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Studying the Adaptive Immune Response: Tools for T and B Cell ResearchMiranda Hanson-Baseler, PhD

Miranda.Hanson-Baseler@qiagen.com

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A four-part webinar series on host responses

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Exploring the first line of defense: research tools for the innate immune system

Toll-like receptors in inflammation

Studying the adaptive immune response: tools for T and B cell research

The crosstalk between cancer inflammation and immunity: exploring cancer immune responses

Explore host responses and defense mechanisms:

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Legal disclaimer

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QIAGEN products shown here are intended for molecular biology

applications. These products are not intended for the diagnosis,

prevention or treatment of a disease.

For up-to-date licensing information and product-specific

disclaimers, see the respective QIAGEN kit handbook or user

manual. QIAGEN kit handbooks and user manuals are available at

www.QIAGEN.com or can be requested from QIAGEN Technical

Services or your local distributor.

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Agenda

Overview of adaptive immunity

• T cells

• B cells

• Antigens and antibodies

Solutions provided by QIAGEN

• Gene expression and regulation

• Signal transduction

• Protein

• Single cells

Questions

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Agenda

Overview of adaptive immunity

• T cells

• B cells

• Antigens and antibodies

Solutions provided by QIAGEN

• Gene expression and regulation

• Signal transduction

• Protein

• Single cells

Questions

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2

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Innate vs adaptive immunity

• Nonspecific• PAMPs/DAMPs• Rapid induction (hours)• Short-lived

• Specific• Antigenic peptides• Slow induction (days)• Memory

Innate immunity Adaptive immunity

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Why is adaptive immunity necessary?

Shortcomings of innate immunity:

• Non-specific◦ Similar pattern of response to all pathogens

• Poorly regulated◦ Control mechanisms are poor or lacking

• Poor amplification◦ Response magnitude same for all insults

• Lack of self discrimination◦ Harm to self due to lack of specificity

• Short duration

• No memory

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Three important features of adaptive immunity

Specificity: An antibody or reactive T cell will react specifically with the antigen that induced its formation – it will not react with other antigens

Memory: Once an immunological response has produced a specific type of antibody or reactive T cell, it is capable of producing more of the antibody or activated T cells rapidly and in larger amounts

Tolerance: Immunological systems are able to distinguish between “self” and “non-self” antigens

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Components of the adaptive immune system

Lymphocytes• T cells• B cells

Antibodies

Antigens

Antigen presenting cells• Dendritic cells (DCs)• Macrophages• B cells

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The two arms of adaptive immunity

• Antibodies – produced by lymphocytes

• Bind temporarily to target cells

◦ Temporarily inactivate

◦ Mark for destruction by phagocytes or complement

• Has extracellular targets

• Lymphocytes act against target cells◦ Directly by killing infected cells

◦ Indirectly by releasing chemicals that enhance inflammatory response or by activating other lymphocytes or macrophages

• Has cellular targets

Cellular immunity Humoral immunity

Adaptive immunity

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Antigens

• The term antigen is derived from two words: antibody generator

• Most antigens are proteins or large polysaccharides

• Both arms of the immune system can recognize antigens

• Not all antigens trigger an immune response; only those considered foreign do so (virus, bacteria, fungus, protozoa, parasitic worms)

• An antigen is not directly recognized as foreign; antigenic determinants are recognized by cells/antibodies◦ When an antigen elicits an immune response, it is referred to as an immunogen◦ Epitope: the reactive portion of an antigen that reacts chemically with an antibody

to form an antigen-antibody complex

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MHC molecules: the basics

Major histocompatibility complex• Body cell surface proteins coded by a family of highly polymorphic

genes• MHC class I: found on all nucleated cells• MHC class II: found only on antigen presenting cells (APCs)

T cell receptors recognize antigenic peptide/MHC complexes• CD4+ T cells: restricted by class II• CD8+ T cells: restricted by class I

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Antigen presenting cells (APCs)

Antigen presenting cells • Are a heterogeneous group of immune cells (classical APCs include

dendritic cells, macrophages and B cells)

• Mediate cellular immune responses by processing and presenting antigens for recognition by certain lymphocytes such as T cells

• Express high levels of MHC I and II molecules

• Express co-stimulatory molecules

• Trap and capture antigen in the periphery

• Process antigens into peptides

• Store antigens

• Transport antigens to peripheral lymphoid tissues

• Present antigenic peptides to T cells

• Co-stimulate T cells

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Overview of T cells and B cells

T cells B cells

Type of immune response Cellular Humoral

Antibody secretion No Yes

Primary targets Intracellular pathogens (e.g. virus-infected cells) and cancer cells

Extracellular pathogens (e.g., bacteria, fungi, parasites)

Site of origin Bone marrow Bone marrow

Site of maturation Thymus Bone marrow

Effector cellsHelper T (Th) cells (CD4+)Regulatory T (Treg) cellsCytotoxic T (Tc) cells (CD8+)

Plasma cells

Memory cell formation Yes Yes

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Types of T cells

Conventional Non-conventional

Uses ab TCR Uses gd TCR

Helper or regulatory (CD4+) and cytotoxic (CD8+)

More abundant and highly specific Primitive with broad specificity

Restricted by classical MHC (I and II) molecules Restricted by non-classical molecules

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T cell receptors and the CD3 complex

A TCR includes:• An alpha and beta chain heterodimer• Two CD3-Epsilon chains• A CD3-Gamma chain• A CD3-Delta chain • A zeta chain homodimer

This complex mediates antigen recognition, which ultimately results in T cell activation.

Activation requires 2 signals:• Signal 1: specific recognition of antigen

(peptide-MHC complex) via antigen receptor• Signal 2: costimulatory signals from APC• Signal 1 alone leads to unresponsiveness

◦ Anergy, deletion, apoptosis

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Roles of helper T cells (Th)

Th cells play a central role in the adaptive immune response:

• 70% of T cells in periphery are Th cells

• Activate both humoral and cellular arms

• Once primed by antigen from APCs, Th cells:

◦ Help activate T and B cells

◦ Induce T and B cell proliferation

◦ Release cytokines that recruit other immune cells

• Without Th cells, there would be no immune response

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Th1 and Th2

CD4+ T helper cells can be classified into two types based on their cytokine profiles:

• T helper cell type 1 (Th1)

• T helper cell type 2 (Th2)

Cytokine profile is influenced by several factors:

• Nature and dose of antigen

• Route of infection

• Initial cytokine environment

• Type of antigen presenting cell/co-stimulation

• Genetic background

CD4+ T cell

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Differences between Th1 and Th2 cells

Th1 cells Th2 cells

Produces type 1 cytokines (IL-2, IFN-g, TNF-a, TNF-b)

Produces type 2 cytokines(IL-4, IL-5, IL-10, IL-13)

Activates macrophages and DCs to kill intracellular pathogens

Provides help to B cells in antibody response

Mediates CMI Mediates allergy and immunity to extracellular pathogens, including parasites

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Th17 cells

• Th17 cells produce and secrete IL-17

• Lineage differentiation of Th17 cells is specified by the ROR gamma t transcription factor

• They play a role in host defense against extracellular pathogens

• There is significant evidence that aberrant regulation of Th17 cells is involved in the pathogenesis of multiple inflammatory and autoimmune disorders

• Polarization promoted by IL-1 and IL-23; inhibited by IL-4 and IFN-g

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Regulatory T cells (Tregs)

• Are needed to keep the immune system in check◦ Help avoid immune-mediated pathology ◦ Prevent the unrestricted expansion of effector T cell populations

• Can be classified into natural or adaptive (induced) Tregs◦ Natural Tregs are CD4+CD25+ T cells, which develop and emigrate from the thymus

to perform their key role in immune homeostasis◦ Adaptive Tregs are non-regulatory CD4+ T cells, which acquire CD25 expression

outside of the thymus, and are typically induced by inflammation and disease processes, such as autoimmunity and cancer

• Express the transcription factor FOXP3 and develop in the thymus or can be induced in peripheral sites, including mucosa-associated lymphoid tissue

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Cytotoxic T cells (Tc)

CD8+ T cell

• Directly attack and kill other cells by:◦ Releasing perforins and granzymes by exocytosis◦ Binding specific membrane receptors on target cells and

stimulating apoptosis

• Comprise 30% of T cells in the periphery

• When activated, circulate in blood, lymph and lymphoid organs in search of body cells displaying antigens they recognize

• Target:◦ Virus-infected cells◦ Cells with intracellular bacteria or parasites◦ Cancer cells◦ Foreign cells (from transfusions or transplants)

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Humoral immunity

• Humoral immunity is mediated by B cells

• B cells can produce 5 classes of antibodies◦ IgA, IgD, IgE, IgG and IgM◦ Basic structure is a Y-shaped molecule with 2 functional parts:

− Fab region (binds to the antigen) − Fc region (notifies the rest of the immune system)

• Antibody production may/may not depend on T cells◦ Stimulated by T-dependent antigens (help from Th cells)◦ APCs with class II MHC proteins process and present antigen to CD4+ T cells ◦ Helper T cell become activated◦ Activated T cell secretes cytokines that in turn activate B cell◦ B cell differentiates into effector and memory (plasma) cells and produce antibodies

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Classes of antibodies

B cells can switch antibody classes but retain antigen specificity• IgM at first; then IgG• Almost all secondary responses are IgG

IgM• Pentamer• First antibody

released • Readily fixes

and activates complement

IgA• Monomer or

dimer• In mucus or

other secretions

• Helps prevent pathogen entry

IgD• Monomer• Attached to

surface of B cells

• Functions as B cell receptor

IgG• Monomer• 75-85% of

antibodies in plasma (most abundant)

• From secondary and late primary responses

IgE• Pentamer• Active in some

allergies and parasitic infections

• Causes mast cells and basophils to release histamines

Antibodies

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Functions of antibodies in host defense

Antibodies defend the body against pathogenic microbes through several means:• Neutralization

◦ Toxin-neutralization antibodies react with a soluble bacterial toxin ◦ Subsequently, they block the interaction of the toxin with its specific target cell or substrate

• Immobilization and prevention of adherence◦ Antibodies combine with the surfaces of microorganisms and may block or prevent their attachment

to susceptible cells or mucosal surfaces ◦ Antibodies against a viral component can block attachment of the virus to susceptible host cells

• Agglutination and precipitation◦ Antibodies combine with the surfaces of microorganisms or soluble antigens and cause them to

agglutinate or precipitate. ◦ This reduces the number of separate infectious units and makes them more readily phagocytosed

because the clumps of particles are larger in size• Opsonization

◦ Antibodies enhance phagocytic engulfment of microbial antigens◦ IgG and IgM antibodies have a combining site for antigens as well as a site for cytophilic

association with phagocytes◦ Bacteria and viral particles are ingested with increased efficiency

• Complement activation◦ Antibodies combine with the surface antigens of microbes to activate the complement cascade

• Antibody-dependent cellular cytotoxicity (ADCC)◦ IgG antibodies can enable NK cells to recognize and kill opsonized target cells

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B cell activation

Antigenic structure determines the rules

T cell dependent antigen: • Signal 1: specific recognition of native antigen via

receptor◦ Antigen processed into peptides and presented

to helper T cells via MHC class II• Signal 2: costimulatory signals from helper T cell

◦ Results in affinity maturation and isotype switching

T cell independent antigen:• Repetitive epitopes • Signal 1: specific recognition of native antigen via

receptor• Cumulative binding energy is enough to trigger

activation • Signal 2 is not required• Poor inducer of affinity maturation and isotype

switching

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Summary

The adaptive immune response:

• Uses lymphocytes, APCs and specific molecules to identify and destroy non-self substances, such as pathogens

• Depends upon ability of immune cells to:

◦ Recognize antigens by binding to them

◦ Communicate with one another so that whole system mounts a specific response

• Has three main features:

◦ Memory

◦ Specificity

◦ Tolerance

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Agenda

Overview of adaptive immunity

• T cells

• B cells

• Antigens and antibodies

Solutions provided by QIAGEN

• Gene expression and regulation

• Signal transduction

• Protein

• Single cells

Questions

1

2

3

Sample to Insight

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Agenda

Overview of adaptive immunity

• T cells

• B cells

• Antigens and antibodies

Solutions provided by QIAGEN

• Gene expression and regulation

• Signal transduction

• Protein

• Single cells

Questions

1

2

3

Sample to Insight

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Tools for your T cell and B cell research needs

Gene expression with qPCR• mRNA• lncRNA• miRNA

Cell-signaling with reporter assays

Protein expression with ELISArrays

Single cell analysis

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Gene expression with qPCR: mRNA and lncRNA

RT2 Profiler Arrays• 84 of the most relevant genes in biological and disease pathways• Gene lists identified through state-of-the-art bioinformatics and text-mining tools• Integrated controls for genomic DNA contamination, normalization and PCR processes• Web-based data analysis software at no additional cost• Compatible with most real-time PCR instruments

T and B cell-related pathways:• T Helper Cell Differentiation• TH17 Response • TH1 and TH2 Responses• T Cell Anergy and Immune Tolerance• T cell and B cell Activation

Species: human, mouse and rat

cDNA conversion RNA QC Data

analysisData

interpretationAssays

and arrays

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Application data

Human PBMCs • Were treated with PMA and ionomycin• Analyzed using the Common Cytokines RT2 Profiler PCR Array

This volcano plot • Shows both fold-change and the statistical significance• Demonstrates that, in response to treatment

◦ 23 genes, including IL-10, IFN-gamma, IL-2 and TNF were upregulated ◦ 6 genes, including IL-1beta, were downregulated

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Experimental question: which cytokines alter expression after PMA-ionomycin treatment?

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Gene expression with qPCR: miRNA

miScript miRNA PCR Arrays• microRNAs (miRNAs) are naturally-occurring noncoding RNAs – around 22 nucleotides

long – which mediate post-transcriptional gene regulation• The content of each array is selected using proprietary methodology, which ensures that

every array is up-to-date and biologically relevant• miRNA PCR arrays available for pathway, disease or miRNome profiling• Compatible with most real-time PCR instruments

T and B cell-related pathways:• T cell and B Cell Activation

Other immunology-related arrays: • Inflammatory Response and Autoimmunity• Immunopathology

Species: human, mouse and rat

miRNA isolation

Functional testing

Data analysis

qPCR detection

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miRNA gene list

T cell differentiation

• Double negative (CD4-/CD8-)

• Double positive (CD4+/CD8+)

• CD4+ naïve

• CD8+ naïve

• CD8+ effector

• CD8+ memory

Differentially expressed in Tregs

T cell activation

B cell differentiation

• Naïve

• Germinal center

• Memory

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Total RNA Discovery Workflow

miScript and RT2 systems can be used to simultaneously quantify mRNA, lncRNA and miRNA in a simple, complete workflow

Why use QIAGEN PCR Arrays for Total RNA Discovery ?

Content, Controls and Custom

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Cignal Reporter Assays & Arrays

Functionally verified assays for 45 pathways:• Type I IFN• IFN-g• NFkB• MAPK• PI3K/AKT• STAT3• TGF-b• And more…

Cignal Finder 10-Pathway Arrays:• Immune Signaling• Cancer• Development• Stem Cell & Differentiation• Nuclear Receptors• Stress & Toxicity

Cignal 45-Pathway Array

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Multi-Analyte ELISArrays

• Th1/Th2/Th17 cytokines (mouse, human)◦IL2, IL4, IL5, IL6, IL10, IL12, IL13, IL17A, IFNg, TNFa, G-CSF,

TGFb1

• Single analyte in T and B cells (mouse, human)

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Application data

Experimental question: are Th1 or Th2 cytokines being produced?

• Time-dependent (0, 6, 18, 24, and 48 h) patterns of Th1/Th2 cytokine induction by human PBMCs in response to PMA (50 µg/ml) and ionomycin (1 µg/ml) were monitored

• The relative amount of each cytokine was profiled simultaneously using the ELISArray Kit

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Single cell analysis enables new insights

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CTC - Circulating tumor cells; PGD - Pre-implantation genetic diagnosis

Cellular heterogeneity

Detection and analysis of rare cells (example: CTC from liquid biopsy)

Identification of cell subpopulations based on genomic structure or gene expression (tumors, tissues, immune cells, cell cultures)

Limited availability of

cellsAnalysis of limited sample material (example: embryo biopsy for PGD, fine-needle aspirates)

ApplicationReason

Biological insights instead of average results

No Data

Bulk result Single-cell data

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Single Cell Multiple Cells

TissueBloodgDNA RNA

Single-cell DNA Sequencing

Single-cell RNA sequencing

REPLI-g Single Cell DNA

Library Kit

REPLI-g Single Cell RNA

Library Kit

NGSLibrary

NGS

Single-cell DNA analysis

Single-cell RNA analysis

Comparative analysis of DNA and RNA

(>25 cells)

REPLI-g Single Cell

Kit

REPLI-g WTA Single

Cell Kit

REPLI-g Cell WGA and WTA

Kit

Amplified WTA-DNA or WGA-DNA

NGS

Microarray

qPCR

Choose a REPLI-g Single Cell Kit for your application

Starting material Application Q solution Kit output Analysis

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Array and PCR-based analysis

REPLI-g Single Cell Kits are the starting point to achieve superior results in various applications:

Immune cells• REPLI-g Single

Cell Kit• REPLI-g Single

Cell WTA Kit

WGA or WTA

qPCR or qRT-PCR

based analysis

QIAGEN's qPCR/qRT-PCR portfolio (enzyme kits and arrays)

MicroarraysCGH arraysSNP arrays

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Summary

• The adaptive immune system comprises a complex network of cellular and molecular components

• Research tools that allow simultaneous analysis of many immunity-related players at once are an effective way to characterize adaptive responses to microbes

• RT2 Profiler PCR Arrays profile expression of 84 or 370 genes simultaneously, and are available for over 170 pathways. Many of these are related to adaptive immunity and host defense, and custom arrays are also available.

• miScript miRNA PCR Arrays enable pathway, disease or miRNome profiling

• Cignal Finder Reporter Arrays (10-Pathway and 45-Pathway) permit simultaneous cell-based reporter analysis of several signaling pathways through DNA-based or lentiviral vectors, using either GFP or luciferase.

• ELISArrays are multiplex cytokine or chemokine analysis assays using a traditional ELISA format. Custom Mix-n-Match Multi-Analyte ELISArray Kits also available.

• REPLI-g Single Cell analysis tools enable study of cellular heterogeneity during immune or stem cell development

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How can you learn more about these research tools?

Assays and arrays:

• GeneGlobe portal◦ Browse by research◦ Biology stores◦ Complementary data analysis tool

Single cell analysis:

• Single cell resource center

• Single cell webinars

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GeneGlobe: your new research companion!

www.qiagen.com/geneglobe

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Browse by research areas: immunology

eBiology Stores

• Cytokines & Chemokines

• Dendritic & Antigen Presenting Cell

• Inflammatory Response & Autoimmunity

• Innate & Adaptive Immune Responses

• NFkB Signaling

• Toll-like Receptor Signaling

• And more!

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Single cell resource center

www.qiagen.com/singlecellanalysis

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Agenda

Overview of adaptive immunity

• T cells

• B cells

• Antigens and antibodies

Solutions provided by QIAGEN

• Gene expression and regulation

• Signal transduction

• Protein

• Single cells

Questions

1

2

3

Sample to Insight

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Agenda

Overview of adaptive immunity

• T cells

• B cells

• Antigens and antibodies

Solutions provided by QIAGEN

• Gene expression and regulation

• Signal transduction

• Protein

• Single cells

Questions

1

2

3

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Thank you for attending

Thank you for attending today’s webinar!

Contact QIAGENCall: 1-800-426-8157

Email: BRCsupport@QIAGEN.com

Miranda Hanson-Baseler, PhDMiranda.Hanson-Baseler@QIAGEN.comQIAwebinars@QIAGEN.com

Questions?

Sample to Insight

A four-part webinar series on host responses

50

Exploring the first line of defense: research tools for the innate immune system

Toll-like receptors in inflammation

Studying the adaptive immune response: tools for T and B cell research

The crosstalk between cancer inflammation and immunity: exploring cancer immune responses

Explore host responses and defense mechanisms:

3

2

1

4