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Immunology

Innate immunity- most primitive- cytokines, phagocytes, and

complement; barriers to infection (skin, mucous membranes,

pH, temp), inflammation (acute phase proteins, complement,

phagocytic cells)

Adaptive immunity- LYMPHOCYTES, phagocytes,

cytokines, Ab, and complement

• Active- convalescence and vaccine

• Passive- transplacental and serum therapy

• Humoral- Ab and complement

• Cellular- lymphocytes, cytokines and

phagocytes

Memory response- if exposed again much greater immune

response- controlled by lymphocytes

3 phases of Adaptive immunity

• Cognitive phase- recognition

• Activation phase- proliferation and

differentiation of lymphocytes

• Effector phase- memory, elimination of Ag

(phagocytes and complement)

Acute phase proteins- induced by stress for inflammation and

healing (C-reactive protein, serum amyloid)

Complement- activation by immune complexes for immune

regulation (over 20 proteins)

Cytokines- all cells in the body to regulate immune and other

responses (interleukins and interferons, TNF

Ab- produced by B cells and plasma cells IgM,A,G,E,D

Clonal selection- lymphocytes, but not all T and B cells

respond to the same Ag, each clone responds to only one Ag

determinant

• Clones against self are deleted or

suppressed for the most part

• Get autoimmunity if those cells are not

suppressed

• Get quicker response when re-exposed by

activation of the clonal cells

Hematopoiesis

• Stem cells- blood cells are short lived and

continuously renewed from pluripotent stem

cells found in the bone marrow;

• Memory lasts for a long time but a given

lymphocyte does not last for yrs and yrs –

progeny are generated

• Under influence of cytokines progenitor cells

become a megakaryocyte or granulocyte of

some kind

• IL-7 important in B cell development

• Megakaryocyte makes platelets

• Colony forming units can produce- rbc’s,

basophils, eosinophils, neutrophils, or

monocytes

T lymphocytes

• TCR- T cell receptor- binds Ag to T cell

o TCRa/b receptor- blood, spleen, lymph

nodes, CD4, CD8,

o TCR g/d receptor- mucosal surfaces, CD8

• Subtypes and surface markers:

o Th- CD4

o Tc- CD8

o Ts- CD4 and CD8

o NK- CD16 (no TCR)

• Surface markers- proteins functioning as

receptors/ligands

o Common to all T cells- CD3, TCR, CD2,

CD28, CD5, and CD7

• CD4 has an affinity for MHCII

• CD8 has an affinity for MHCI

B lymphocyte-

• Surface markers- surface immunoglobulin,

complement receptors, and Fc receptors

• Plasma cells- mature B cells, make bulk of Ab and

secretes them into blood (lives for about 1 week)

• Markers: HLA-D, CD22, CD19, CD20, Igb, Iga,

sIg, CD40, CR1, CR2, CD72, CD5, Fc_RII,

Mononuclear phagocytes- large cells, amoeboid, granular,

endosomes, and kidney shaped nuclei

• Involved in innate immunity (phagocytosis and

wound healing) and active immunity (Ag

presentation, regulatory cytokines)

• Markers: MHCII, Fc receptors (Fc_RI, Fc_RII,

Fc_RIII), complement receptors (CR1, CR3)

• Releases many enzymes and prostaglandins

Granulocytes- neutrophils, eosinophils, basophils,

• Blood is 70% neutrophils, 5% eosinophils, and 1%

basophils

Antigen presenting cells- express MHCII

• ALL nucleated cells express MHCI

• Only APC present MHCII

• Langerhans cells in skin, interdigitating cells in

thyroid, follicular dendritic cells in neurons, B cells

in blood, macrophages in blood

Lymphoid organs

• Primary organ- thymus, bone marrow- foster

maturation of stem cells; cells mature to T or B cells

• Secondary organ- lymph node, spleen; contain

mature cells and foster immune response

Thymus- cortex and medulla; macrophages and dendritic

cells present self-Ag to differentiating T cells leading to

positive and negative selection

• Positive selection- selecting cells that

respond to non-self

• Negative selection- selecting cells that

respond to self (at corticomedullary

junction)

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Bone marrow- contains all cells and cytokines necessary for

B cell development

Spleen- filters Ag out of blood and promotes immune

response

• No spleen- more susceptible to blood-borne

infections- esp. Strep. pneumo

• Composed of capsule and reticular fiber and cellular

framework

• Lymphocytes, macrophages, interdigitating cells,

follicular dendritic cells, and reticular cells

• Red pulp surrounds white pulp which surrounds the

central artery and contains the PALS w/ T cells and

B cell zones (T cells surround the germinal centers

of B cells)

Lymph node- filters Ag from lymph;

• Capsule, medulla, cortex; contains lymphocytes and

other cells

• Cortex- outer region B cells inner region T cells

• Medulla- plasma cells

Structure of Ag and Ab

Hapten- small moiety such as DNP that induces immunity

only when attached to carrier such as bovine serum albumin

(BSA)

• B cells recognize hapten

• T cells recognize carrier protein

Proteins are best immunogens.

Epitope- chemical group that confers Ag specificity- proteins

have more than 1 to engage a variety of clones to increase

chance of stimulation of immune response

Need an APC to present non-peptide to T cells

Do not need APC to present non-peptide to B cell

Lipopolysaccharides:

• Lipid A- endotoxin- binds to serum proteins and

ligates to CD14 which stimulates B cells

• O antigen- binds to B cell receptors- if receptor is

clone w/ affinity for O Ag

• Core polysaccharide

• B cells get several signals from a single molecule of

LPS- enough to have it develop into plasma cell-

independent of T cells

Ig Basic structure: 2 Light and 2 Heavy chains both with

variable and constant regions

Ig Properties

• 4 IgG subclasses- different heavy chains

• sIgA- secretory IgA

• IgG- most common

• 3 subclasses of IgA

Isotype- gene product that every member of a species has

Allotype- gene products that vary among individuals

Idiotype- unique gene products for each individual

H chain- 3 C regions and 1 V region

L chain- 1 C and 1 V region

Ag binds to V region- 2 binding sites (bivalent)

Fab- Ag binding region (V regions of H and L chains)

Fc- terminal piece (most glycosylation sites)

J chain- helps link 5 subunits of IgM together to form

pentamer

IgM can bind 10 Ag.

Only 1 IgM pentamer is needed to activate complement since

2 Fc receptors are close to each other.

IgA also has a J chain

Secretory piece- helps protect IgA from proteolytic digestion

in secretion

COMPLEMENT:

Complement is involved in cytolysis, Opsonization, and

inflammation, enhancement of humoral response, and

clearance

Inappropriate firing of complement system can lead to shock

like state.

Insufficiency of complement can cause more susceptibility to

bacteria and have immune complexes that are not cleared.

Classical system- starts with Ab/Ag complex

• C3b critical to both classical and alternative

pathways

• C5 splitting- entering MAC complex (C5b, C6, C7,

C8, C9)- cascading is now creating small holes in

bacterial cell walls to cause cell lysis

• See pg 69 bottom slide

• C3a, C4a, and C5a

o Smooth muscle contraction

o Increased vascular permeability

o C3a and C5a- degranulation of eosinophils,

and platelet aggregation

• When there is a classical pathways deficiency (C1q,

C1r, C1s, C4, C2) can’t have cell lysis and also have

problems clearing immune complexes leading to

autoimmunity, vasculitis, etc

• Short lasting for fine control

• Receptors : CR1, CR2, CR3, CR4

• Specific factors- regulatory factors that have very

specific actions in limiting the complement system

o CCP and DAF inhibit C3 convertase

o MCP- regulated C4b

• Missing MAC complex can’t undergo Opsonization

but can have inflammatory cells brought in

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Alternative system- no Ab needed

• C3b can start alternative system on bacterial cells

• C3b can enter the self-amplification loop

• Tickover- C3 hydrolyzed into C3a and C3b

Complement Deficiencies:

• Early components- absent

o Immune complex disease - increased

incidence

o Pyogenic infections when missing C1, C4,

C2– increased incidence-

• Late components

o Neisseria- chronic infections

• Regulatory factors missing

o C1H- most common autosomal problem

(controls C1)

HANE- hereditary angioneurotic

edema – when deficiency in C1H-

complement system always firing

leading to inflammation

• Complement receptors

o LAD- leukocyte adhesion deficiency-

inflammatory cells do not enter area since

they are missing complement receptor

Ag/Ab Interactions and Functions:

CDR- complementary determining region- has many

substitutions- diff aa sequences in CDR determine Ag

specificity

Overall affinity for Ag is higher after proliferation of Ab

Each clone has a different specificity for each Ag.

Carboxy end motifs of Ig to activate complement:

• 3 aa in 2nd

region of IgG found to activate C1q

• 3 aa in 3rd

region of IgM activates C1q

• IgE Fc_RI- can bind between 2nd

and 3rd

region for

immune complex removal

IgD- helps B cell respond to Ag (w/ IgM)

Fc receptors are involved in binding Fc regions of Ab and

leading to phagocytosis of the Ab/Ag complex, also for

degranulation and allergy response

Transport of IgA- Ab produced on abluminal surface of

mucosa and are transported to luminal surface by poly Ig

receptor

Ab-based lab testing:

Primary interaction- bivalent Ab and univalent Ag

Secondary interaction- multivalent Ag- lattice formation

Tertiary- involves secondary interaction leading to activation

of 3rd

component such as complement

Ab tests- ppt, aggultination, complement fixation, EIA, RIA,

ELISA, flow cytometry

Can’t measure Ag/Ab union kinetically- must measure at

equilibrium

Primary union- direct and indirect

Secondary union- ppt- reaches max then decreases (Ab>Ag,

Ab=Ag, Ab<Ag)

Immunodiffusion- secondary union- where lattice formation

occurs size of ring formation is proportional to [Ag]

Agglutination- secondary union- add Ab and see if

agglutination takes place (blood typing)

Tertiary union- complement fixation-

• Ag + Ab and just Ag

• Add complement to both

• Add rbc to both

• If complement still active then get lysis of rbc

Sandwich ELISA:

• Coat plate w/ Ab

• Add specimen containing Ag

• Add enzyme linked Ab

• Add substrate

• Measure color change

Flow cytometry- used to analyze cells in suspension

Generation of Ab diversity and Ig genes:

Clonal selection- individual lymphocyte express membrane

receptors specific for distinct Ag. Receptor specificity

determined prior to Ag exposure.

Somatic mutation- method by which point mutations are

elicited; responsible for affinity maturation

L chain- kappa and lambda genes- constant regions plus V

and J regions

V chain- V, D, and J regions and a number of different

constant regions

RSS- sequence that allows for cutting and recombining

sequences

RAG1 and 2- important in enhancing or making recombinase

enzymes

Class switching-

• Occurs only in heavy chains

• Changes effector function of Ig

• Cannot reverse switching

• Cytokines can control

• Does not change specificity

• Removes introns rather than exons

• Accompanied by somatic mutations in Ig

hypervariable region caused by point mutation in

DNA

• Seeing Ag again can lead to switching from IgM to

other Ig via Th cell cytokines

• Hyper IgM immunodeficiency- can’t switch from

IgM to IgG- X-linked; susceptible to infection; treat

with IV IgG

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T Cell Receptors and MHC

MHC- in humans found on short arm of chromosome 6; HLA

Class I-

• Co-dominant inheritance

• ALL nucleated cells

• 2 chains (alpha and beta) that are not covalently

linked; Ag binds to looped domains alpha1 and

alpha2. Beta chain does not bind Ag

• recognize self vs. non self (transplants,

autoimmunity)

• recognize self + foreign Ag (viral infections,

malignancies)

• CD8

Class II- found on APC- alpha and beta chains coded for by

D gene regions

• binding zone made by alpha 1 and beta 1

• recognize processed Ag in context of self

• CD4

TCR

• Near CD3

• Binds Ag and CD3 sends signal

• CD8 recognizes alpha 3 domain on class I

• CD4 recognizes alpha 2 domain of class II

T cell activation

Most Ag are monomeric

Capping of B cells leads to their activation.

IL-2 induces the release of other cytokines.

ANTIGENS ARE PROCESSED BEFORE THEY ARE

PRESENTED TO T CELLS!!!!

Class I- endogenous protein processing

Class II- exogenous protein processing

TAP- transporter necessary for moving peptides into RER for

MHC I.

Invariant chain- MHCII binding domain that prevents the

binding of endogenous peptides.

Co-stimulation reaction between B7 on APC and CD28 on T

cell that leads to T cell activation for proliferation.

Binding proteins:

• T cell- LFA-1, CD4, CD3(zeta chain), TCR, CD2,

CD28

• APC- ICAM-1, MHCII, LFA-3, B7

APC make IL-1 which causes Th cells to make IL-2 receptor

and APC cells to make more MHCII receptors.

IL-2 helps Tc cells in killing target cells- does not require co-

stimulatory event.

APC cytokines- IL-1, IL-6, TNFa, IL-12, IL-15

T cell cytokines- IFNg, GMCSF, IL-4, TNFb

Redundancy in cytokines so if one not made proper course

still occurs.

CYTOKINES:

Cytokines are soluble mediators involved in many cellular

processes.

Generalities:

• Low molecular weight

• Glycosylated protein

• Surface receptor

• Mode of action- autocrine, paracrine, endocrine

Function and Properties:

• Lots of overlap in function and tasks

• Produced in novo

• Made when contact with Ag is made

• Non-specific mediators of immunity

• Regulatory of lymphocyte activation, growth, and

differentiation

• Activators of nonspecific cells

• Stimulators of immature leukocytes

Major cytokines of inflammation: IFN, TNF, IL-1, IL-6

TNFa leads to necrosis and inflammation- why cancer pts

with bacterial infections do better.

TNF can also result in shock and death

Can make Ab to cytokines.

TNF more problematic at higher concentrations.

IL-1: Co-stimulator of inflammation and T cell function;

produced by macro, epithelial, and endothelial cells;

increases cAMP, nuclear factors, other cytokines, and

prostaglandins, can slow cells down with adhesive properties

(low concentrations)

• High concentrations- fever, APR, cachexia, but NO

TISSUE DAMAGE OR NO TUMOR NECROSIS

LPS causes increases in TNF, IL1 and IL6

Interferon- (alpha [mac] and beta [fibroblasts])- inhibits viral

replication, increases MHC class I, decreases MHCII,

activates NK cells

Chemokines- small cytokines

• Produced by leukocytes, endothelial cells, and

fibroblasts

• Stimulate neutrophils, basophils, eosinophils, and

lymphocytes

Interferon (Type II (gamma))- produced by T cells

• Function- MAF, increased class I expression,

activates T and B cells, neutrophils, NK cells etc

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IL-2

• Produced by T cells (CD4)

• Major autocrine growth factor for T cells

• Leads to synthesis of other cytokines

• Acts on B, T, and NK cells

• Can live w/out IL-2

IL-4: produced by T cells (CD4) and acts on B cells

IL-10: Produced by T cells, decreases IFNg, increases B

cells

Hematopoiesis:

• CSF- colony stimulating factor

• IL-3- early progenitor

• GM-CSF: granulocyte/monocytes colony

stimulating factor

• M-CSF, G-CSF

• Can be used by chemo pts and bone marrow

transplant patients

B CELL ACTIVATION

Major APC:

• Interdigitating dendritic cells- induces T cell

proliferation most effectively

• Macrophages- produce proliferation of T cells and

helper functions

• B cells- most effective APC when Ag conc. is low-

high affinity receptor on cell surface

IgM binds complement the best

Ab mediation is very important need B cell control.

Plasma cells are terminally differentiated. Memory B cells

have a long _ life

Cross-linking is important w/ monomeric Ag and receptors

on B cells.

C3d- degradation product of C3b, binds CR2 on B cells to

lower B cell activation threshold.

CD40- important for B cell activation and class switching.

No CD40 ligand on T cell when hyper IgM immunodeficient.

CELL MEDIATED IMMUNITY:

Innate CMI;

• Chemotaxis- activate macrophages or PMN directly

or through C5a

• Phagocytosis- enhanced by C3b deposition on

microbes and binding to complement receptors on

phagocytes.

• Acute phase cytokines- binding and uptake of

microbes by phagocytes also induces them to

synthesize and release cytokines.

Inflammatory mediators: His, 5-HT, PAF, NCF, IL-8

(chemokines), C3a, C5a, bradykinin, fibropeptides, PG, LT,

(mast cells and macrophages produce many of these)

TNF causes PMN to be more adhesive.

Macrophages release IL-12, and TNFa

NK- IFNgamma

Adhesion and Migration

• ICAM- intracellular adhesion molecule- allows cell

to move through endothelium

• CD15

• E selectin- slows movement of phagocytic cells in

blood

ADCC- Ab dependent cellular cytotoxicity- killer cell w/ Fc_

receptor (IgG) becomes active if it senses an immune

complex to kill target cell

Th0- makes IL12, IFN gamma, and IL4; proliferate into Th1

and Th2

Th1- controls classical side of immunity- activates

macrophages- secretes IFNgamma, TNFa, and IL2

Th2- controls allergy side of immunity- activates mast cells,

B cells and eosinophils (makes Ab)- produces IL4,5,6,10

MHC restricted- as T cells develop recognize self from non-

self

Perforin- in cytoplasmic granules Tc and NK cells – induces

lysis by making channels in target cell surfaces

Tc and NK lytic mechanisms- lytic granules store and release

perforin and granzymes- to form pores and induce apoptosis

(respectively)

When natural DNA repair is inhibited by TNF and granzyme

can lead to apoptosis.

Macrophages- phagocytize and present Ag to T cells and

activate immune cytokines to release more cytokines, kill

microbes and tumors

Macrophage products-

• Inflammation and fever- IL-1, IL-6, and PG

• Lymphocyte activation- processed Ag and IL-1

• Tissue rebuild- angiogenesis factor, collagenase

• Microbicidal- reactive O2 and NO, lysozyme

• Tissue damage- H2O2 and TNF

VACCINES:

Vaccination- infection w/ agent (Ag) that induces an immune

response- doesn’t protect against infection, just the disease

Passive immunity- immediate but transient; administration of

preformed Ab; can result in serum sickness

Adoptive immunity- T cells transferred into animal to impart

immunity

Active immunity- delayed but more permanent (vaccines)

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Types of Vaccines:

• Killed organism- inactivated or killed vaccine

• Ag part of disease causing organism

• Attenuated or weakened preparation

• Toxoid vaccines- toxins treated w/ or absorbed w/

aluminum salts; usually must add adjuvant to

increase immunogenicity

• Organisms similar to virulent organisms but that

does not cause human disease

• Subunit vaccines- utilizes techniques of genetic

engineering

• DNA plasmid vaccines- circular DNA plasmids

expressing specific proteins are injected w/

presentation of the protein to the immune system

Ways to give vaccines:

• Subcutaneous

• Intramuscular

• Intradermal

• Oral

Live attenuated vaccines: Polio, measles, mumps, rubella,

yellow fever, varicella zoster, hep A, TB

Killed vaccines: polio, rabies, typhoid, cholera, influenza,

plague, and pertussis

Toxin Based Vaccines: Clostridium tetani (inactivated toxin),

Corynebacterium diptheria (inactivated toxin), vibrio

cholerae (toxin B subunit), Clostridium perfringes

(inactivated toxin)

Vaccines based on subcellular microbial fragments: Nisseria

meningitis, Strep pneumoniae, H. influenzae B, hep B virus

Safety problems with vaccines:

• Attenuated vaccines- reversion to wild type, severe

disease in immunocompromised

• Killed vaccines- vaccines not killed, yeast, animal

viruses, or endotoxin contaminant

Organisms that have no vaccine: HIV, staphylococcus,

Candida, malaria, schistomlasis

Adjunvants: inorganic salts, delivery systems, bacterial

products, natural products

DNA plasmid based vaccines- live attenuated vaccines, make

Ab and cellular immunity but risk infection; DNA seen as

endogenous Ag

REGULATION AND TOLERANCE:

Regulation by antigens

• Proteins are best

• Too much or too little can lead to immune

unresponsiveness or tolerance

• Subcutaneous or intradermal induce immunity

• Adjuvant augments immunity

Th1- not susceptible to IFN gamma

CR2- augment immune response by binding to complement

receptor

Anti-Ids- produced to either non-antigen binding idiotyopes

or to Ag binding idiotyopes

Anti-Ids can either increase or decrease immune response

Changes in the alpha chains can lead to changes in the

binding cleft of MHCI and II.

Polymorphism of MHC allows us to respond to some

idiotopes and not others can respond to only a certain set of

Ag due to genetic coding

IMMUNITY TO TUMORS

Tumors- breakdown of normal growth regulatory

mechanisms; changes that occur in surface Ag of malignant

cells can sometimes be recognized by the immune system

Often a number of subclinical tumors.

Tumor specific antigens- unique to cancerous cells and are

not found on normal counterparts

Tumor associated antigens- expression is greatly increased on

tumors