T CELL RECEPTOR: STRUCTURE AND GENETIC BASIS

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1 T CELL RECEPTOR: STRUCTURE AND GENETIC BASIS 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. 6 th edition, Chapter 9; 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. 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

2 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, a and b, and the associated signal transduction complex, CD3 (γ, δ and ε chains) plus ζ (zeta) or η (eta) or θ (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 [α] and beta [β] chains on their surface. Cells that express gamma [γ] and delta [δ] chains comprise only 5% of the normal circulating T cell population in healthy adults. Each chain (α, β, γ or δ) represents a distinct protein with approximate molecular weight of 45 kda. An individual T cell can express either an αβ or a γδ 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 between heterodimers create three hypervariable regions called complementarity determining regions (CDRs 1, 2, and 3).

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 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.

4 TCR-CD3-complex. The TCR heterodimer is tightly associated with six independently encoded CD3 subunits (δ, γ, ε, ζ, η and θ) 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. α and γ uses only V and J gene segments. β and δ use V, D, and J gene segments. There are many more Vα and Vβ genes (50-100) than Vγ and Vδ genes (5-10) present in germ line. The a and d chain genes are mixed together in one locus. The genes encoding the d chain are entirely located between the cluster of Vα and Jα 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 α and β loci. During T- cell development, a V-region sequence for each chain is assembled by DNA recombination. For the α chain (top), a Vα gene segment rearranges to a Jα gene segment to create a functional gene encoding the V domain. For the β chain (bottom), re-arrangement of a Dβ, a Jβ, and a Vβ gene segment creates the functional V-domain exon. Order of TCR Gene Rearrangement. The earliest cell entering the thymus has its TCR genes in the germ line configuration (unrearranged). Both γ and β chain genes then begin to rearrange, more or less simultaneously. If the γ chain genes rearrange successfully, then δ chain genes also start to rearrange. If both γ and δ genes rearrange functionally, no further gene rearrangement takes place and the cell remains a γδ T cell. If γ and/or δ rearrangements are not functional, then β gene rearrangement continues followed by α gene rearrangement. In this manner, a αβ product appears, and the cell becomes an αβ T cell.

5 The Process of Recombination. Recombination of V, D, and J gene segments is coordinated by recombinaseactivating 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. 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 binding sites.

6 So, while the center of the binding site is highly variable, the remaining portion of the heterodimer is subject to relatively little variation. Number of V gene pairs Junctional diversity Total Diversity Immunoglobulins ~2-3.4 x 106 ~3 x 107 ~1014 T cell a:b Receptors 5.8 x 106 ~2 x 1011 ~1018 Development of T lymphocytes During differentiation in the thymus, immature T cells undergo rearrangement of their TCR α and β 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+). The majority of peripheral blood T lymphocytes express the α and β form of the TCR. In healthy adults, less than 5% express a heterodimer comprised of the γ and δ chains. Virtually all the cells that express the TCRabare CD4+CD8- (T helper) or CD4-CD8+ (T cytotoxic or T suppressor). Almost all cells expressing TCR-γδ are CD4-CD8- (double negative). While the TCR-αβ expressing lymphocytes are known to function as helper and cytotoxic cells, the function of the TCR-γδ cells is not well understood. Figure. Changes in surface molecules allow thymocytes at different stages of maturation. Figure. Main stages in the development of a T lymphocyte.

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8 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-b and provide help for IgA production, and have suppressive properties for Th1 and Th2 cells. Th17 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 antigenspecific 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+, TGFb secretors. γδ T Cells: Not all T cells express αβ TCRs. An alternative is to express γδ chains of the TCR. Generally, γδ cells lack CD4, although some γδ cells do express CD8. The functions of γδ cells are not well understood. γδ 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. γδ expressing cells have been found to be active towards mycobacterial antigens and heat shock proteins. They have the ability to secrete cytokines like their αβ 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. hese 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 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 between 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

9 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 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

10 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 α and β 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. T Cell Receptor: Structure and Genetic Basis 1. Mature T cells express antigen-specific TCR in a complex with CD3 molecules. The TCR is a disulfidelinked heterodimer composed of either αβ or γδ chains. T cells express either αβ or γδ chain heterodimers, but never both. 2. 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. 3. 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. γ chains of the TCR have only V and J segments, and join to δ chains. δ 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 If gd rearrangements are unsuccessful on both chromosomes, α chains join to β chains to give αβ phenotypic T cells. α chains have only V and J segments; β chains have V, D, and J segments.