T Lymphocytes

Key Points

  • T cells develop primarily in the thymus. The importance of the thymus is underscored by the absence of T cells in patients in whom the thymus has failed to develop (e.g., DiGeorge syndrome).

  • Thymic selection consists of a positive phase in which T cells must recognize self-major histocompatibility complex (MHC) molecules and a negative phase in which thymocytes bearing high-affinity T cell receptors for self-peptide MHC are deleted through apoptosis.

  • Only a small proportion of T cells emerge from the thymus as naïve T cells that are quiescent and produce relatively low levels of cytokines. After they acquire memory phenotype (CD45RO + ), they can produce high levels of cytokines.

  • Naïve T cells can undergo homeostatic proliferation to self-peptide/MHC in peripheral lymphoid tissues to generate a critical number of T cells. This process requires IL-7 and IL-15 and results in the upregulation of several cytolytic genes that can provoke inflammation. Lack of regulation of this process may contribute to various autoimmune disorders.

  • Th1 and Th17 cells accumulate at sites of inflammation such as rheumatoid synovium, whereas Th2 cells accumulate at sites of allergic responses such as asthma.

  • T cells undergo a tremendous change in metabolism as they transition from resting naïve T cells (oxidative phosphorylation) to proliferating effector T cells (glycolysis) to memory T cells (oxidative phosphorylation).


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.

Fig. 12.1

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.

Fig. 12.2

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.

Fig. 12.3

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.

Fig. 12.4

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.

Immunodeficiencies Resulting From Defects in T Cell Development

Given the vast number of events in T cell development, it is not surprising that multiple causes can underlie human T cell immunodeficiencies. The influence of the thymic stroma on thymocyte ontogeny is underscored in the DiGeorge anomaly in which development of the pharyngeal pouches is disrupted and the thymic rudiment fails to form. This results in the failure of normal T cell development. Less severe T cell deficiencies are associated with a failure to express class I and/or class II MHC (the “bare lymphocyte syndrome”), which are directly involved with interactions required to induce the positive selection of, respectively, CD8 + and CD4 + mature T cells.

Metabolic disorders can affect thymocytes more directly. The absence of functional adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) results in the buildup of metabolic byproducts that are toxic to developing T and B lymphocytes. This ultimately produces forms of severe combined immunodeficiency disorders (SCIDs).

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.

Activation of T Cells

Metabolic Switch

T cell activation initiates an intra-cellular signaling cascade that ultimately results in proliferation, effector function, or death, depending on the intensity of the TCR signal and associated signals. This requires a dramatic change in metabolism from largely oxidative phosphorylation to glycolysis to provide the synthetic machinery for proliferation and effector molecules. This is detailed in Chapter 24 . To guard against premature or excessive activation, T cells have a requirement of two independent signals for full activation. Signal 1 is an antigen-specific signal provided by the binding of the TCR to antigenic peptide complexed with MHC. Signal 2 is mediated by either cytokines or the engagement of co-stimulatory molecules such as B7.1 (CD80) and B7.2 (CD86) on the antigen-presenting cell (APC). Receiving only Signal 1 without co-stimulation results in T cell unresponsiveness or anergy, a process known as peripheral tolerance.

TCR Signal Regulation

TCR αβ and γδ have very short cytoplasmic domains and by themselves are unable to transduce signals. The molecules of the noncovalently associated CD3 complex couple the TCR to intra-cellular signaling machinery (see Fig. 12.4 ). The CD3 complex contains nonpolymorphic members known as CD3ε, CD3γ, CD3δ, as well as the ζ chain, which is a separate gene not genetically linked to the CD3 complex. Although the functional stoichiometry of the TCR complex is not completely defined, current data indicate that each TCR heterodimer is associated with 3 dimers: CD3εγ, CD3εδ, and ζζ or ζ. CD3ε, -γ, and -δ have an immunoglobulin-like extra-cellular domain, a transmembrane region, and a modest cytoplasmic domain, whereas ζ contains a longer cytoplasmic tail. The transmembrane domains of ζ and the CD3 chains contain a negatively charged residue that interacts with positively charged amino acids in the transmembrane domain of the TCR.

None of the proteins in the TCR/CD3 complex has intrinsic enzymatic activity. Instead, the cytoplasmic domains of the invariant CD3 chains contain conserved activation domains that are required for coupling the TCR to intra-cellular signaling molecules. These ITAMs contain a minimal functional consensus sequence of paired tyrosines (Y) and leucines (L): (D/E)XXYXXL(X) 6-8 YXXL. ITAMs are substrates for cytoplasmic protein tyrosine kinases (PTKs), and upon phosphorylation they recruit additional molecules to the TCR complex. Each ζ chain contains three ITAMs, whereas there is one in each of the CD3ε, -γ, and -δ chains. Thus, each TCR/CD3 complex can contain as many as 10 ITAMs.

Activation of PTKs is one of the earliest signaling events following TCR stimulation. Four families of PTKs are known to be involved in TCR signaling: Src, Csk, Tec, and Syk. The Src family members Lck and Fyn T have a central role in TCR signaling and are expressed exclusively in lymphoid cells. Src PTKs contain multiple structural domains, including: (1) N-terminal myristylation and palmitoylation sites that allow association with the plasma membrane, (2) an Src homology (SH)3 domain that associates with proline-rich sequences, (3) an SH2 domain that binds phosphotyrosine-containing proteins, and (4) a carboxy-terminal negative regulatory site. Their catalytic activity is regulated by the balance between the actions of kinases and phosphatases. Activity is repressed by phosphorylation of a conserved carboxy-terminal tyrosine, and thus its dephosphorylation by the phosphatase CD45 is critical for the initiation of TCR-mediated signal transduction. In addition, autophosphorylation of other tyrosines within the kinase domain enhances catalytic activity. Lck is physically and functionally associated with CD4 and CD8. Fifty percent to 90% of total Lck molecules is associated with CD4 and 10% to 25% with CD8. CD4 and CD8 physically associate with the TCR/CD3 complex during antigen stimulation as a result of their interaction with class II and class I MHC molecules, respectively, and thus enhance TCR-mediated signals by recruiting Lck to the TCR complex. Lck phosphorylates the CD3 chains, TCRζ, ZAP-70, phospholipase C-γ1 (PLC-γ1), Vav, and Shc. Fyn binds TCRζ and CD3ε and, although its substrates are less well defined, T cells lacking Fyn have diminished response to TCR signals. In addition, the SH2 and SH3 domains of Src PTKs can mediate their association with, respectively, phosphotyrosine- and proline-containing molecules.

Somewhat less is known about the Csk and Tec PTKs. Csk negatively regulates TCR signaling by phosphorylating the carboxy-terminal tyrosine of Lck and Fyn. Dephosphorylation of this negative regulatory tyrosine is mediated by the transmembrane tyrosine phosphatase CD45. CD45 activity is essential for TCR signaling as CD45-deficient T cells fail to activate by TCR stimulation. The Tec family member Itk is preferentially expressed in T cells and regulates PLC-γ. T cells from Itk-deficient mice have diminished response to TCR stimulation.

Phosphorylation of the ITAM motifs on the CD3 complex recruits the Syk kinase family member ZAP-70 by its tandem SH2 domains. ZAP-70 is expressed exclusively in T cells and is required for TCR signaling. Following TCR stimulation, ZAP-70 is activated by phosphorylation of tyrosine 493 by Lck. Loss-of-function hypomorphic alleles of ZAP-70 result in reduced TCR signaling and a propensity for autoimmune phenomena.

Adaptor Proteins

Phosphorylation of tyrosine residues in ITAMs and PTKs following TCR stimulation creates docking sites for adaptor proteins. Adaptor proteins contain no known enzymatic or transcriptional activities but mediate protein-protein interactions or protein-lipid interactions. They function to bring proteins in proximity to their substrates and regulators, as well as sequester signaling molecules to specific subcellular locations. The protein complexes formed can function as either positive or negative regulators of TCR signaling, depending on the molecules they contain.

Two critical adaptor proteins for linking proximal and distal TCR signaling events are SLP-76 and LAT (see Fig. 12.4 ). Loss of these adaptor proteins has profound consequences for T cell development. Mice deficient for LAT or SLP-76 manifest a block in T cell development at the CD4 8 CD25 + CD44 + stage. LAT is constitutively localized to lipid rafts and, following TCR stimulation, is phosphorylated on tyrosine residues by ZAP-70. Phosphorylated LAT then recruits SH2-domain–containing proteins including PLCγ1, the p85 subunit of phosphoinositide-3 kinase, IL-2 inducible kinase (Itk), and the adaptors Grb2 and Gads. Because the SH3 domain of Gads is constitutively associated with SLP-76, this brings SLP-76 to the complex where it is phosphorylated by ZAP-70. SLP-76 contains three protein binding motifs: tyrosine phosphorylation sites, a proline-rich region, and an SH2 domain. The N-terminus of SLP-76 contains tyrosine residues that associate with the SH2 domains of Vav, the adaptor Nck, and Itk. Vav is a 95 kDa protein that acts as a guanine nucleotide exchange factor for the Rho/Rac/cdc42 family of small G proteins. The complex of LAT, SLP-76/Gads, PLCγ1, and associated molecules results in the full activation of PLCγ1 and activation of Ras/Rho guanosine triphosphatases (GTPases) and the actin cytoskeleton.

In addition to acting as positive regulators for TCR signaling, adaptors can also mediate negative regulation. As described previously, the activity of the Src family kinases is regulated by the interaction of kinases (Csk) and phosphatases (CD45) specific for inhibitory C-terminal phosphotyrosine, which is determined by the subcellular localization of these regulatory molecules. A second mechanism by which adaptor proteins can negatively regulate TCR stability is through regulation of protein stability. Proteins can be targeted for degradation by the conjugation of ubiquitin to lysine residues via a series of enzymatic reactions by E1, E2, and E3 ubiquitin ligases. Cbl-b, c-Cbl, ITCH, and GRAIL are E3 ligases, and mice lacking these proteins manifest T cell hyperproliferation and autoimmune phenotypes. For example, Cbl-b binds and ubiquitinates ZAP70, resulting in its degradation and reduced TCR signaling.

Downstream TCR Signaling

The aforementioned signaling events couple TCR stimulation to downstream pathways that culminate in changes in gene transcription that are required for proliferation and effector function (see Fig. 12.4 ). One of the best-characterized genes induced following T cell activation is the T cell growth factor IL-2. Transcription of the IL2 gene is regulated in part by the transcription factors AP-1, nuclear factor of activated T cells (NFAT), and nuclear factor (NF)-κB, all of which are activated following TCR stimulation. Proximal signaling events lead to the activation of Ras and PLCγ. Ras initiates a cascade of kinases including Raf-1, MEK, and the MAPK ERK, which leads to the production of the transcription factor Fos. Ligation of the co-stimulatory molecule CD28 results in the activation of another member of the MAPK family, c-Jun N-terminal kinase (JNK), and phosphorylation of the transcription factor c-Jun. c-Jun and Fos associate to form AP-1. PLCγ hydrolyzes membrane inositol phospholipids to generate phosphoinositide second messengers including inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol. IP 3 stimulates the mobilization of calcium from intra-cellular stores. Diacylglycerol activates protein kinase C (especially PCKθ in T cells) and, along with CARMA, connects with the NF-κB pathway.

Increased intra-cellular calcium is central to many forms of cellular activation. Calcium activates the calcium/calmodulin-dependent serine phosphatase, calcineurin, which dephosphorylates NFAT. Dephosphorylated NFAT translocates to the nucleus and, together with AP-1 and NF-κB, activates the IL2 gene. The immunosuppressive agents Cyclosporin-A and FK-506 specifically inhibit the calcium-dependent activation of calcineurin, thereby blocking activation of NFAT and the transcription of NFAT-dependent cytokines such as IL-2, IL-3, IL-4, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Recently it has been appreciated that differences in the amplitude and duration of calcium signals mediate different functional outcomes. Although high spikes of calcium are easily measured in lymphocytes during the first 10 minutes after antigen stimulation, sustained low-level calcium spikes over a few hours are necessary for full activation. These latter more subtle calcium fluxes are controlled by cyclic adenosine phosphate (ADP)-ribose and ryanodine receptors. Selective inhibitors exist for these molecules, opening the potential for new specific blockers of T cell activation.


Signal 2 is mediated either by growth factor cytokines or through a co-stimulatory molecule, of which the prototype is CD28 on T cells interacting with CD80 (B7-1) or CD86 (B7-2) on APC. CD28 is a disulfide-linked homodimer constitutively expressed on the surface of T cells. Virtually all murine T cells express CD28, while in human T cells, nearly all CD4 + and 50% of CD8 + cells express CD28. The CD28 subset of T cells appears to represent a population that has undergone chronic activation and can manifest suppressive activity. Increased levels of CD28 T cells have been reported in several inflammatory and infectious conditions, including granulomatosis with polyangiitis, RA, and certain viral infections such as cytomegalovirus (CMV) and mononucleosis. The cytoplasmic domain of CD28 has no known enzymatic activity but does contain two SH3 and one SH2 binding sites. CD28 interacts with PI 3 kinase and GRB2 and promotes JNK activation. CD28 ligation alone does not transmit a proliferative response to T cells, but in conjunction with TCR engagement augments the production of several cytokines, including IL-2, IL-4, IL-5, IL-13, IFN-γ, and TNF, as well as the chemokines IL-8 and RANTES at the level of both transcription and translation.

The ligands for CD28, CD80 (B7-1), and CD86 (B7-2) are expressed in a restricted distribution on B cells, dendritic cells, and monocytes. CD80 and CD86 have similar structures but share only 25% amino acid homology. They each contain rather short cytoplasmic tails that may signal directly and bind to CD28 with different avidities. Increased levels of soluble co-stimulatory molecules CTLA-4, CD28, CD80, and CD86 have been reported in systemic lupus erythematosus (SLE) patients and correlate with disease activity.

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.

May 20, 2021 | Posted by in RHEUMATOLOGY | Comments Off on T Lymphocytes

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