitosis and proliferation.

Cross-Reactivity: Binding of antibody to a epitope or molecule similar in structure to the

antigen used to elicit antibody response.

Cyclosporine A: Powerful immunosuppressive agent.

Cytokine: Class of small molecule immune mediator secreted by leukocytes as a mechanism of

immune regulation and cross-talk. Cytokines produced by lymphocytes are called “lymphokines”

or “interleukins”.

Cytokine Storm: A cascade event that is a potentially fatal immune reaction consisting of a

positive feedback loop between cytokines and immune cells, with highly elevated levels of

cytokines made in response to infectious assault, disease state, or trauma.

Spring Semester, 2015

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK. Glossary6

Cytotoxic T cell: T lymphocytes bearing CD8 cell surface molecules that respond to antigenic

stimulation through elicitation of toxic mediators. Critical for anti-viral responses.

Defensins: Natural molecules able to limit growth of microorganisms.

Degranulation: Process by which myeloid leukocytes release digestive proteins stored in

cytoplasmic vesicles.

Delayed Type Hypersensitivity (DTH): See: Hypersensitivity, Type IV.

Dendritic Cell: Primary phagocytic antigen-presenting cell capable of initiating immune

response and lymphocytic activation, accomplished by cytokine secretion.

Enzyme-Linked Immunoadsorbent Assay (ELISA): Assay used to detect antigens bound to

solid wells in a plate format. Labeled reagents are used for quantitation, by linking enzymes to an

antibody to allow substrate color change for recognition of antigenic detection.

Endocytosis: Mechanism utilizing receptors or pinocytosis whereby materials are up-taken from

solution into plasma membrane vesicles by cells.

Eosinophil: Polymorphonuclear granulocytic cell involved in the innate response to parasitic

infections.

Epitope: Antigenic determinant; portion of antigen capable of interacting with antibody or

eliciting a lymphocytic response.

Extravasation: Movement across blood endothelial barriers into tissue.

Fab fragment/F(ab)’2: Portion of the antibody heavy and light chains which combine to make

up the antigen binding region.

Fas-FasL: Cell surface molecule interactions involved in activation of apoptosis.

Fc Fragment: Portion of the antibody heavy chain that comprise regions able to interact with

cellular receptors; confers biological function to the immunoglobulin.

Spring Semester, 2015

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK. Glossary7

Foreign: Non-self.

Germ Line: Genetic material in original configuration, representing non-rearranged

chromosomes.

Germinal Center: Secondary lymphoid tissues sites where lymphocytic populations can

proliferate and mature in response to antigen.

Graft-versus-Host Disease (GVHD): Clinical state where donor immune cells develop

pathological reactions to recipient post transplantation.

Granulocyte: General term for phagocytic leukocyte containing granular particles.

Granzyme: Protein involved in cytotoxic reactions; involved in cell lysis.

Hapten: Small low-weight molecule that can only elicit immune responsiveness when attached

to a larger carrier molecular, thus rendering it immunogenic.

Heavy Chain: Larger protein associated with the antibody molecule; confers biological

functions, associated with the constant portion of the chain.

Helper T Cell: Class of CD4+ T lymphocytes that respond to antigens by secreting cytokine

subsets to give help to cells to become effectors of cellular immunity, or to stimulate B cells to

make antibodies.

Hematopoietic Stem Cell: Precursor cell found in bone marrow. Can give rise to leukocytes.

Herd Immunity: Social concept to preventing spread of infection within a community;

vaccination of a significant portion of a population provides a measure of protection for

individuals who have not developed immunity, due to limitation of infection spread.

Heterograft: Graft in which the donor and recipient are of different species. See: Xenograft.

Histamine: Compound released from neutrophils during immunological and allergic reactions

causing vasodilation and smooth muscle contraction.

Spring Semester, 2015

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK. Glossary8

Histocompatability: Tissue compatability between individuals based on presence of

polymorphic major histocompatability molecules present on cellular surfaces.

Human Leukocyte Antigen (HLA): Genetic designation for the human major

histocompatibility complex (MHC) molecules. Class I molecules are represented by gene loci

HLA-A, HLA-B, and HLA-C. Class II molecules are represented by gene loci HLA-DR, HLA-

DP, and HLA-DQ. See: MHC.

Human immunodeficiency virus (HIV): Lentiviral family member retrovirus with an RNA

genome that forms a DNA intermediate incorporated into the host cell genome. Infection leads

to loss of CD4+ lymphocytes and an eventual state of acquired immune deficiency.

Humoral Immunity: Refers to antibody mediated immunity.

Hyperacute Graft Rejection: Reaction representing immediate recipient antibody reactivity to

antigens present in donor tissue during transplantation.

Hypersensitivity: Immune reactivity to antigen at levels higher than normal, often leading to

clinical states. Reactions are classified by mechanism: Type I (allergic) reactions involve IgE

triggering mast cells; Type II (cytotoxic) reactions involve IgG against cell surfaces resulting in

cytolytic events; Type III (immune complex) reactions involve destructive deposition of

antibody and antigen complexes; Type IV (delayed type hypersensitivity; DTH) reactions are T-

cell mediated.

Hypervariable Regions: Portions of the antibody light and heavy chains that represent the most

variable amino acid sequences coding for contact with the epitopes on the antigen. Encoded by

“complementarity determining regions”.

Immunodeficiency: Relative decrease in immune responsiveness due to lack of components

(innate or adaptive) capable of responding to a foreign influence. Immune deficiency disease is

the resultant clinical state when parts of the immune system are missing or defective.

Immune Deficiency Disease: Resultant clinical state when one or more parts of the immune

system are missing or defective.

Spring Semester, 2015

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK. Glossary9

Immunogenic: Capable of eliciting an immune response.

Immunoglobulin: Any of 5 classes of antibodies (IgM, IgD, IgG, IgE, or IgA) that function in

immune regulation through specific binding of antigens. See: Antibody.

Immune/Immunity: Protection against a specific disease or pathogen, normally as a result of

effective innate and adaptive resistance.

Immunization: Induction of adaptive immunity by pre-exposure of antigen or by active

infection, thereby a generating a memory lymphocytic response.

Immunology: Branch of biological science concerned with the study of all components

associated with function and structure of the immune system.

Inflammation: Buildup of fluid and cells that occurs in responses to acute injury or trauma.

Innate Immunity: Component of the immune system consisting of genetically encoded

constitutive factors readily able to respond to pathogens on short notice. Factors involved do not

change or adapt during the lifetime of the organism; no associated memory response.

Interferons: Specialized subset of cytokine originally discovered as having properties that

interfere with viral replication. Mediators of cellular immune function.

Interleukin: See: Cytokine.

Isograft: Tissue graft between individuals of genetic identity. Similar to “syngraft”.

Isohemagglutinin: Naturally occurring IgM molecules that recognize ABO antigenic

determinants on red blood cells.

Isotype: Antigenic marker that distinguishes members of an immunoglobulin class.

Immunoglobulin isotypes include IgG, IgA, IgE, IgD, and IgM.

Isotype Switching: Genetic rearrangement in B lymphocytes to allow change in production of

immunoglobulin isotypes.

Spring Semester, 2015

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK. Glossary10

Kinins: Polypeptides released during inflammatory responses which increase vascular

permeability and smooth muscle contraction.

Killer cell Immunoglobulin-like Receptors (KIRs): Found on NK cells; recognize MHC class

I molecules to either inhibit or activate the killer cells.

Lactoferrin: Innate iron binding component that has bactericidal and bacteriostatic activity, as

well as immune modulating properties. Found secreted onto mucosal surfaces.

Leukocyte: Any white blood cell (myeloid or lymphoid) that plays a functional role in either

innate or adaptive responses. The myeloid population includes neutrophils, eosinophils,

basophils, mast cells, as well as monocytes and macrophages, and dendritic cells. The lymphoid

group includes the lymphocyte populations.

Leukotrienes: Products of arachidonic acid which promote inflammatory processes such as

chemotaxis and increased vascular permeability; produced by mast cells, basophils and

macrophages.

Light Chain: Smaller protein associated with the antibody molecule; can be either of the kappa

() or lambda () variety.

Lipopolysaccharide (LPS): Endotoxin component of gram-negative bacteria cell wall that

elicits mitogenic activity.

Lymph Nodes: Small, rounded secondary lymphoid organs where mature leukocytes, especially

lymphocytes, interact with antigen presenting cells.

Lymphatics: Endothelial lined network of vessels permitting flow of lymph to lymph nodes.

Lymphocyte: Lymphoid derived leukocyte expressing an antigen specific receptor. There are

two broad categories, T cells and B cells. Lymphocytes function as an integral part of the body’s

adaptive defenses and are critical for distinguishing self from foreign antigens.

Lymphoid: Tissue responsible for producing lymphocytes and antibodies, including regions in

the lymph nodes, thymus, and spleen.

Spring Semester, 2015

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK. Glossary11

Lymphokine: See: Cytokine.

Lymphotoxin: A T-cell cytokine cytotoxic for tumor cells; also called TNF-β.

M cells: Microfold cell found in the follicle-associated epithelium of the Peyer's patch. Function

to sample antigen from the small intestine lumen to deliver via transcytosis to presenting cells

and lymphocytes located on the basolateral side.

Macrophage: Myeloid derived cell involved in phagocytosis and intracellular killing of

microorganisms, and antigen presentation to T lymphocytes.

Major Histocompatibility Complex (MHC) Molecule: Polymorphic molecules to allow the

immune system to distinguish between self and foreign substances. Class I molecules present

antigen to CD8+ cytotoxic T lymphocytes, and are on all nucleated cells. Class II molecules

present to CD4+ helper T lymphocytes, and are found on antigen presenting cells.

Mast Cell: Large myeloid cell found in connective tissues which mediates allergic reactions.

Membrane Attack Complex (MAC): Terminal product of complement cascade, whereby

components C5 through C9 self assemble on a membrane to form a cytolytic pore.

Mitogen: Agent capable of stimulating cellular activation and division.

Molecular Mimicry: Cross reactive occurrence during development of autoimmune disorder

where a microorganism contains antigenic determinants that resemble those on self tissues.

Monoclonal Antibodies: Antibodies derived from a single B cell specific for a single antigen.

Monocyte: Part of the innate leukocyte population; blood precursor to the tissue macrophage.

Mucosa Associated Lymphoid Tissue (MALT): Diffusion system of concentrated lymphoid

tissue found in the gastrointestinal tract, thyroid, breast, lung, salivary glands, and skin. Related

to Gut Associated Lymphoid Tissue (GALT) which represents Peyer's patches found in the

lining of the small intestines, and the Bronchus-Associated Lymphoid Tissue (BALT)

representing aggregations of immune cells in the lower respiratory tract.

Spring Semester, 2015

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK. Glossary12

Myeloid: Derived from granulocyte precursors stem cells in bone marrow.

Natural Killer Cell: Small granular innate cell derived from lymphoid progenitors. Able to

rapidly destroy tumor cell targets by antibody-dependent cell cytotoxicity which permits target

destruction in a non-phagocytic manner. They do not express a T cell receptor.

Natural Killer T Cell: Small subpopulation of T cells that express a limited T cell receptor

repertoire; receptors recognize bacterial lipids or glycolipids bound to non-classical

histocompatability class I-like molecules.

Neutrophil: Polymorphonuclear, phagocytic granulocytic cells involved in the acute

inflammatory response to pathogens.

Nitric oxide: Molecule important in intra-cellular signaling; free radical and regulator of

hydrogen peroxide in phagosomes within phagocytic cells.

Opsonization: Process by which a molecule or pathogen is targeted for ingestion and subsequent

destruction by phagocytic cells, mediated through complement or antibody interactions. An

“opsonin” is a molecule that enhances directed phagocytosis.

Paratope: Portion of the antibody that contacts the epitope on the antigen.

Passive Immunity: State of immunity acquired through transfer of factors (serum or antibodies),

allowing a protective state in the absence of active immunity.

Pathogen Associate Molecular Patterns (PAMPs): Conserved molecular motifs associated

with infectious agents that are able to trigger innate immune function. Cellular receptors on

monocytes that recognized conserved molecular motifs associated with infectious agents are

called “Pattern Recognition Receptors (PPRs)”.

Pattern Recognition Receptors (PPRs): Cellular receptors on monocytes that recognized

conserved molecular motifs associated with infectious agents. See: Pathogen Associate

Molecular Patterns (PAMPs).

Perforin: Protein involved in cytotoxic reactions; involved in cell lysis.

Spring Semester, 2015

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK. Glossary13

Peyer’s Patches: Lymphatic nodules located along the small intestine.

Phagocyte: Any mobile leukocyte that engulfs foreign material. The process of directed uptake

is called phagocytosis.

Phagolysosome: Internal digestive compartment within phagocytic cells where phagosome and

lysosomal enzymes destroy engulfed pathogenic invaders and digest engulphed protein.

Plasma: Fluid component of blood containing water, electrolytes, proteins and molecular

mediators, but no cells.

Plasma Cell: Terminally differentiated antibody-secreting B lymphocyte.

Polymorphonuclear Cells (PMNs): Group of white blood cells (neutrophils, basophils and

eosinophils) with multi-lobed nuclei and cytoplasmic granules.

Primary Immune Response: Adaptive immune response representing initial exposure to

antigen, predominantly comprised of IgM followed by later presence of other antibody isotypes.

“Priming” is the activation of lymphocytic response to antigen for the first time, initiated by

antigen presenting cells.

Primary Lymphoid Tissue: Immune organs where lymphocytes develop and mature; organs

where antigen-specific receptors are first expressed.

Priming: Activation of lymphocytic response to antigen for the first time, initiated by antigen

presenting cells.

Pus: Protein rich liquid inflammatory accumulation of cellular debris and necrotic factors.

Regulatory T Cells (Treg cells): Specialized T lymphocyte subgroup able to regulate immune

responses; effective post thymic development.

Respiratory Burst: Phagocytic metabolic activity resulting in formation of superoxide anion

and hydrogen peroxide.

Spring Semester, 2015

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK. Glossary14

Rheumatoid Factor: IgM isotype antibodies reactive with IgG molecules.

Rhesus Antigens (Rh): See: Blood Group Antigens.

Severe Combined Immunodeficiency (SCID): Disease state in which defects in maturation

pathways for both B and T lymphocytes result in lack of functional adaptive immunity.

Secondary Immune Response: Immune response induced by repeated antigen exposure, often

of higher affinity and with greater speed than elicited by primary response. Has characteristic

maturation of antibody isotype.

Secondary Lymphoid Tissue: Immune organs where antigen-driven proliferation of

lymphocytes occur in response to antigenic stimulation.

Secretory Component: Portion of the dimeric IgA molecule critically involved in release across

mucosal barriers.

Seroconversion: Indicates when antibody can be first detected against antigen, following

infectious challenge or immunization.

Severe Combined Immune Deficiency (SCID): Disease state in which defects in maturation

pathways for both B and T lymphocytes result in lack of functional adaptive immunity.

Somatic Hypermutation: Change in affinity maturation of the antigen binding site in an

antibody following antigenic stimulation.

Superantigen: Molecule able to elicit T lymphocyte responses by circumventing normal antigen

processing and presentation functions.

Syngeneic: Being from individuals that are genetically identical.

T Cell/T Lymphocyte: Derived from bone marrow lymphoid progenitor stem cells, possessing

specific cell-surface antigen receptors; types include helper T cells of different cytokine secreting

subsets, as well as cells that confer regulatory and cytotoxic function.

Spring Semester, 2015

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK. Glossary15

T-dependent Antigen: An antigen that requires helper T-cells to elicit an antibody response.

T-independent Antigen: An antigen able to elicit an antibody response in the absence of T-

cells; usually not able to drive maturation of B cells to induce antibody class switching.

TAP-1/TAP-2: Transporters of antigen processing molecules that transfer antigenic peptides

from cytoplasm into lumen of the endoplasmic reticulum for incorporation onto MHC class I

molecules.

Thymocyte: Hematopoietic progenitor cell present in the thymus.

Titer: Method to express relative antibody concentration.

Tolerance: State of less responsiveness to a substance or a physiological insult; instrumental in

prevention of autoimmunity.

Toll-like Receptors: Subset of pattern recognition receptors recognizing conserved molecular

motifs associated with infectious agents; initiate strong innate immunity when triggered.

Toxoid: Chemically or physically modified toxin that retains immunogenicity without harmful

effects of native toxin molecule.

Transplantation: Grafting of tissue from one individual to another.

Tumor Necrosis Factor: Substance secreted by multiple cell phenotypes; member of a group of

cytokines that stimulate a proinflammatory response.

Vaccine: Immunogenic substance used to stimulate production of protective immunity

(antibodies or T cell based) to provide protection against clinical disease.

Vaccination: Artificial induction of adaptive immunity by pre-exposure of antigen or pathogen

to generate a memory lymphocytic response.

Variable Domain/Variable Region: End portion of the antibody or T cell receptor which

comprises the antigen binding region.

Spring Semester, 2015

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK. Glossary16

Vasoactive Amines: Substances including histamine thatincrease vascular permeability and

smooth muscle contraction.

Western Blot: Diagnostic antigen identification of a mixture separated by electrophoresis

through a gel matrix. Proteins are transferred to a solid matrix (usually nitrocellulose) and probed

with specific immune reagents.

Xenograft: Tissue graft in which donor and recipient are of different species. Similar to

“heterograft”.

Table 1-5. Selected CD Markers and Associated Functions CD Marker Biological Function CD1 Presentation of glycolipids to NKT cells CD2 T cell adhesion molecule CD3 Signaling chains associated with the TCR CD4 Co-receptor for Class II MHC on T cells CD8 Co-receptor for Class I MHC on T cells CD11 Leukocyte adhesion CD18 β2 Integrin CD19 B cell signal transduction CD20 B cell calcium channel activation CD21 B cell activation CD25 IL-2 receptor α chain CD28 T cell co-stimulatory molecule CD32 IgG receptor CD34 Hematopoietic stem cell marker CD40 Class switching on B cells CD44 Lymphocyte adhesion CD54 Adhesion molecule CD58 Adhesion molecule CD59 Regulator of complement MAC assembly CD62L T cell adhesion to high endothelial venules CD69 Early T cell activation marker CD80 Co-stimulatory receptor on APCs CD86 Co-stimulatory receptor on APCs CD95 Induction of apoptosis CD152 Negative regulator for T cells CD154 Involved in B cell proliferation and class switching

Spring Semester, 2015

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK. Glossary17

Medical Puns (source unknown) Alimentary: What Sherlock Holmes said to Dr. Watson. Antibody: Against everyone. Barium: What you do when the patient dies. Benign: What we want when we are eight. Bunion: Paul's surname. Carpal: Someone you drive to work with. Castrate: The going price for setting a fracture. CAT Scan: Searching for ones lost kitty. Cauterize: What the intern did before he winked at his date. Colic: A sheep dog. Coma: A punctuation mark. Constipation: Endangered feces. Cystogram: A wire sent to your sister. Denial: Where Cleopatra used to swim. Dilate: To live long. Elixir: What a dog does to his owner when she gives him a bone. Fibrillate: To tell a small lie. Genes: Blue denim slacks. G.I. Series: Baseball games between teams of soldiers. Hernia: Pertaining to a female's knee. Hormones: What a prostitute does when she doesn't get paid. Humerus: To tell us what we want to hear. ICU: Peek-a-boo. Impotent: Distinguished, well known. Inbred: The best way to have your jam. Inpatient: Tired of waiting. Intern: One after the other. Intestine: Currently taking an exam. Migraine: What a farmer says about his harvest. Nitrate: Lower than day rate. Outpatient: A person who has fainted. Pap Smear: To slander your father. Paradox: Two doctors. Paralyze: two far-fetched stories. Pathologcial: a reasonable way to go. Rectum: Dang near killed him. Saline: Where you go on your boyfriend's boat. Scar: Rolled tobacco leaf. Seizure: Roman Emperor. Terminal Illness: Getting sick at the airport. Testes: What you order when you don't know what the patient has. Tolerance: What you get if you give growth hormone to ants. Urinate: What a nurse would say if a patient asked her what room he's in. Urine: The opposite of "You're out!" Vertigo: How foreigners ask for directions.

Appendix I

APPENDIX

Nomenclature of Immune System Cells.

Table 2-2. Lymphoid Leukocytes and Their Properties

Total Lymphocytes 1.3-3.5 × 109/L Effector Function B cell Monocytic Adaptive Humoral immunity

Plasma cell Monocytic Adaptive Terminally differentiated, antibody secreting B cell

T cell Monocytic Adaptive Cell-mediated immunity

Natural killer cell Monocytic Innate Innate response to microbial or viral infection

Table 2-1. Myeloid Leukocytes and Their Properties

Phenotype Morphology Circulating Differential Count* Effector Function Neutrophil PMN granulocyte 2-7.5 ×109/L Phagocytosis and digestion of microbes

Eosinophil PMN granulocyte 0.04-0.44 ×109/L Immediate hypersensitivity (allergic) reactions, defense against helminths

Basophil PMN granulocyte 0-0.1 ×109/L Immediate hypersensitivity (allergic) reactions

Mast cell PMN granulocyte Tissue specific Immediate hypersensitivity (allergic) reactions

Monocytes Monocytic 0.2-0.8 ×109/L Circulating macrophage precursor

Macrophage Monocytic Tissue specific Phagocytosis and digestion of microbes, antigen presentation to T cells

Dendritic cell Monocytic Tissue specific Antigen presentation to naïve T cells, initiation of adaptive responses

*Normal range for 95% of population, +/- 2 standard deviations. PMN, polymorphonuclear.

Appendix II

Antibodies

Table 3-1. Classes of Antibody Isotypes and Functional Properties*

Immunoglobulin Class Isotype IgM IgD IgG IgE IgA Structure Pentamer Monomer Monomer Monomer Monomer, dimer

Heavy chain designation

μ δ γ ε α

Molecular weight (kDa) 970 184 146-165 188 160 × 2

Serum concentration(mg/mL)

1.5 0.03 0.5-10.0 <0.0001 0.5-3.0

Serum half-life (days) 5-10 3 7-23 2.5 6

J chain Yes No No No Yes

Complement activation Strong No Yes, except IgG4

No No

Bacterial toxin neutralization

Yes No Yes No Yes

Antiviral activity No No Yes No Yes

Binding to mast cells and basophils

No No No Yes No

Additional properties Effective agglutinator of particulate antigens, bacterial opsonization

Found on surface of mature B cells, signaling via cytoplasmic tail

Antibody-dependent cell cytotoxicity

Mediation of allergic response, effective against parasitic worms

Monomer in secretory fluid, active as dimer on epithelial surfaces

Appendix III

Table 3-2. Unique Biological Properties of Human IgG Subclasses

IgG1 IgG2 IgG3 IgG4 Occurrence (% of total IgG) 70 20 7 3

Half-life (days) 23 23 7 23

Complement binding + + Strong No

Placental passage ++ ± ++ ++

Receptor binding to monocytes Strong + Strong ±

Appendix IV

Table 1-5. Selected CD Markers and Associated Functions CD Marker Biological Function CD1 Presentation of glycolipids to NKT cells CD2 T cell adhesion molecule CD3 Signaling chains associated with the TCR CD4 Co-receptor for Class II MHC on T cells CD8 Co-receptor for Class I MHC on T cells CD11 Leukocyte adhesion CD18 β2 Integrin CD19 B cell signal transduction CD20 B cell calcium channel activation CD21 B cell activation CD25 IL-2 receptor α chain CD28 T cell co-stimulatory molecule CD32 IgG receptor CD34 Hematopoietic stem cell marker CD40 Class switching on B cells CD44 Lymphocyte adhesion CD54 Adhesion molecule CD58 Adhesion molecule CD59 Regulator of complement MAC assembly CD62L T cell adhesion to high endothelial venules CD69 Early T cell activation marker CD80 Co-stimulatory receptor on APCs CD86 Co-stimulatory receptor on APCs CD95 Induction of apoptosis CD152 Negative regulator for T cells CD154 Involved in B cell proliferation and class switching

Adapted from: Introductory Immunology, 1st Edition. Basic Concepts for Interdisciplinary Applications. Academic Press, Elsevier. July, 2014. Actor, JK.

Appendix V

T Cells

Appendix VI

Intracellular transcription factors regulate T cell phenotype development.

Appendix VII

Cytotoxic T Cells

Effector Cells in Cytotoxic Cell Mediated Immunity Effector Cell CD markers Effector Molecules MHC

recognition

Antigen recognition

CTL TCR,CD3,CD8,CD2 Perforin, cytokines (TNF-β, IFN-)

required Class I

specific TCR

NK cell CD16,CD56, CD2 Perforin, cytokines (TNF-β, IFN-)

no nonspecific

NK cell ADCC

CD16,CD56, CD2 Perforin, cytokines (TNF-β, IFN-)

no specific IgG

LAK cell CD16,CD56, CD2 Perforin, cytokines (TNF-β, IFN-)

no nonspecific

Macrophage CD14 TNF-α, enzymes, NO, O radicals

no nonspecific

Appendix VIII

Complement Cascade

Appendix IX

Biological Functions of Complement.

Appendix X

Interleukins are the cytokines that act specifically mediate activity between leukocytes.

Major Cell Source Major Functions

IL-1 Macrophages Stimulation of T cells and antigen-presenting cells B-cell growth and antibody production Promotes hematopoiesis (blood cell formation)

IL-2 Activated T cells Proliferation of activated T cells IL-3 T lymphocytes Growth of blood cell precursors

IL-4 T cells and mast cells, B cells,

stromal cells

Promotes TH2 cell development B-cell proliferation IgE production

IL-5 T cells and mast cells Eosinophil growth

IL-6 Activated T cells, B cells,

Monocytes and PMNs Promotes granulomatous response Induces fever and shock, synergistic effects with IL-1 or TNF-

IL-7 thymus and bone marrow

stromal cells Development of T cell and B cell precursors.

IL-8 (CXCL8) Macrophages Chemoattractant for neutrophils IL-9 Activated T cells Promotes growth of T cells and mast cells

IL-10 Activated T cells, B cells and

monocytes Inhibits inflammatory and immune responses Inhibits TH1 cell responses

IL-11 Stromal cells Synergistic effects on hematopoiesis Promotes TH2 cell response

IL-12 Macrophages, B cells Promotes TH1 cells while suppressing TH2 functions IL-13 TH2 cells Similar to IL-4 effects, attenuates macrophage function IL-15 Epithelium and monocytes Similar to IL-2 effects IL-16 CD8 T cells Chemoattractant for CD4 T cells IL-17 Activated memory T cells Promotes T cell proliferation IL-18 Macrophages Induces IFN- production IL-19 Monocytes and B cells Inflammatory response, induction of IL-6 and TNF- IL-20 Monocytes and Keratinocytes Involved in inflammatory skin diseases

IL-21 NK, B, T, and dendritic cells,

macrophages, and endothelial cells

Modulates B, T, and NK cell function

IL-22 T cells (CD4+) and NK cells IL-10 homologue

IL-23 Activated dendritic cells Stimulate IFN- production and proliferation in blast T cells and activated (memory) T cells

IL-24 B cells, CD4+ (naïve) T cells,

TH2 cells, epithelium and fibroblasts, NK cells,.

Inhibition of endothelial cell differentiation and migration of endothelial cells (anti-tumor)

IL-25 Bone marrow stromal and Mast

cells Possible mediator of allergic disease (TH2 responses)

IL-26 CD4+ (mature) T cells and NK

cells IL-10 homologue

IL-27 Activated APCs Inhibits hyperactive T cells (CD4+)

Appendix XI

Interferons were first recognized for their ability to confer resistance to viral infection.

Major Cell Source Major Functions

IFN-; 24 distinct species identified

Leukocytes

Anti-viral, Anti-tumor Regulate differentiation Modulates lipid metabolism Inhibits angiogenesis Immunoregulates (monocyte/macrophage activation) Enhances MHC expression Class I: IFN- and IFN- β beta Class II: IFN- Increases cytotoxic T-cell activity, activates NK cell activity.

IFN-β Fibroblasts

IFN- T cells (TH1), macrophages (rare)

Properties of selected immune mediators, growth factors and chemokines.

Major Cell Source Major Functions

CCL2 (MCP-1) Monocytes and Macrophages,

Fibroblasts Chemoattractant for monocytes Proliferation/activation of Chemokine-activated killer cells

CCL3 (MIP-1) Monocytes, T cells, Fibrobalsts,

Mast cells Chemoattractant for neutrophilic granulocytes Stimulates TNF secretion by macrophages

CCL5 (Rantes) T cells, Endothelium

Chemoattractant for Eosinophils and Basophils, Monocytes and Dendritic cells, and T cells Increases monocyte adherence to endothelial cells Activates Basophils (degranulation)

CCL11 (Eotaxin) Monocytes and Macrophages,

Endothelium and Epithelium Chemoattractant for Eosinophils Mediator of allergic response

CXCL1 CXCL2 CXCL3

Monocytes, Fibroblasts, Epithelium

Chemoattractant for Neutrophils Activates Neutrophils (degranulation)

CXCL8 (IL-8) Monocytes and Macrophages,

Fibroblasts, Endothelial cells Chemoattractant for Neutrophils Activates Neutrophils (degranulation)

Granulocyte-Macrophage Colony Stimulating Factor

(GM-CSF)

T cells and macrophages, endothelial cells and fibroblasts

Stimulates growth of macrophages and granulocytes Stimulates differentiation: Monocytes, Neutrophils, EosinophilsStimulates release of arachidonic acid metabolites from granulocytes and increased generation of reactive oxygen species, granulocytes

Transforming Growth Factor-beta (TGF-β); 5 isoforms

Platelets, Macrophages, T cells, Endothelial cells, Keratinocytes

Growth inhibitor for Lymphocytes, epithelium, endothelium, fibroblasts, neuronal cells, hepatocytes, keratinocytes, and hematopoietic cell types. Inhibits MHC Class II expression

Tumor Necrosis Factor-alpha (TNF-); Lymphotoxin B, Cachectin

Moncytes and Macrophages Activates vascular endothelium, increases vascular permeabilityInduces fever and shock Induces acute-phase responses

Tumor Necrosis Factor-beta (TNF-β)

Tcells, Fibroblasts, Astrocytes, Endothelium and Epithelium

Cytolytic or cytostatic for tumor cells Induces reactive oxygen species from Neutrophils Critical component of wound healing

Appendix XII

Toll-like receptors and their ligands.

Appendix XIII

Type I Hypersensitivity (also called immediate hypersensitivity) is due to aberrant production and activity of IgE against normally nonpathogenic antigens (commonly called allergy). The IgE binds to mast cells via high affinity IgE receptors. Subsequent antigen exposure results in crosslinking of mast cell bound IgE with activation of mast cells that release preformed mediators (e.g. histamine, leukotrienes, etc.) and synthesize new mediators (e.g. chemotaxins, cytokines). These mediators are responsible for the signs and symptoms of allergic diseases.

Appendix XIV

Type II Hypersensitivity is due to antibody directed against cell membrane-associated antigen that results in cytolysis. The mechanism may involve complement (cytotoxic antibody) or effector lymphocytes that bind to target cell-associated antibody and effect cytolysis via a complement independent pathway (Antibody Dependent Cellular Cytotoxicity, ADCC). Cytotoxic antibodies mediate many immunologically-based hemolytic anemias while ADCC may be involved in the pathophysiology of certain virus-induced immunological diseases.

Coico, Sunshine, Benjamini, 2003. Fig. 15.11

Appendix XV

Type III Hypersensitivity results from soluble antigen-antibody immune complexes that activate complement. The antigens may be self or foreign (e.g. microbial). Such complexes are deposited on membrane surfaces of various organs (e.g. kidney, lung, synovium, etc). The byproducts of complement activation (C3a, C5a) are chemotaxins for acute inflammatory cells. These result in the inflammatory injury seen in diseases such as rheumatoid arthritis, systemic lupus erythematosus, postinfectious arthritis, etc).

Appendix XVI

Type IV Hypersensitivity (also called Delayed Type Hypersensitivity, DTH) involves macrophage-T cell-antigen interactions that cause activation, cytokine secretion and potential granuloma formation. Diseases such as tuberculosis, leprosy and sarcoidosis as well as contact dermatitis are all clinical examples where the tissue injury is primarily due to the vigorous immune response rather than the inciting pathogen itself.

Appendix XVII

Type IV Hypersensitivity (continued)

Figure 16.1. DTH reaction. (A) Stage of sensitization by antigen involves presentation of antigen to T cells by APC, leading to the differentiation of TH0 T cells to TH1 and TH17 cells. (B) Challenge with antigen (the elicitation stage) involves antigen presentation to TH1cells by APC, leading to TH1 and TH17 activation, release of cytokines, and recruitment and activation of macrophages.

Appendix XVIII

Table 8-1. Autoimmunity and Disease Autoimmune Disease Mechanism PathologyAutoimmune hemolytic anemia Autoantibodies to RBC antigens Lysis of RBCs and anemia

Autoimmune thrombocytopenia purpura

Autoantibodies to platelet integrin Bleeding, abnormal platelet function

Myasthenia gravis Autoantibodies to acetylcholine receptor in neuromuscular junction Blockage of neuromuscular junction transmission and muscle weakness

Graves' disease Autoantibodies to receptor for thyroid- stimulating hormone (TSH) Stimulation of increased release of thyroid hormone (hyperthyroidism)

Hashimoto's thyroiditis Autoantibodies and autoreactive T cells to thyroglobulin and thyroid microsomal antigens

Destruction of thyroid gland (hypothyroidism)

Type I diabetes (insulin-dependent diabetes mellitus; IDDM)

Autoantibodies and autoreactive T cells to pancreatic islet cells

Destruction of islet cells and failure of insulin production

Goodpasture's syndrome Autoantibodies to type IV collagen Glomerulonephritis

Rheumatic fever Autoantibodies to cardiac myosin (cross-reactive to streptococcal cell wall component)

Myocarditis

Pemphigus vulgaris Autoantibodies to epidermal components (cadherin, desmoglein) Acantholytic dermatosis, skin blistering

Multiple sclerosis T-cell response against myelin basic protein Demyelination, marked by patches of hardened tissue in the brain or the spinal cord; partial or complete paralysis and jerking muscle tremor

Systemic lupus erythematosus (SLE)

Circulating immunocomplexes deposited in skin, kidneys, etc, formed by autoantibodies to nuclear antigens (antinuclear antibodies, or ANA), including anti-DNA

Glomerulitis, arthritis, vasculitis, skin rash

Rheumatoid arthritis Autoantibodies to IgG (rheumatoid factors); deposition of immunocomplexes in synovium of joints and elsewhere; infiltrating autoreactive T cells in synovium

Joint inflammation, destruction of cartilage and bone

AUTOIMMUNE DISEASE CLINCAL PHENOTYPE

Systemic Lupus Erythematosus Rash; inflammation of joints and serosal linings; glomerulonephritis; hemolytic anemia, systemic symptoms

Rheumatoid Arthritis Inflammation of synovium of diarthroidal joints, systemic inflammation

Scleroderma Inflammation, dermal fibrosis, internal organ fibrosis, vasculopathy

Ankylosing Spondylitis Inflammation of spine, joints, and tendon insertions; uveitis

Multiple Sclerosis Demyelination, optic neuritis, neurological deficits

Myasthenia Gravis Skeletal muscle weakness, diplopia, dysarthria, dysphagia Hashimoto’s Thyroiditis Hypothyroidism

Graves Disease Hyperthyroidism, opthalmopathy

Celiac Disease Diarrhea and malabsoprtion

Autoimmune hemolytic anemia Anemia through lysis of red blood cells

Type I diabetes Failure of insulin production and glycemic control

Sjorgren’s Syndrome Disorder of the moisture-producing glands

Appendix XIX

Figure 8-5 Primary immunodeficiencies. Manifestation of immunodeficiency is dependent upon the etiology of response. B-cell deficiency is marked by recurrent infections with encapsulated bacteria. T-cell deficiency manifests as recurrent viral, fungal, or

protozoal infections. Phagocytic deficiency with associated inability to engulf and destroy pathogens usually appears with recurrent bacterial infections. Complement disorders demonstrate defects in activation patterns of the classical, alternative, and/or lectin-binding

pathways, which augment adaptive host defense mechanisms.

Appendix XX

Tissue Rejection and Host Response to Transplantation.

Appendix XXI

Mechanisms of Tolerance.

Appendix XXII

Interventions to Boost Anti-Cancer Immune Function.

Figure 10.2. Introductory Immunology: Basic Concepts for Interdisciplinary Applications. Elsevier/Academic Press. Actor, JK. 2014.

Appendix XXIII

http://www.cdc.gov/vaccines/schedules/downloads/child/0-18yrs-child-combined-schedule.pdf