adaptive immunology overview w. robert fleischmann, ph.d. department of urologic surgery university...

Adaptive Immunology Overview

W. Robert Fleischmann, Ph.D.

Department of Urologic Surgery

University of Minnesota Medical School

rfleisch@umn.edu

(612) 626-5034

Objectives

• Provide an overview of immunological principles

• Provide a framework for future lectures

• Introduce immunological terminology

At age 3 months, Andrew Anderson begins to suffer from a series of bacterial infections that never seem to quite resolve with antibiotic treatment. Consequently, he has had nearly continual diarrheal and respiratory infections. As a result, he has not gained much weight since birth. At age 4 months, his parents bring him to the clinic with a white growth on the mucosal surface of his mouth.

What do you think is wrong with Andy?What tests might you wish to request?

Andy AndersonTotal WBC count: 4,800/µl (4,100-10,900/µl)

Differential count:

Neutrophils 79% (35-80%)

Lymphocytes 10% (20-50%)

Macrophages 10% (2-12%)

Eosinophils 1% (0-7%)

Basophils 0% (0-2%)

Immunoglobubin Levels:

IgM 10 mg/100 ml (25-60 mg/100 ml)

IgG 100 mg/100 ml (275-600 mg/100 ml)

IgA 3 mg/100 ml (10-45 mg/100 ml)

Adaptive Immunity Differs From Innate Immunity In Important Ways

• Innate Immunity– Pre-existing defenses that are non-specific– Pre-existing defenses that do not change

with repeated exposure

• Adaptive Immunity– Reactive defenses that are specific– Reactive defenses that have memory and

can give a greater level of 2° response

Cells of the Adaptive Immune System

• B and T Lymphocytes

• Monocytes/Macrophages/Dendritic Cells

• Basophils and Mast Cells

• Eosinophils

Lymphoid Organs

• B cells and T cells are produced in the bone marrow.

• B cells are released from the bone marrow as mature cells, while T cells must pass through the thymus to become mature cells.

• Mature B cells and T cells can be in the blood or resident in the lymph nodes and spleen.– The spleen filters the blood– The lymph nodes filter the lymph

• Mature B cells and T cells can travel from one lymph node to another and to and from the spleen (trafficking or homing).

There Are Two Arms To Adaptive Immunity

• Cell-Mediated Immunity

• Humoral Immunity

Cytotoxic T cells

Antibodies

Adaptive Immunity - Theory

• Specific response for each antigen– Can recognize 107 to 109 different antigenic

sequences– Only a few T and B lymphocytes recognize any

given antigenic sequence

• Must be induced – Requires 7-10 days for activation because rare B

and T lymphocytes with identical antigen recognition sequences must find each other

• Generates memory – Takes 1-5 days additional days for development of

memory

Adaptive Immunity - Overview

• Antigen is phagocytosed and processed by professional antigen-presenting cells, such as macrophages, dendritic cells, and B cells.

• An epitope of the antigen is bound to an MHC class II molecule and presented to the helper T cell.

• The helper T cell produces cytokines and stimulates the activation of cytotoxic T cells and B cells.

Functions of the Thymus• Immature T cells migrate from the bone marrow to

the thymus.• The T cells mature to become either CD4+ helper T

cells or CD8+ cytotoxic T cells.• During the maturation process selection occurs.

– Positive selection: T cells must recognize MHC class I or MHC class II molecules in order to be stimulated to mature (self-restricted).

– Negative selection: T cells that recognize self-antigens bound to MHC class I or MHC class II on thymus epithelial cells are driven to apoptose (tolerant to self-antigens.

• Mature T cells that are self-MHC restricted and tolerant to self-antigens leave the thymus to settle in lymph nodes or the spleen.

Functions of the Cells Involved in Adaptive Immunity

Antigen Presentation

• The first step in responding to an antigen is to be able to “see” it.

• Macrophages and dendritic cells (also B cells) are professional antigen-presenting cells. – Macrophages are found in the tissues and lymph

nodes.• Monocytes are undifferentiated macrophages that are

found in the blood.

– Dendritic cells are found in the tissues and lymph nodes.

• Macrophages and dendritic cells produce important cytokines and lymphokines that activate T and B cells.

Antigen Presentation

• Antigens are presented as peptides (epitopes) bound to antigen-presenting molecules.

• In the mouse, the antigen-presenting molecules are called major histocompatibility antigens or MHC.

• In man, the antigen-presenting molecules are called human leukocyte antigens or HLA.

What Is an Epitope?

• Epitopes are regions on an antigen that can be recognized by an antibody or by T cell receptor.

• Epitopes are also called antigenic determinants.

• In the picture, epitopes on a whole poliovirus virion and on the isolated poliovirus VP1 protein are shown in white.

Two Types of HLA Molecules Are Used for Antigen Presentation

• HLA Class I Antigen Presentation– Antigens that are synthesized within a cell– Self-antigens or antigens from cell infection– Recognized by CD8+ cytotoxic T cells

• HLA Class II Antigen Presentation– Antigens that are products of phagocytosis

are expressed on HLA Class II antigens– Recognized by CD4+ helper T cells

Antigen Presentation

What Cells Express HLA?

• HLA Class I– All cells except RBCs– Lack of expression on RBCs may play a role in

persistence of malarial parasite (Plasmodium)

• HLA Class II– Monocytes/macrophages– Dendritic cells– B cells– Epithelial cells of thymus

Class I versus Class II HLA

• Class I (one unique chain plus common chain [2-microglobulin])– Expresses epitopes of antigens (8-10 aa) that have been digested

in the endoplasmic reticulum (endogenous antigen); 8-10 aa bound to 1 and 2 domains

– Recognized by cytotoxic T cells– Important for killing virus-infected cells and for tumor surveillance– Mediates transplant rejection

• Class II (two unique chains: chain plus chain)– Expresses epitopes of antigens (12-28 aa) that have been

digested in phagolysosomes (exogenous antigen); 12-28 aa bound to 1 and 1 domains

– Recognized by helper T cells to trigger adaptive immunity

Characteristics of T Cells

• All T cells express CD3.• All T cells express a T cell receptor that

recognizes their cognate antigen epitope.• T cell receptors are created through DNA

rearrangement; 1010 to 1012 paratopes.• There are two main subtypes of T cells.

– CD4+ Helper T cell– CD8+ Cytotoxic T cell

T Lymphocytes

• T lymphocytes (T cells)– One subset expresses CD4 on their surface

• CD4 = for something = help• CD4+ helper T cells play the central role in

adaptive immunity as activators of both cell-mediated and humoral immunity

– One subset expresses CD8 • CD8 = ate• CD8+ cytotoxic T cells are effectors of cell-

mediated immunity that are HLA-restricted (see antigen in context of HLA)

Activation of Helper T Cells

• Signal 1– Requires T cell receptor recognition of HLA bound

antigen– Requires CD4+ binding to Class II or CD8+ binding to

Class I

• Signal 2– B7-1 or B7-2 on the antigen presenting cells binds to T

cell surface protein CD28

• Other co-stimulation molecules– CD2 binding to leukocyte functional antigen-3 (LFA-3)– Intercellular adhesion molecule (ICAM) binding to LFA-1

• Cytokine signals

Cytokine Signals That Activate Helper T Cells

• IL-2 and IL-15 are general activators of T cells.

• IL-12 and IFN- drive T helper cells to Th1 helper cell subtype.

• IL-4 drives T helper cells to Th2 helper cell subtype.

• IL-10 down-regulates Th1 and TGF- down-regulate Th1 and Th2 subtypes.

T Cell: APC Contacts

Helper T Lymphocytes

• CD4+ Helper T cells mature in the thymus.• CD4+ Helper T cells are activated by

macrophage-produced or dendritic cell-produced cytokines.

• Differential cytokine production by macrophages induces different types of helper T cells.– Th1 cells are induced by exposure of naïve CD4+

T cells to IL-1 + IL-12. – Th2 cells are induced by exposure of naïve CD4+

T cells to IL-1.

Th1 Helper T Lymphocytes

• CD4+ Th1 Helper T cells– Activated by IL-1 + IL-12– Produce IL-2, IFN-– Activate CD8+ cytoxic T cells to divide

and differentiate– Some CD4+ Th1 helper T cells become

memory cells

Th2 Helper T Lymphocytes

• CD4+ Th2 Helper T cells– Activated by IL-1– Produce IL-4, IL-5, IL-6, IL-9, IL-10, IL-13– Activate B cells to divide and differentiate to

become plasma cells that produce antibodies– Some CD4+ Th2 helper T cells become

memory cells

Regulatory T Lymphocytes

• CD4+ Regulatory T cells (Tregs)– Produce IL-10 and TGF- – Down-regulate Th1 and Th2 cells

functions, respectively– Some CD4+ Treg cells become memory

cells

Cytotoxic T Lymphocytes

• CD8+ T cells mature in the thymus.

• CD8+ T cells recognize target cells by antigen bound on MHC class I molecules.

• CD8+ T cells kill target cells by several different mechanisms.

• Some CD8+ T cells become memory cells.

Killing by Cytotoxic T Lymphocytes

• Kill by FAS - FAS ligand interaction– T cells expressing FAS ligand bind to FAS, a protein

on the target cell, inducing caspase activation and apoptosis

• Kill by secreting toxic agents– TNF, a cytokine, binds to TNF receptor on the target

cell, inducing caspase activation and apoptosis – Perforin, a pore-forming protein, related to

complement C9 is inserted into the target cell membrane causing lysis

– Granzymes, serine esterases that activate apoptosis in a manner similar to that of caspases, are passed to target cells

Complement Pores Versus Perforin Pores

B Lymphocytes

• B lymphocytes (B cells)– Express CD19, CD20, and CD22 on their surface– Differentiate to plasma cells that produce

antibodies– Some B cells become memory B cells

B Lymphocytes

• Rearrange their DNA as they mature in bone marrow– Each B cell has a unique VDJ rearrangement (idiotype)– Antibody repertoire permits recognition of “all” antigens

• When activated, divide and differentiate to become plasma cells– Undergo class switching from IgM or IgD by

recombination• Change from IgM or IgD isotype production to IgG, IgE, or IgA

production

– Express increased affinity for antigen (affinity maturation)• Undergo somatic cell mutations as they differentiate• Undergo sloppy recombination during isotype switching

Antibody Structure

Antibody Rearrangement

Terms Related to Antibodies• Epitope: part of antigen that binds to antibody• Paratope: part of antibody that binds antigen• Idiotype: unique antigen-binding site on each

antibody; antibodies with same antigen binding sites have the same idiotype

• Isotypes: antibodies with different types of Fc regions (i.e., IgM, IgD, IgG, IgE, IgA)

• Allotypes: antibodies from different individuals that have slightly different Fc regions (i.e., different IgM)

B Cell Proliferation

• Each B cell expresses specific antibody on its surface that recognizes a specific antigen.

• B cells are triggered to proliferate and differentiate to plasma cells by exposure to that specific antigen.

• Triggering this proliferation also requires help by exposure to CD4+ Th2 cytokines.

• Plasma cells then secrete this specific antibody.• For memory B cells, exposure to antibody alone is sufficient.

Types of Antibodies

IgD: Membrane-associated Ig that is found on mature but unactivated B cells

IgM: Pentameric Ig that is found on mature but unactivated B cells; first secreted Ig made; found in blood

IgG: Monomeric Ig that is most abundant Ig in blood and most important Ig for memory response

IgE: Monomeric Ig that is associated with mast cells and basophils; mediator of allergic reactions

IgA: Dimeric Ig that is associated with J chain and secretory component; most important secretory Ig

Stylized Structures

Of Different

Antibody Classes

Summary of Acquired Immunity

MIL-1IL-12

CD4 Th1IL-2IL-3IFN-

CD8 T cell

IL-1CD4 Th2

IL-4IL-5IL-6IL-9IL10IL-13

B cell

Cytolysis: FAS/FAS ligand TNF Granzyme B Perforin Antibody

Andy Anderson Culture of the white growth showed the presence of Candida albicans. Lymphocyte stimulation test:

Phytohemaglutinin1 4.28 134.3Concanavalin A1 1.33 48.8Pokeweed mitogen2 1.56 46.5

NK cell test: normal activity of NK cells

What do these results indicate?What treatment can be performed?1 T cell mitogen2 B cell mitogen

Andy Anderson Andy has severe combined immunodeficiency disorder. Causes of SCID:No T cells:

IL-7 -chain deficiencyCD3 -chain deficiency

No T cells or B cells:RAG1 or RAG2 deficiency (no TCR/Ab rearrangement)

No T cells or NK cells:X-linked IL-3R -chain deficiencyJAK-3 deficiencyCD45 deficiency

No T cells, B cells, or NK cells:Adenosine deaminase (ADA) deficiencyPurine nucleoside phosphorylase deficiency

Andy Anderson

Andy is found to have a RAG1 deficiency that is causing his severe combined immunodeficiency disease. Andy is given a bone marrow transplant from his father.

He recovers from the bone marrow transplant to have a reconstituted immune system.

Note: Since 2010, testing for SCID is part of the neonate genetic testing program in every state in the U.S.

Additional Topics

• Most important disorders of the adaptive immune system

• Hypersensitivities

• Tolerance

B Cell Immunodeficiencies

• Disorders of B cells– Bruton’s agammaglobulinemia (x-linked

agammaglobulinemia)– Transient hypogammaglobulinemia of

infancy– Common variable immunodeficiency

(actually defect in T cell signaling of B cell)– IgA and IgG subclass deficiencies

T Cell Immunodeficiencies

• Disorders of T cells– Severe combined immunodeficiency syndrome (SCID)

(common chain lack of IL-7 signaling causes failure to mature)

– Purine nucleoside phosphorylase deficiency (causes severe combined immunodeficiency)

– Adenosine deaminase deficiency (causes severe combined immunodeficiency)

– MHC class II deficiency– DiGeorge’s syndrome (congenital thymic aplasia)– Wiscott-Aldrich Syndrome (x-linked, few platelets,

cytotoxic T cell malfunction)– Ataxia-telangiectasia (wobbly gait, T cell deficiency)

Deficiencies of Innate Immunity• Congenital neutropenia

– Lack of GM-CSF – Frequent bacterial infections

• Glucose-6-phosphate dehydrogenase deficiency (G6PD) – Unable to produce NADPH by pentose phosphate pathway, buildup of reduced

glutathione– RBC denaturation and hemolysis

• Chronic granulomatous disease– Inability to produce hydrogen peroxide and hypochlorous acid– Inability to kill phagocytosed bacteria

• Leukocyte adhesion deficiency (LAD)– Lack of integrin subunit, the common chain– Inability to recruit innate immune cells to site of inflammation– Increased susceptibility to bacterial, fungal, and viral infections.

• Complement defects– Increased susceptibility to bacterial infections– Reduced ability to remove immunocomplexes

• Chediak-Higashi Syndrome– Defect in gene LYST (CHS1), a lysosomal trafficking gene that affects lysosomes

and melanosomes (microtubule disorder)– Increased susceptibility to bacterial infections.

Hypersensitivities

• Type I Hypersensitivity– Allergy– Mediated by IgE bound to mast cells and to

basophils causes degranulation (early phase)

– Release of histamine– Synthesis of prostaglandins and

leukotrienes– Recruitment of Th1 cells and basophils

(late phase)

Hypersensitivities

• Type II Hypersensitivity– Antibodies against cell surface and/or

extracellular matrix components– Causes complement activation – Examples

• hemolytic disease of the newborn (maternal antibodies to fetal blood group antigens cross the placenta to destroy the fetal RBCs)

• Myasthenia gravis (antibodies to acetylcholine receptors cause problems with nerve conduction)

• Goodpasture’s syndrome (antibodies to basement membranes causes nephritis)

Hypersensitivities

• Type III Hypersensitivity– Immune complex disease– Deposition of antigen:antibody complexes

• Kidney, joints, lungs, arteries, skin• Damage occurs by activation of complement

– Examples• Post-streptococcal glomerulonephritis• Autoimmunity such as Systemic Lupus

Erythrematosus

Hypersensitivities

• Type IV Hypersensitivity– Cell-mediated hypersensitivity caused by activated CD4

cells– Examples

• Contact hypersensitivity such as reaction to nickel – Dendritic Langerhans cells react recruiting CD4 cells that

ultimately mediate this hypersensitivity

• Tuberculin reaction– Macrophages react recruiting CD4 cells (2/3) and CD8 cells (1/3)

that ultimately mediate this hypersensitivity

• Granulomatous hypersensitivity– Macrophages wall off mycobacterium, undergo changes to

become epitheloid (that may form giant cells), and recruit CD4 cells.

– Crohn’s disease = granulomas containing macrophages and CD4 cells in the ileum and colon

Tolerance

• Central Tolerance– Self-reactive T cells recognize antigens presented by

thymus epithelial cells and are deleted in the thymus by negative selection

– Self-reactive B cells are deleted in the bone marrow

• Peripheral Tolerance– Not all self-antigens can be presented in the thymus– Thus, tolerance must be established in mature T and

B cells

Possible Mechanisms of Peripheral Tolerance

• Clonal exhaustion– High levels of self-antigen expression trigger a rapid

proliferative response in responding T cells that cannot be sustained in the absence of cytokines and results in apoptosis of the responding T cells (no cytokine-mediated activation of T cells)

• Clonal anergy– Non-professional antigen presenting cells or resting

(non-activated) antigen presenting cells provide only signal one but not signal two (B7 molecules), so no proliferation (no cytokine-mediated activation by APCs)

• Regulatory T cells– CD4+ T cells that also are CD25+ inhibit Th1 and Th2

cells (IL-10- and TGF--mediated inhibition)