The T cell receptor for MHC-associated peptide antigens

The T cell receptor for MHC-associated peptide antigens T lymphocytes have a dual specificity: they recognize polymporphic residues of self MHC molecules, and they also recognize residues of peptide antigens displayed by these MHC molecules. The receptor that recognizes these peptide-mhc complexes is T cell receptor (TCR). T cell receptor is a heterodimer consisting of two transmembrane polypeptide chains, and covalently linked to each other by disulfide bonds. The extracellular portions of both and chains contain an N-terminal variants (V) domain and a membrane-proximal constant (C) region. T cells also express other membrane receptors that do not recognize antigen but participate in responses to antigens; these are collectively called accessory molecules. 2

T cell receptors and accessory molecules. The principal T cell membrane proteins involved in antigen recognition and in responses to antigens are shown. The functions of these proteins fall into three groups: antigen recognition, signal transduction, and adhesion. 3

Antigen recognition and signaling functions of lymphocyte antigen receptors. The antigen recognition and signaling functions of antigen receptors are mediated by distinct proteins of the antigen receptor complex. When TCR or Ig molecules recognize antigens, signals are delivered to the lymphocytes by proteins associated with the antigen receptors. The antigen receptors and attached signaling proteins form the T and B cell receptor complexes. Note that single antigen receptors are shown recognizing antigens, but signaling requires the cross-linking of two or more 4 receptors by binding to adjacent antigen molecules.

Antigen and MHC molecules bind here A hybrophobic region anchors the chain in the plasma membrane 7

Structure of the T cell receptor. The schematic diagram of the ab TCR (left) shows the domains of a typical TCR specific for a peptide-mhc complex. The antigen-binding portion of the TCR is formed by the Va and Vb domains. The ribbon diagram (right) shows the 8 structure of the extracellular portion of a TCR as revealed by x-ray crystallography.

Role of the αβ TCR in MHC-restricted antigen recognition. The TCR a and b genes from a T cell clone of known specificity are expressed in a T cell tumor line. Transfection of both a and b genes is required to give the tumor line the antigen specificity and MHC 9 restriction of the original T cell clone.

Antigen-binding site is formed by CDR loops (labeled 1,2,3) from the variable regions T cell receptor 10

TCR complex The TCR heterodimer provides t cells the ability to recognize peptide antigens bound to MHC molecules. Both the cell surface expression of TCR molecules and their function in activating T cells are dependent on up to five other transmembrane proteins that noncovalently associate with the heterodimer. TCR complex Three proteins in the complex are called CD3 molecules and include highly homologous Ig superfamily members designated,, and In addition, 80 to 90 percent of TCR complexes contain a disulfide-linked homodimer of a protein called the chain. 11

Components of the TCR complex. The TCR complex of MHC-restricted T cells consists of the αβ TCR noncovalently linked to the CD3 and z proteins. One possible stoichiometric combination is shown, but this may vary. 12

Binding of a TCR to a peptide-mhc complex. The V domains of a TCR are shown interacting with a human class I MHC molecule, HLA-A2, presenting a viral peptide (in yellow). 13

The expression of the TCR complex required synthesis of all its components Assembly and surface expression of the TCR complex. In the absence of any one component (in this case, the CD3g protein), the TCR complex is not assembled and all its proteins are degraded within the cell, probably in the endoplasmic reticulum. Introduction of the missing component by gene transfection allows the complex to be assembled and transported to the cell surface. 14

Accessory molecules on T cells In addition to TCR complex, T cells express several other integral membrane proteins (accessory molecules) that are important in antigen recognition. 1. Accessory molecules on T cells specifically bind other molecules present on the surface of other cells, target cells or vascular endothelium. 2. Accessory molecules are nonpolymorphic and invariant. This implies that these molecules have no capacity to specifically recognize many different, variable ligands, such as antigens. 3. As a consequence of binding their specific ligands on the surface of other cells, many Accessory molecules increase the strength of adhesion between a T cell and an APC or target cell. 4. Accessory molecules binding to endothelial cell surfaces and extracellular matrix ligands contributes to T cell homing and retention in tissues. 5. Many Accessory molecules may transduce biochemical signals to the interior of the T cell that are important in regulating functional responses. 6. T cell Accessory molecules are useful cell surface markers that facilitate immunocytochemical identification of T cells Antibodies against one or more of these molecules can block T cell responses to antigens suggested that the importance of accessory molecules in antigen presentation. 15

Accessory molecules of T lymphocytes. The interaction of a CD4+ helper T cell with an APC (A), or of a CD8+ CTL with a target cell (B), involves multiple T cell membrane proteins that recognize different ligands on the APC or target cell. 16

Structure of CD4 and CD8. The models of CD4 and CD8 binding to class II MHC and class I MHC molecules, respectively, are based on the structures defined by x-ray crystallography. Both CD4 and CD8 bind to nonpolymorphic regions of MHC molecules (CD4 to the class 18II b2 domain and CD8 to the class I a3 domain) away from the peptide-binding clefts.

Role of the CD4 coreceptor in T cell responses to antigens. During a T cell response to an MHCassociated peptide, CD4 binds to the MHC molecule. An antibody against CD4 blocks its binding to the MHC molecule and prevents T cell activation (A). A CD4-negative T cell does not respond to antigen, and introduction of CD4 into the T cell restores responsiveness (B). An MHC-negative antigen-presenting cell (APC) fails to activate T cells. Antigenpresenting function is restored by expressing normal MHC molecules in the APC (C) but not by expressing mutant 19MHC molecules that cannot bind CD4 (D).

Regulation of integrin avidity. Integrins are present in a low-affinity state in resting T cells. Chemokines produced by the antigen-presenting cell (APC) and signals induced by the TCR when it recognizes antigen both act on integrins and lead to their clustering and to conformational changes that increase the affinity of the integrins for their ligands. As a result, the integrins bind with high avidity 20 to their ligands on APCs and thus promote T cell activation.

The nature of T cell responses Because T cells recognize only MHC-associated peptide antigens, they can respond only to antigens associated with other cells and are unresponsive to soluble or circulating proteins. 1. The helper T cells activate B cells and macrophages. B lymphocytes and macrophages are two of the principal cell types that express class II MHC genes, function as APCs for CD4+ helper T cells, and focus helper T cell effects to their immediate vicinity. 2. CTLs can lyse any nucleated cells express class I MHC molecules, which are the restricting elements for antigen recognition by CD8+ CTLS Extracellular antigens usually end up in the endosomal pool and activate class IIrestricted CD4+ T cells. These cells function as helpers to stimulate effector mechanisms. Endogenously synthesized antigens are present in the cytoplasmic pool of proteins and usually activated class I-restricted CD8+ CTLS 21

Cytotoxic T cells and MHC I proteins contribute to the cellular immune response 1. Like class II MCH molecules, class I MHC molecules also present processed antigen to T cells. 2. Every nucleated cell has proteasomes that degrade cellular proteins to small peptide fragments. 3. Some of these protein fragments are bound by class I MHC molecules and carried to the plasma membrane, where CT cells can check them. 4. Fragments of normal proteins are presented but are generally ignored by the immune system. 5. If a cell has been infected by a virus, or if it has mutated, it may present protein fragments that are not normally found in the body. 23

6. If a TC cell binds to the MHC/processed antigen complex, the TC cell is activated to proliferate and differentiate. 7. In the effector stage, TC cells once again bind to the MHC/processed antigen complex on a cell and secrete molecules that lyse the cell. 8. TC cells can also bind to specific target cell receptors called Fas. 9. This binding initiates apoptosis in the target (for example, virusinfected) cell. This system helps rid the body of virus-infected cells. It also helps to destroy some cancer tumors. 24

Interleukin-1 activates a TH cell 4. TH cells proliferate and form a clone 1. The antigen is taken up by phagecytosis and degrades in a lysosome 2.A T cell receptor recognizes processed antigen bound to a class II MHC protein on the macrophage 3. Cytokines released by the TH cell stimulate it to proliferate 25

6. Cytokines activate B cell proliferation 8. B cells proliferate and differentiate 5. The binding of antigen to a specific IgM receptor triggers receptor-mediated endocytosis, degradation, and display of the processed antigen 7. A T cell recpetor recognized processed antigen bound to a class II MHC protein on a B cell 9. The plasma cell produces antibodies 26

1. A viral protein made in an infected cell, or a cellular protein, is degraded to fragments and picked up by a class I MHC protein 2. A T cell receptor recognizes processed antigen bound to an class I MHC protein on an infected cell 3. TC cells proliferate and form a clone 27

5. The T cell releases perforin, which lyses the infected cell. 4. A T cell receptor again recognizes processed antigen bound to a class I MHC protein 28

General features of T lymphocyte responses to antigen 1. Proliferation of T cells is mediated primary by an autocrine growth pathway, in which the responding T cell secretes its own growth-promoting cytokines (IL-2) and also expresses cell surface receptors for these cytokines. Clonal expansion 2. Differentiation is the process that converts naïve T lymphocytes to effector cells that perform various functions. 3. Effector functions of T cells are initiated by antigen stimulation of differentiated effector cells generated by antigen-induced differentiation of naïve T cells. The major effector function of CD4-expressing helper T cells is the secretion of cytokines, which act on T cells and on other cell types, including B lymphocytes, macrophages, and vascular endothelium. 4. Some of the progeny of antigen-responsive cells develop into antigen-specific memory T cells 29

Activation of naive and effector T cells by antigen. Antigens that are transported by dendritic cells to lymph nodes are recognized by naive T lymphocytes that recirculate through these lymph nodes. The T cells are activated to differentiate into effector and memory cells, which may remain in the lymphoid organs or migrate to nonlymphoid tissues. At sites of infection, the effector cells are again activated by antigens and perform their various functions, such as macrophage activation. 31

Phases of T cell responses. Antigen recognition by T cells induces cytokine (e.g., IL-2) secretion, clonal expansion as a result of IL-2-induced autocrine cell proliferation, and differentiation of the T cells into effector cells or memory cells. In the effector phase of the response, the effector CD4+ T cells respond to antigen by producing cytokines that have several actions, such as the activation of macrophages and B lymphocytes, and CD8+ CTLs respond by32 killing other cells. APC, antigen-presenting cell; CTL, cytolytic T lymphocyte.

Functions of costimulators in T cell activation. The resting antigen-presenting cell (APC) expresses few or no costimulators and fails to activate naive T cells (A). (Sometimes antigen recognition with-out costimulation may make the T cells anergic; this phenomenon will be discussed in Chapter 10.) Microbes and cytokines produced during innate immune responses activate the APCs to express costimulators, such as B7 molecules (B). The APCs then become capable of activating naive T cells. Activated APCs also produce cytokines such as IL-12, which stimulate the differentiation of naive T cells into effector cells. 33

Role of B7 and CD28 in T cell activation. A costimulator-deficient antigen-presenting cell (APC) does not stimulate responses of CD4+ T cells or may induce T cell anergy (A). The expression of B7 molecules in the APCs by gene transfection (B) or the provision of a costimulatory signal with an anti- CD28 antibody (C) leads to T cell activation. Similarly, cross-linking of the TCR complex with an antibody specific for the TCR/CD3 complex does not activate the T cells (D), but the addition of an34 activating anti-cd28 antibody elicits T cell responses (E).

Role of CD40 in T cell activation. Antigen recognition by T cells induces the expression of CD40 ligand (CD40L). CD40L engages CD40 on the antigen-presenting cell (APC) and stimulates the expression of B7 molecules and the secretion of cytokines that activate T cells. Thus, CD40L on the T cells makes the APCs "better" APCs. T cells can express CD40L on antigen recognition even without costimulation, but sustained expression of CD40L requires B7:CD28 costimulation as well as antigen. Thus, the B7 and CD40 pathways stimulate each other. 35

Intracellular signaling events during T cell activation. Binding of the TCR and coreceptors to peptide-mhc complexes on the antigen-presenting cell (APC) initiates proximal signaling events, which result in phosphorylation of the z chain, binding and activation of ZAP-70, phosphorylation of adapter proteins, and activation of various cellular enzymes. These enzymes then activate transcription 36 factors that stimulate the expression of various genes involved in T cell responses.

Formation of the immunological synapse. Before antigen recognition, various receptors on T cells and their ligands on APCs are dispersed in the plasma membranes of the two cells. When the T cell recognized antigen presented by the antigen-presenting cell (APC), selected receptors on the T cells and their respective ligands are redistributed to a defined area of cell-cell contact, forming the synapse. The molecules in the central portion of the synapse from the central supramolecular activation cluster (csmac), and the molecules in the periphery form the peripheral supramolecular activation cluster (psmac). 38

Early tyrosine phosphorylation events in T cell activation 39

Ras-MAP kinase pathway in T cell activation 40

T cell signaling through membrane inositol phospholipid metabolism 41

Activation of transcription factors in T cells 42

Phases of the humoral immune response. The activation of B cells is initiated by specific recognition of antigens by the surface Ig receptors of the cells. Antigen and other stimuli, including helper T cells, stimulate the proliferation and differentiation of the specific B cell clone. 43

Signal transduction by the B lymphocyte antigen receptor complex The activation of B lymphocytes is initialed by the binding of antigen to membrane Ig molecules on specific B cells. This interaction can lead to two responses. 1. The B lymphocyte antigen receptor delivers biochemical signals to the cells that initiate the process of activation. 2. The antigen is internalized into endosomal vesicles, and if it is a protein, it is processed and peptides are presented on the B cell surface for recognition by helper T cells. 44

Membrane IgM and IgD, the antigen receptors of resting mature B cells, have short cytoplasmic tails consisting of only three amino acids (Lysine, valine, and lysine). Singling via Ig is actually transduced by Ig and Ig. Ig and Ig contain tyrosine-rich motifs to be required fro signal transduction. 46

most helper T cells are CD4+CD8- and class II MHC-restricted in their recognition of foreign protein antigens. 47

Helper T cell contact-mediated activation of B lymphocytes The physical contact between B cells specific for the native antigen and helper T lymphocytes specific for processed peptides is mediated by multiple ligand-receptor pairs, some of which deliver signals to the B cells that are required for the full development of the humoral immune response. The two critical ligand-receptors, are B7 molecules:cd28 and CD40:CD40 ligand. Antigen recognition by B cells enhances the expression of costimulators, B7-2 and B7-1. These costimulators are recognized by CD28 on helper T cells, which are interacting with peptide-class II MHC complexes on the B cells. The helper T cell thus received the two sets of stimuli for their activation. Upon the activation, helper T cells express a 35-39 kda surface molecule called CD40 ligand. The interaction between CD40 and CD40 ligand not only stimulates B cell proliferation and differentiation but also leads to enhanced expression of B7 molecules, thus causing more T cell activation. 48

Kinetics of primary and secondary humoral immune responses. In a primary immune response, naive B cells are stimulated by antigen, become activated, and differentiate into antibody-secreting cells that produce antibodies specific for the eliciting antigen. Some of the antibody-secreting plasma cells survive in the bone marrow and continue to produce antibodies for long periods. Long-lived memory B cells are also generated during the primary response. A secondary immune response is elicited when the same antigen stimulates these memory B cells, leading to more rapid proliferation and differentiation and production of greater quantities of specific antibody than are produced 49 in the primary response.

B cell antigen receptor complex. Membrane IgM (and IgD) on the surface of mature B cells is associated with the invariant Iga and Igb molecules, which contain ITAMs in their cytoplasmic tails that 50 mediate signaling functions. Note the similarity to the TCR complex

Signal transduction by the BCR complex. Antigen-induced cross-linking of membrane Ig on B cells leads to clustering of the Iga and Igb molecules and tyrosine phosphorylation of the ITAMs in the cytoplasmic tails of these molecules. This leads to docking of Syk and subsequent tyrosine phosphorylation events as depicted. Several signaling cascades follow these events, as shown, leading 51 to the activation of several transcription factors.

Role of complement in B cell activation. B cells express a complex of the CR2 complement receptor, CD19, and CD81. Microbial antigens that have bound the complement fragment C3d can simultaneously engage both the CR2 molecule and the membrane Ig on the surface of a B cell. This leads to the initiation of signaling cascades from both the BCR complex and the CR2 complex, because of which the response to C3d-antigen complexes is greatly enhanced compared with the response to antigen alone. 52

Functional responses induced by antigen-mediated cross-linking of the BCR complex. Antigen-mediated cross-linking of the B cell antigen receptor induces several cellular responses, including mitosis, expression of new surface molecules, including costimulators and cytokine receptors, 53 and altered migration of the cells.

B cell antigen presentation to helper T cells. Protein antigens bound to membrane Ig are endocytosed and processed, and peptide fragments are presented in association with class II MHC molecules. Antigen binding to the B cell also stimulates expression of the costimulatory molecules B7-1 and B7-2. Helper T cells recognize the MHC-peptide complexes and costimulators and are activated 54 to then stimulate B cell responses.

Mechanisms of helper T cell-mediated B cell activation. B cells display processed peptides derived from endocytosed protein antigens and express the costimulators B7-1 and B7-2. Helper T cells recognize the antigen (in the form of peptide-mhc complexes) and the costimulators and are stimulated to express CD40 ligand and to secrete cytokines. CD40 ligand then binds to CD40 on the B cells and initiates B cell proliferation and differentiation. Cytokines bind to cytokine receptors on 55 the B cells and also stimulate B cell responses.

B lymphocyte growth and differentiation induced by cytokines Cytokines are soluble proteins secreted by T lymphocytes and by other cell types in response to activating stimuli. Although cytokines are produced as a result of the stimulation of specific T cells by antigens, the cytokines themselves are not antigen-specific and do not bind to antigens. Cytokines serve two principal functions in antibody responses: they determine the types of antibodies produced by selectively promoting switching to different heavy chain isotypes, and they provide amplification mechanisms by augmenting B cell proliferation and differentiation. Different cytokines play distinct, but overlapping, roles in antibody production, and their actions may be synergistic or antagonistic. 60

Cytokines have been identified to act at each stage of B cell activation 1. Many cytokines have been shown to stimulate the proliferation of B cell in vitro. IL-2, IL-4, and IL-5 (derived by T helper cells) all contribute to B cell proliferation and may act synergistically. IL-6 (produced by macrophages and T cells) is a growth factor for already differentiated, antibody-secreting B cells. IL-1, IL-10, and TNF (by macrophages) also promote B cell proliferation. 2. Antibody secretion. In the mouse, IL-4 and IL-5 are potent inducers of antibody secretion by b cells. IL-2, IL-6, or IL-10 have shown to induce the antibody production in human culture cell system. 3. Isotype switching. The most selective, and only obligatory, functions of different cytokines in humoral immune responses are in regulating the pattern of heavy chain isotype switching. IL-4 is the principal switch factor for IgEIL-5, acts in concert with transforming growth factor- to stimulate IGA production in mucosal lymphoid tissues. 61

1. T cells produce several cytokines that serve primary to regulate the growth and differentiation of various lymphocyte populations, and thus play important roles in the activation phase of T-cell dependent immune response. 2. Other T cell-derived cytokines function principally to activate and regulate inflammatory cells, such as mononuclear phagocytes, neutrophils, and eosinophils. These cytokines act in the effector phase of cell-mediated immunity. 3. Colony-stimulating factors are produced by lymphocytes and mononuclear phagocytes produced cytokines which stimulate the growth and differentiation of immature leukocytes in the bone marrow. 63

General properties of cytokines 1. Cytokines are produced during the activation and effector phases of innate and specific immunity and serve to mediate and regulate immune and inflammatory responses. 2. Cytokines secretion is a brief, self-limited event. 3. Many individual cytokines are produced by multiple diverse cell types. 4. Cytokines act upon many different cell types. 5. Cytokines often have multiple different effects on the same target cell. 6. Cytokine actions are often redundant 7. Cytokines often influence the synthesis of other cytokines. 8. Cytokines often influence the action of other cytokines. 9. Cytokines, like other polypeptide hormones, initiate their action by binding to specific receptors on the surface of target cells. 10. The expression of many cytokines receptors is regulated by specific signals. 11. Most cellular responses to cytokines require new mrna and protein synthesis. 12. For many target cells, cytokines act as regulators of cell division, i.e., growth factors. 64