Introduction

The establishment and maintenance of immune responses, homeostasis, and memory depends to a large extent on T lymphocytes. T cells provide more than protection from infection and are involved in tumor surveillance as well as tissue homeostasis. T cells thus confront the dilemma of generating a receptor that can recognize a broad array of antigens from foreign pathogens, tumors, and normal tissue without provoking an autoimmune response to the host. The price for generating an increasingly varied population of antigen receptors needed to recognize a wide spectrum of pathogens is the progressive risk of producing self-reactive lymphocytes. T lymphocytes are thus subjected to a rigorous selection process during development in the thymus to delete self-reactive T cells (i.e., central tolerance). In addition, premature activation of mature peripheral T cells is prevented by requiring two signals for activation (i.e., peripheral tolerance). Finally, the expansion of T cells that occurs during either homeostatic proliferation in the periphery or in response to an infection is resolved by the active induction of cell death. The consequences of inefficient lymphocyte removal at any one of these junctures could provoke an autoimmune diathesis. These issues are discussed in more detail in Chapter 19 on cell survival and death.

The activation of T lymphocytes yields a variety of effector functions that are pivotal to combating infections. Cytolytic T cells can kill infected cells through the release of granules containing perforin and granzymes that induce holes in cell membranes and cleave cellular substrates, respectively, or expression of ligands for death receptors such as Fas (CD95) or TNF receptor 1. Production of T cell cytokines such as interferon-γ (IFN-γ) by T helper (Th)1 cells (see below) can inhibit viral replication, whereas other cytokines such as interleukin (IL)-4, IL-5, and IL-21 by Th2 cells are critical for optimal B cell growth and immunoglobulin production. However, this same armamentarium, if not tightly regulated, can also precipitate damage to host tissues and provoke autoimmune responses, such as in the synovium of inflammatory arthritides, pancreatic islets in type I diabetes, and the central nervous system in multiple sclerosis. Damage in these cases need not be directly the result of recognition of target tissues by the T cells. T cells may be activated elsewhere and then migrate to the tissue and damage innocent bystander cells. T cells may also promote autoimmunity through the augmentation of B cell responses. The following sections detail these events.

T Cell Development

T lymphocytes originate from bone marrow progenitors that migrate to the thymus for maturation, selection, and subsequent export to the periphery. T cells must traverse two stringent hurdles during their development. First, they must successfully rearrange the genes encoding the two chains of the T cell antigen receptor (TCR). Second, T cells must survive thymic selection during which T cells bearing a TCR th0at interacts strongly with self-major histocompatibility complex (MHC) molecules/peptides are eliminated (i.e., negative selection). This minimizes the chances of autoreactive T cells escaping to the periphery and is known as central tolerance . In addition, developing thymocytes must also make moderate interactions with self-MHC/peptides to survive (i.e., positive selection), as those T cells making no meaningful contact are also eliminated. The result of this “Goldilocks” concept of not too strong, not too weak TCR-MHC/peptide interactions is that less than 3% of developing thymocytes actually leave the thymus.

The TCR is an 80 to 90 kDa disulfide-linked heterodimer composed of a 48 to 54 kDa α chain and a 37 to 42 kDa β chain. An alternate TCR composed of γ and δ chains is expressed on 2% to 3% of peripheral blood T cells and is discussed below. The TCR has an extra-cellular ligand binding pocket for MHC/peptide and a short cytoplasmic tail that by itself cannot signal. Consequently, it is noncovalently associated with as many as five invariant chains of the CD3 complex, which relay information to the intra-cellular signaling machinery via immunoreceptor tyrosine activation motifs (ITAMs) (see later). The structure of the TCR gene locus is, not surprisingly, similar to immunoglobulin genes in B cells (see details in Chapter 13 ). To economically package up to 10 ¹⁵ TCR specificities within a genome of fewer than 30,000 genes, the process of gene rearrangement and splicing evolved using machinery similar to what already existed to promote gene translocations. The β- and δ-chain genes of the TCR contain 4 segments known as the V (variable), D (diversity), J (joining), and C (constant) regions. The α and γ chains are similar but lack the J component. Each of the segments has several family members (approximately 50 to 100 V, 15 D, 6 to 60 J, and 1 to 2 C members). An orderly process occurs during TCR gene rearrangement in which a D segment is spliced adjacent to a J segment, which is subsequently spliced to a V segment. Following transcription, the VDJ sequence is spliced to a C segment to produce a mature TCR messenger RNA. Arithmetically, this random rearrangement of a single chain of the TCR locus can give rise to minimally 50V × 15D × 6J × 2C or about 9000 possible combinations. At each of the splice sites, which must occur in-frame to be functional, additional nucleotides not encoded by the genome (so-called N-region nucleotides ) can be incorporated, adding further diversity to the rearranging gene. The combinations from the two TCR chains, plus N-region diversity, yield at least 10 million theoretical possible combinations. The cutting, rearranging, and splicing are directed by specific enzymes. Mutations in the genes for these processes can result in an arrest in lymphocyte development. For example, mutation in the gene encoding a DNA-dependent protein kinase (DNA-PK) required for receptor gene recombination results in a severe combined immunodeficiency known as scid .

Because the developing T cell has two copies of each chromosome, there are two chances to successfully rearrange each of the two TCR chains. As soon as successful rearrangement occurs, further β-chain rearrangements on either the same or the other chromosome are suppressed, a process known as allelic exclusion . This limits the chance of dual TCR expression by an individual T cell. The high percentage of T cells that contain rearrangements of both β-chain genes attests to the inefficiency of this complex event. Rearrangement of the α chain occurs later in thymocyte development in a similar fashion, although without apparent allelic exclusion. This can result in dual TCR expression by a single T cell.

Development of T cells occurs within a microenvironment provided by the thymic epithelial stroma. The thymic anlage is formed from embryonic ectoderm and endoderm and is then colonized by hematopoietic cells, which give rise to dendritic cells, macrophages, and developing T cells. The hematopoietic and epithelial components combine to form two histologically defined compartments: the cortex, which contains immature thymocytes, and the medulla, which contains mature thymocytes ( Fig. 12.1A ). As few as 50 to 100 bone marrow–derived stem cells enter the thymus daily.

Sequence of thymocyte development. (A) The earliest thymocyte precursors lack expression of CD4 and CD8 (CD4 ⁻ CD8 ⁻ ). These can be further divided into four subpopulations based on the sequential expression of CD44 and CD25. It is at the CD44 ⁻ CD25 ⁺ stage that the TCR-β chain rearranges. The scid mutation or deficiencies of the rearrangement enzymes Rag-1 and Rag-2 result in inability to rearrange the β chain and maturational arrest at this stage. Those thymocytes that successfully rearrange the β chain express it associated with a surrogate α chain known as pre-Tα. Concomitant with a proliferative burst, development can then progress to the CD4 ⁺ CD8 ⁺ stage in the cortex where the TCR α chain rearranges and pairs with the β chain to express a mature TCR complex. These cells then undergo thymic positive and negative selection (as diagrammed in Figure 12.3B). Successful completion of this rigorous selection process results in mature CD4 ⁺ or CD8 ⁺ T cells in the medulla, which eventually emigrate to peripheral lymphoid sites. (B) Schematic two-color flow cytometry showing subpopulations of thymocytes defined by CD4 and CD8 expression in their relative proportions.

The stages of thymocyte development can be defined by the status of the TCR gene and the expression of CD4 and CD8, proceeding in an orderly fashion from CD4 ⁻ 8 ⁻ → CD4 ⁺ 8 ⁺ → CD4 ⁺ 8 ⁻ or CD4 ⁻ 8 ⁺ ( Fig. 12.1B ). CD4 and CD8 define, respectively, the helper and cytolytic subsets of mature T cells.

CD4 ⁻ 8 ⁻ thymocytes can be further subdivided based on their expression of CD25 (the high affinity IL-2 receptor α chain) and CD44 (the hyaluronate receptor). Development proceeds in the following order: CD25 ⁻ CD44 ⁺ → CD25 ⁺ CD44 ⁺ → CD25 ⁺ CD44 ⁻ → CD25 ⁻ CD44 ⁻ (see Fig. 12.1 ). These subpopulations correspond to discrete stages of thymocyte differentiation. CD25 ⁻ 44 ⁺ cells contain TCR genes in germline configuration. These cells upregulate CD25 to give rise to CD25 ⁺ CD44 ⁺ thymocytes, which now express surface CD2 and low levels of CD3ε. At the next stage (CD25 ⁺ CD44 ⁻ ), there is a brief burst of proliferation followed by upregulation of the recombination-activating enzymes, RAG-1 and RAG-2, and the concomitant rearrangement of the genes of the TCR β chain. A small subpopulation of T cells rearranges and expresses a second pair of TCR genes known as γ and δ. Productive TCR β-chain rearrangement results in a second proliferative burst, yielding CD25 ⁻ CD44 ⁻ thymocytes.

The TCR β chain cannot be stably expressed without an α chain. Because the TCR α chain has not yet rearranged, a surrogate invariant TCR pre-α chain is disulfide linked to the β chain. When associated with components of the CD3 complex, this allows a low-level surface expression of a pre-TCR and progression to the next developmental stage. Failure to successfully rearrange the TCR β chain results in a developmental arrest at the transition from CD25+CD44− to CD25-CD44−. This occurs in RAG-deficient mice as well as in Scid mice and humans.

A number of transcription factors, receptors, and signaling molecules are required for early T cell development ( Fig. 12.2 ). IKAROS encodes a transcription factor required for the development of cells of lymphoid origin. Notch-1, a molecule known to regulate cell fate decisions, is also required at the earliest stage of T cell lineage development. Cytokines, including IL-7, promote the survival and expansion of the earliest thymocytes. In mice deficient for IL-7, its receptor components IL-7Rα or γ _c , or the cytokine receptor–associated signaling molecule Janus kinase (JAK)-3, thymocyte development is inhibited at the CD25 ⁻ CD44 ⁺ stage. In humans, mutations in γ _c or JAK-3 result in the most frequent form of SCID. Pre-TCR signaling is required for the CD25 ⁺ CD44 ⁻ → CD25 ⁻ CD44 ⁻ transition. Thus, loss of TCR signaling components including Lck, SH2-domain–containing leukocyte protein-76 (SLP-76), and Linker for activation of T cells (LAT)-1 results in a block at this stage of T cell development. TCR signals are also required for differentiation of CD4 ⁺ CD8 ⁺ to mature CD4 ⁺ or CD8 ⁺ cells. Humans deficient in ZAP-70 (see later) have CD4 ⁺ but not CD8 ⁺ T cells in the thymus and periphery.

Sequence of αβ T cell development in the thymus. The earliest thymocyte precursors lack expression of CD4 and CD8 (CD4 ⁻ CD8 ⁻ ). These can be further divided into four subpopulations based on the sequential expression of CD25 and CD44. At the CD25 ⁺ CD44 ⁻ stage, the TCR β chain rearranges and associates a surrogate α chain known as pre-Tα. Concomitant with a proliferative burst, thymocytes progress to the CD4 ⁺ CD8 ⁺ stage, rearrange the TCR α chain, and express a mature TCR complex. These cells then undergo thymic positive and negative selection. Those thymocytes that survive this rigorous selection process differentiate into mature CD4 ⁺ or CD8 ⁺ T cells. Shown also are the various signaling molecules that are involved at specific stages of thymic development.

CD25 ⁻ CD44 ⁻ cells upregulate expression of CD4 and CD8 to become CD4 ⁺ 8 ⁺ , at which point the α chain of the TCR rearranges. Unlike the β chain, allelic exclusion of the α chain is not apparent. Rearrangement of the α chain can occur simultaneously on both chromosomes, and if one attempt is unsuccessful, repeat rearrangements to other Vα segments are possible. Reports exist of dual TCR expression by as many as 30% of mature T cells in which the same T cell expresses different α chains paired with the same β chain. However, in most cases of dual TCR α chains, one is downregulated during positive selection by Lck and Cbl, through ubiquitination, endocytosis, and degradation.

Although the structure of immunoglobulin and TCR are quite similar, they recognize fundamentally different antigens. Immunoglobulins recognize intact antigens in isolation, either soluble or membrane bound, and are often sensitive to the tertiary structure. The TCRαβ recognizes linear stretches of antigen peptide fragments bound within the grooves of either class I or class II MHC molecules ( Fig. 12.3A ). Thymic selection molds the repertoire of emerging TCRs so that they recognize peptides within the groove of self-MHC molecules, ensuring the self-MHC restriction of T cell responses. The MHC structure is described in detail in Chapter 21, Chapter 22 . Pockets within the MHC groove bind particular residues along the peptide sequence of 7-9 amino acids for class I MHC and 9-15 amino acids for class II MHC molecules. As a result, certain amino acids make strong contact with the MHC groove while others are exposed to the TCR.

TCR interaction with the MHC/peptide complex. (A) Polymorphic residues within the variable region of the α and β chains of the TCR contact determinants on the MHC molecule on an antigen presenting cell (APC) as well as with the peptide fragment that sits in the MHC binding groove. (B) A schematic diagram illustrating that during thymocyte development, those TCR conferring either a very low signal intensity (null selection) or high intensity (negative selection) each lead to apoptosis. Only those thymocytes whose TCR can engage MHC/peptides and confer moderate intensity survive by positive selection.

The contact between the TCR and MHC/peptide has been revealed by crystal structure to be remarkably flat, rather than a deep lock and key structure that one might imagine. The TCR axis is tipped about 30 degrees to the long axis of the class I MHC molecule and is slightly more skewed for class II MHC. The affinity of the TCR for MHC/peptide is in the micromolar range. This is a lower affinity than many antibody-antigen affinities, and several logs less than many enzyme-substrate affinities. This has led to the notion that TCR interactions with MHC/peptide are brief, and successful activation of the T cell requires multiple interactions, resulting in a cumulative signal.

Once the T cell has successfully rearranged and expressed a TCR in association with the CD3 complex, it encounters the second major hurdle in T cell development, thymic selection . Selection has two phases, positive and negative, and the outcome is based largely upon the intensity of TCR signaling in response to interactions with self-MHC/peptides expressed on thymic epithelium and dendritic cells. TCR signals that are either too weak (death by neglect) or too intense (negative selection) result in elimination by apoptosis, whereas those with intermediate signaling intensity survive (positive selection) ( Fig. 12.3B ). Successful positive selection at the CD4 ⁺ 8 ⁺ stage is coincident with upregulation of surface TCR, the activation markers CD5 and CD69, and the survival factor Bcl-2. T cells bearing a TCR that recognizes class I MHC maintain CD8 expression, downregulate CD4, and become CD4 ⁻ 8 ⁺ . T cells expressing a TCR that recognizes class II MHC become CD4 ⁺ 8 ⁻ .

An enigma for thymic selection has been how to present the myriad self-proteins to developing thymocytes so that self-reactive thymocytes are effectively eliminated by negative selection. This includes particularly those antigens with tissue or developmentally restricted expression. A solution was found with the discovery of the autoimmune regulator (AIRE) gene. AIRE is a transcription factor expressed by the medullary epithelium of the thymus that induces transcription of a wide array of organ-specific genes, such as insulin, that might otherwise be sequestered from developing thymocytes. This effectively creates a self-transcriptome within the thymus against which autoreactive T cells can be deleted. Gene knockout mice of AIRE , and humans bearing AIRE mutations, manifest various autoimmune sequelae in a syndrome known as autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED).

Not surprisingly, a variety of signaling molecules activated by TCR engagement are important to thymic selection. These include Lck, the RasΠRaf-1ΠMEK1ΠERK kinase cascade, the kinase ZAP-70, and the phosphatases CD45 and calcineurin, which are involved with positive selection. Among these, the Ras ΠERK pathway is particularly important as dominant negative variants of these molecules can disrupt positive selection. Conversely, an activator of Ras known as GRP1 assists with the positive selection of thymocytes expressing weakly selecting signals. These molecules are discussed in more detail in the section on TCR signaling. By contrast, although a number of molecules may promote negative selection, among them the mitogen-activated protein kinases (MAPKs) JNK and p38, there appears to be sufficient redundancy so that only rarely does elimination of any one of these molecules alter the efficient deletion of thymocytes. The few exceptions include CD40, CD40L, CD30, or the proapoptotic Bcl-2 family member, Bim, where preservation of at least some thymocytes bearing self-reactive TCR could be observed in mice deficient in these molecules.

The survivors of these stringent processes of TCR gene rearrangement and thymic selection represent less than 3% of total immature thymocytes. This is reflected by the presence of a high rate of cell death among developing thymocytes. This can be visualized by the measurement of DNA degradation, a hallmark of apoptosis, as shown in Fig. 12.4 . The survivors become either CD4 ⁺ helper or CD8 ⁺ cytolytic T cells and reside in the thymic medulla for 12 to 14 days before emigrating to the periphery. The decision to become a CD4 ⁺ versus CD8 ⁺ T cell parallels the observation that long TCR interactions are required for CD4 progression, whereas shorter TCR engagement favors CD8 progression.

TCR signal pathways. The schema shows the principle signal pathways resulting from TCR activation and how they impinge on the regulatory region of the IL2 gene. See text for details.

Peripheral Migration and Homeostatic Proliferation of T Cells

In early life, the majority of T cells are newly emerged from the thymus and naïve. With age, memory T cells are generated from exposure to antigens, which plateaus in adulthood. This may parallel a shift in function toward tumor surveillance and tissue homeostasis. At this later stage, immunosenescence occurs with a decline in T cell function, which can result in immunodysregulation and inflammation. The migration of naïve T cells to peripheral lymphoid structures or their infiltration into other tissues requires the coordinated regulation of an array of cell adhesion molecules. Entry from the circulation into tissues occurs via two main anatomic sites: the flat endothelium of the blood vessels and specialized postcapillary venules known as high endothelial venules (HEV). A three-step model has been described for lymphocyte migration: rolling, adhesion, and migration. L-selectin expressed by naïve T cells binds via lectin domains to carbohydrate moieties of GlyCAM-1 and CD34 (collectively known as peripheral node addressin ), which are expressed on endothelial cells, particularly high-endothelial venules (HEVs). The weak binding of CD62L to its ligand mediates a weak adhesion to the vessel wall which, combined with the force of blood flow, results in rolling of the T cell along the endothelium. The increased cell contact facilitates the interaction of a second adhesion molecule on lymphocytes, the integrin LFA-1 (CD11a/CD18), with its ligands, intercellular adhesion molecule (ICAM)-1 (CD54) and ICAM-2 (CD102). This results in the arrest of rolling and firm attachment. Migration into the extra-cellular matrix of tissues involves additional lymphocyte cell surface molecules such as the hyaluronate receptor (CD44) or the integrin α4β7 (CD49d/β7), which binds the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on endothelium of Peyer’s patches and other endothelial cells.

Additional cytokines known as chemokines contribute to lymphocyte homing. Chemokines are structurally and functionally related to proteins bearing an affinity for heparan sulfate proteoglycan and promote migration of various cell types. The chemokines RANTES, MIP-1α, MIP-1β, MCP-1, and IL-8 are produced by a number of cell types including endothelium, activated T cells, and monocytes and are present at inflammatory sites such as rheumatoid synovium (see Chapter 74 ).

Once mature T cells have reached the peripheral lymphoid tissues of lymph node and spleen, they undergo a slow rate of proliferation known as homeostatic proliferation in response to self-MHC/peptide complexes and the cytokines IL-7 and IL-15. This serves to maintain the number of peripheral T cells and can become enhanced in response to lymphopenia, such as after chemotherapy or HIV infection. Because homeostatic proliferation is driven by self-MHC/peptides and is thus inherently autoimmune, its acceleration could precipitate an autoimmune syndrome. In this regard, it is of interest that one of the standard murine models of autoimmunity is day 3 thymectomy, which results in lymphopenia and autoimmunity of various organs. In addition, nonobese diabetic (NOD) mice have chronic lymphopenia that contributes to their diabetes, and evidence of augmented homeostatic proliferation has been suggested to occur in rheumatoid arthritis (RA). Homeostatic proliferation is also regulated by the death receptor Fas (CD95). In the absence of Fas in mice or humans, T cells accumulate that express a gene profile that includes upregulation of pro-inflammatory molecules such as Fas ligand and Granzyme B, as well as the immunoinhibitory molecules programmed cell death 1 (PD-1) and LAG3. This might serve to explain the clinical immunology paradox of sudden autoinflammatory features in immunodeficient individuals, such as the development of sudden psoriasis and psoriatic arthritis (PsA) in HIV-infected individuals.

The Immunologic Synapse

Antigen-specific interaction between the T cell and APC results in the formation of a specialized contact region called the immunologic synapse or supramolecular activation cluster (SMAC) ( Fig. 12.5 ). Synapse formation is an active, dynamic process that requires specific antigen and MHC. The synapse also overcomes the obstacles resulting from interactions of tall molecules (e.g., ICAM-1, LFA-1, and CD45) to promote T cell/APC contact that is mediated by short molecules (e.g., TCR, MHC, CD4, and CD8). Two stages of assembly have been described. During the nascent stage, cell adhesion molecules such as ICAM-1 on APC and LFA-1 on T cells make contact in a central zone, surrounded by an annulus of close contact between MHC and TCR. Within minutes the engaged TCR migrates to the central area, resulting in a mature synapse in which the initial relationships are reversed; the central area (cSMAC) now contains TCR, CD2, CD28, and CD4 and is enriched for Lck, Fyn, and PKC-θ. Surrounding the central domain is a peripheral ring (pSMAC) that contains CD45, LFA-1, and associated talin. T cell activation also leads to compartmentalization of TCR signaling molecules to plasma membrane microdomains called rafts . Rafts are composed primarily of glycosphingolipids and cholesterol and are enriched in signaling molecules, actin, and actin-binding proteins. Src family kinases, Ras-like G proteins, LAT, and phosphatidylinositol-anchored membrane proteins all localize to raft domains.

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