T lymphocytes develop in the marrow from a common lymphoid progenitor that also gives rise to B lymphocytes. While B-lymphocyte precursors remain in the marrow, T-cell precursors migrate to the thymus, where they undergo distinct maturation steps and immunologic education. This is accompanied by characteristic TCR gene and surface expression changes of the CD3 complex, CD4, and CD8. At the early stage, thymocytes are double-negative and express neither CD4 nor CD8. This is a highly heterogeneous population, which includes γδ T cells, αβ T cells that also express the NK1.1 receptor commonly found on natural killer (NK) cells, and immature thymocytes that do not yet express a complete TCR molecule, but are thought to be precursors to the αβ lineage. The latter start to express CD8 and CD4 and enter the double-positive stage, where they undergo positive/negative selection events and CD4/CD8 cell fate choice. This results in the generation of mature thymocytes and peripheral T cells that express either CD4 or CD8, but not both (Chap. 74).33
HELPER AND CYTOTOXIC T CELLS
The mutually exclusive expression of CD4 or CD8 on mature T cells defines two major blood T-cell subsets: blood T cells that express CD8 normally constitute 25 to 35 percent of the peripheral T-cell population. They recognize antigens presented by MHC class I molecules and differentiate into cytotoxic CD8 T cells. Their main function is lysis of the target cell bearing the surface antigens for which a cytotoxic T cell is specific. Within this subset, there are a range of phenotypes defined both by function and expression of markers with an immunoregulatory function. Blood T cells that solely express the CD4 surface antigen are designated helper T cells. They normally comprise approximately 65 percent of blood T cells. Generally, their function is the production of lymphokines upon activation by foreign antigens presented by MHC class II molecules, regulating and/or assisting in the active immune response. Helper T cells can differentiate into several subtypes, each secreting different cytokines to facilitate a different type of immune response.
Based on distinct cytokine patterns upon activation, mature CD4+ T cells may be divided into subsets–the first of which identified were named T-helper type 1 (Th1) and T-helper type 2 (Th2).34 In general, Th1 cells are a major source of interferon-γ (IFN-γ), and also the major T-cell population involved in activating macrophages and clearing intracellular pathogens. Th2 cells are a major source of IL-4 and are important for the generation of immunoglobulin (Ig) E, the production of eosinophils, and the immune defense against infections by parasites. Both subsets are produced from a noncommitted population of precursor T cells. The process by which commitment develops is called polarization.
In addition to IFN-γ, Th1 cells produce lymphotoxin β, IL-2, and IL-12, whereas Th2 cells also produce IL-5, IL-13, and IL-25. Human Th1 and Th2 cells can also differ in the array of surface antigens or cytokine receptors they express. Th1 cells preferentially express CD26, membrane IFN-γ, the chemokine receptors CCR1 (CD191), CCR2 (CD192), CCR5 (CD195), CXCR3 (CD183), and CXCR6 (CD186), and the receptor for IL-12 (IL-12R or CD212). Higher levels of the lymphocyte activation gene 3 (LAG-3 or CD223), a ligand for MHC class II antigens that is structurally related to CD4, have also been described. Th2 cells preferentially express CD62L, the α chain of the IL-4 receptor (IL-4Rα), the α chain of the IL-33 receptor (IL-33Rα), CD30, and the chemokine receptors CCR3 (CD193), CCR4 (CD194), CCR8 (CDw198), and, to some extent, CXCR4 (CD184).35,36,37 Distinctive expression levels of these cytokine and chemokine receptors, along with distinctive binding activities for various endothelial selectins, most likely account for the differences in the response to cytokines and tissue-specific migration of these helper T-cell subsets.
The cytokines produced by each subset also stimulate polarization of additional T cells to the same subset, while inhibiting the polarization of the other subset. In naïve CD4+ T cells, the Th1 cytokine IFN-γ induces or activates the signal transducer and activator of transcription (STAT) 1, STAT4, and the T-box transcription factor T-BET, while simultaneously modulating IL-2 and Th2 cytokines, resulting in an attenuation of Th2 cell development (Fig. 76–3).38 Several other studies demonstrate that T-BET also physically interacts with other transcription factors important for alternative T helper cell developmental decisions to functionally repress the opposing subtype specific gene expression programs and promote Th1 development.39 Similarly, IL-4 activates or enhances the expression STAT5, STAT6, and GATA3, transcription factors that play important roles in Th2 cell development (Fig. 76–3).40 Another Th2 cytokine, IL-10, inhibits Th1 cell activation, thereby limiting the production of Th1-type cytokines. Because of these self-amplifying and mutually excluding feedback loops, an immune response becomes increasingly polarized once it develops along a Th1 or Th2 pathway, particularly upon protracted stimulation by chronic infection or prolonged exposure to environmental antigens. Other factors that drive polarization are chemokines (explaining the differential expression of chemokine receptors on Th1/Th2 cells), eicosanoids, oxygen free radicals, various inflammatory mediators, and direct cell-to-cell interaction with APCs.
Differentiation of CD4+ T-cell subsets. During the course of the immune response, a naïve CD4+ T cell (top of figure) can differentiate into any one of several distinctive CD4+ T cells, as indicated beneath each differentiated cell type. Beneath the name of each T-cell subset is listed the transcription factor (if known) that is critical for the differentiation and maintenance of the subset. Interleukin (IL)-4 is critical for development of T-helper (Th)-2 cells (green), which triggers activation and/or induction of signal transducer and activator of transcription (STAT)5, STAT6, and GATA3, transcription factors that are important in Th2 differentiation. On the other hand, interferon-γ (IFN-γ) and IL-12 pattern the development of Th1 cells (light red) through the activation and/or induction of transcription factors STAT1, STAT4, and T-BET. IL-6 along with transforming growth factor beta (TGF-β) can induce blood CD4+ T cells to express the transcription factors STAT3 and RORγt (retinoic acid-related orphan receptor γ thymus isoform), which programs differentiation into Th17 cells, whereas TGF-β, retinoic acid (RA), and IL-2 induces these cells to express the transcription factors forkhead box P3 (FOXP3) and STAT5, which are required for differentiation into CD4+CD25+ regulatory T cells (TREG) cells (red). IL-21 favors differentiation of naïve CD4 T cells into follicular helper T cells (TFH [gold]). Th9 cells (dark blue) are induced by IL-4 and TGF-β, express a combination of transcription factors and secrete IL-9. There is some plasticity in these differentiated T-cell subsets. IL-6 in combination may induce TREG cells to differentiate into Th17 cells, whereas B cells stimulated via CD40-CD40-ligand (CD40L or CD154) may induce their differentiation into TFH cells, as indicated by the dashed horizontal arrows.
Functionally, Th1 cells predominantly drive cellular immunity to fight viruses and other intracellular pathogens, eliminate cancerous cells, and stimulate delayed-type hypersensitivity (DTH) skin reactions. This is mostly achieved by stimulating macrophage Fc receptor expression, phagocytosis, and antigen presentation, enhancing the capacity of macrophages to kill intracellular pathogens. Th2 cells drive humoral immunity and upregulate antibody production to fight extracellular organisms. They initiate the antibody response to antigen by activating naïve antigen-specific B cells to produce IgM antibodies, subsequently stimulate the production of switched immunoglobulin isotypes, including IgA, IgE, and neutralize and/or weakly opsonize subtypes of IgG (Chap. 75), probably via IL-4 as a B-cell stimulatory/growth factor. In addition to stimulating the production of IgE antibodies, the cytokines made by Th2 cells induce differentiation of mast cells and eosinophils.34
Several studies have indicated that Th2 polarization and accumulation at inflammatory sites is likely to trigger the hypersensitivity reaction in allergic diseases.41 On the other hand, these responses are protective against metazoan parasite infections such as helminths: Th2 responses are host protective while extracellular parasites migrate through the body, and reduce the number of parasites either through direct killing in the tissues or expulsion from the intestines.42 Other studies demonstrate that eosinophilia and elevated IgE that accompany infection with Schistosoma mansoni are caused by the induction of Th2-type cells in the immune response to parasite ova.43 The Th2 polarization in these diseases/conditions is most likely a result of minimal IL-4 secretion during initial activation. If an antigen is present at high concentrations but does not trigger acute inflammation and attendant production of IL-12, the local concentration of IL-4 increases over time and induces a Th2 polarization of cells. On the other hand, pathogens that induce acute inflammation and/or engage toll-like receptors on accessory cells and macrophages can promote production of IFN-γ and IL-12, thereby stimulating development of the immune response along the Th1 pathway.38 Immune responses restricted to Th1 cells, for example, are observed in patients with leprosy who have developed cellular immunity to Mycobacterium leprae44 or M. tuberculosis,45 in patients with Yersinia enterocolitica46 or arthritis triggered by infection with Borrelia burgdorferi.47
However, it is probably overly simplistic to view the Th1 pathway as being the more aggressive of the two, generating acute organ-specific autoimmune diseases and inflammations, while the Th2 pathway predisposes to atopic diseases and systemic autoimmune disease. For example, Helicobacter pylori-associated peptic ulcer can be regarded as a Th1-driven immunopathologic response to some H. pylori antigens, while deregulated and exhaustive H. pylori-induced T-cell–dependent B-cell activation can support the onset of low-grade B-cell lymphoma.48 In addition, many chronic inflammatory and autoimmune conditions such as rheumatoid arthritis (RA), type 1 diabetes, and multiple sclerosis (MS) are mixed Th1/Th2 conditions, and a clear Th1/Th2 bias has also not been identified yet for many types of cancer.34
CD4+CD25+ Regulatory T Cells
Another type of CD4 cells that suppresses rather than provides helper activity are regulatory T (TREG) cells. TREGs possess potent suppressive capacity and can exert diverse suppressive mechanisms allowing them to influence a very broad range of cell populations in a variety of anatomical locations and disease scenarios, both by direct cell–cell contact and the secretion of cytokines.49 The cardinal phenotypic features of CD4+ TREG cells include their constitutive expression of the transcription factor forkhead box P3 (FOXP3), their cell surface expression of CD25 (the low-affinity receptor for IL-2 and their cell surface and cytoplasmic expression of the coinhibitory receptor cytotoxic T-lymphocyte antigen 4 (CTLA-4 or CD152).50
FOXP3 is an essential transcription factor required to manifest the TREG cell phenotype,51 but it does not function alone and requires the expression of additional transcription factors to define the TREG cell phenotype and to establish its characteristic transcriptional programme.52 Patients with the immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX syndrome) are found to have germline mutations in the gene encoding FOXP3, which maps to the long arm of the X chromosome at Xp11.23 (Chap. 80).53 TREG developmental deficiency or dysfunction is a hallmark of IPEX, leading to severe, multiorgan, autoimmune phenomena. Patients typically have autoimmune skin conditions, such as bullous pemphigoid or alopecia universalis, and autoimmune endocrinopathies similar to those seen in patients with the autoimmune polyendocrine candidiasis ectodermal dystrophy syndrome (APECED syndrome), which is associated with genetic defects in the autoimmune regulator (AIRE) gene responsible for the generation of T-cell tolerance in the thymus (Chaps. 6 and 80).54,55 The IPEX syndrome demonstrates the importance of TREG cells in maintaining tolerance to self-antigens and in preventing runaway immune responses to environmental antigens that might evolve into cross-reactive autoimmunity.
Additional key factors and signals required for TREG cell development and survival include IL-2, transforming growth factor-β (TGFβ) and co-stimulatory molecules.56 It is also becoming clear that unique epigenetic changes that are partly induced by TCR signalling are typical of the TREG cell lineage, and that they can be used to differentiate TREG subpopulations such as thymus-derived TREG (tTREG) cells and peripherally derived TREG (pTREG) cells.57,58 These terms have replaced “natural FOXP3+ TREG cells” and “induced or adaptive TREG cells” to more accurately describe the anatomical location of their differentiation. tTREG cells differentiate from CD4+CD8– T cells that have undergone positive and negative selection to self-antigens presented in the thymus (Chap. 6) and are thought to play a role in maintaining tolerance to self-antigens. pTREG cells, on the other hand, differentiate upon antigen encounter under certain conditions and during normal homeostasis of the gut.59 As such, pTREGs are thought to play an important role in the development and maintenance of mucosal immune tolerance and in the control of severe chronic allergic inflammation and “altered” self-antigens of inflamed tissues or neoplastic cells. They are also required to minimize tissue damage in inflammatory settings such as viral infection60 or mediate tolerance to allografts.61 tTREG and pTREG cells have subtle differences in the methylation status of conserved noncoding sequence 2 (CNS2; also known as the TREG cell-specific demethylated region [TSDR]) in the FOXP3 locus, potentially influencing the stability of the cells under inflammatory or pathogenic conditions.62
TREG cells can specifically suppress immune responses via contact-dependent and cytokine-mediated mechanisms.63 Upon activation through their TCR, TREGs can (1) produce antiinflammatory cytokines (e.g., IL-10, TGF-β, or IL-35), (2) reduce the availability of IL-2 via absorption by the CD25 receptor, (3) lyse other immune effector cells via granzyme secretion or CD95-CD95L–mediated cell killing, (4) modulate the activation state and/or function of APCs and other immune effector cells, and/or (5) release suppressor factors, such as galectin-164 (a β-galactoside–binding protein that can bind and inhibit the function of many glycoproteins, including CD7, CD43, and CD45) and fibrinogen-like protein 2 (FGL2; a member of the fibrinogen family that mediates it suppressive effect through binding to low affinity Fcγ receptors expressed on APCs).65 Consequently, TREG cells can act directly against specific target antigens, while activating suppressive functions of other types of immune effector cells such as CD4+ and CD8+ cells.
Naïve CD4+ T cells also can differentiate into Th17 T cells that play an important role in immune responses to certain extracellular pathogens and fungi.66 These cells produce IL-17 (sometimes referred to as IL-17A) and a closely related cytokine, IL-17F, which can form biologically active homodimers or heterodimers and induce substantial tissue reactions because of the broad distribution of the IL-17 and IL-22 receptors. Principal cytokines involved in the differentiation of naïve blood CD4+ T cells into Th17 cells are IL-23 and IL-1β (see Fig. 76–3), but a combination of TCR stimulation and the cytokines TGF-β and IL-6 are also required.67 Prostaglandins, most notably prostaglandin E2, can synergize with IL-23 and IL-1β to drive differentiation of CD4+ T cells into Th17 cells.68 These cytokines and factors can induce activation and/or expression of transcription factors that are distinct from those used by Th1 or Th2 cells, including the retinoic acid-related orphan receptor γ (RORγt) and STAT3 (see Fig. 76–3),69,70 which, in turn, can induce expression of IL-17 and IL-17F.71,72 However, for full commitment of precursors to the Th17 lineage, RORγt and STAT3 must act in cooperation with other transcription factors, including RORα, interferon regulatory factor 4 (IRF4), and runt-related transcription factor 1 (RUNX1). Th17 cells also express high levels of the IL-23R, CCR4, CCR6, CXCR4, CD161, and multiple CD49 integrins, but not CCR2, CCR5, or CCR7.37,73,74 In contrast to Th1 or Th2 cells, Th17 cells do not elaborate IFN-γ or IL-4, both of which can inhibit expression of IL-17.75
Th17 cells play a central role in inflammation and defense against intestinal bacteria, extracellular pathogens, and fungal infections, mostly via activation of neutrophils. Common pathogens that induce mainly Th17 responses include Gram-positive Propionibacterium acnes, gram-negative Citrobacter, Klebsiella pneumoniae, bacteroides and Borrelia species, and fungi such as Candida albicans.76 Th17 cells are abundantly found in the intestinal lamina propria, where they are induced and stimulated by commensal bacteria, maintain epithelial integrity, and clear extracellular pathogens.77
Th17 cells are the principal producers of IL-17 in response to specific immune stimulation, but NK and natural killer T (NKT) cells are also able to produce IL-17. IL-17 is a proinflammatory cytokine that has pleiotropic effects on multiple target cells, resulting in enhanced antigen presentation, antibody production, macrophage activation, cellular extravasation, and neutrophil migration.78 In addition to IL-17 and IL-17F, Th17 cells elaborate other proinflammatory factors, including chemokines (e.g., CXCL8 [IL-8] and CCL20), cytokines (e.g., IL-6, tumor necrosis factor-α, IL-21, and IL-22), growth factors (e.g., granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor), acute phase proteins (e.g., C-reactive protein), and antimicrobial peptides and mucins.76
The importance of Th17 cells in the defense against certain microorganisms is reflected in the rare primary immunodeficiency disorder called autosomal dominant hyper-IgE syndrome, in which a mutation in STAT3 abrogates Th17 differentiation (Chap. 80).79,80 Patients lack Th17 cells and have an increased susceptibility to infection with the various species of Staphylococcus or Candida. Furthermore, the loss of intestinal commensal bacteria that are essential for the induction of Th17 cells through the use of antibiotics can cause depletion in intestinal Th17 cells, and might account in part for the increased incidence of gastrointestinal infections with C. albicans or Clostridium difficile observed in patients subjected to long-term, broad-spectrum antibiotic therapy.81 Because of their capacity to enhance inflammation in an antigen-specific manner, Th17 cells also have been implicated in the development and/or propagation of several autoimmune disease, such as rheumatoid arthritis,82 systemic lupus erythematosus (SLE),83 MS,84 inflammatory bowel disease,85 glucocorticoid-resistant asthma,86 and psoriasis.87
T follicular helper cells (TFH cells) constitute another subset of CD4+ T cells that regulates the development of antigen-specific B-cell immunity in the germinal center of secondary lymphoid follicles. TFH cells express the CXCR5 chemokine receptor, allowing them to home to the CXCL13-rich B-cell zones of lymphoid follicles where they engage antigen-specific B cells in cognate intercellular interactions, and cell-surface proteins such as programmed cell death-1 (PD-1), inducible T-cell costimulator (iCOS), B- and T-lymphocyte attenuator (BTLA), and CD40L, which allow them to form stable contacts with antigen-primed B cells.88 Such interactions play a critical role in B-cell differentiation into plasma cells or memory B cells in response to antigenic stimulation. In addition, TFH cells secrete cytokines such as IL-4, IFN-γ, IL-10, and/or IL-21, which partly overlap with cytokines characteristic of other T-effector cells, helping to modify the differentiation fate of B lymphocytes (Chap. 75).
TFH cells also express the cytoplasmic adaptor protein signal lymphocyte activation molecule (SLAM)-associated protein (SAP), required for lymphocyte interactions, and the transcription factor B-cell lymphoma 6 (BCL-6), required for TFH cell differentiation. Several lines of evidence suggest that TFH differentiation is a multistage process, but that dendritic cells (DCs) are crucial for CD4+ T cell priming and initial acquisition of TFH cell characteristics, including the induction of BCL-6 expression.89 The flexibility and plasticity of TFH cells, which is mostly mediated by chromatin modifications, is underlined by the expression of a constellation of transcription factors, including BCL-6, BATF, STAT3, IRF4, c-Maf, and GATA-3, many of which are expressed by other Th effector subsets (see Fig. 76–3).
Another helper T-cell subset that has recently emerged are Th9 cells. In contrast to Th17 cells and TREGs, they are induced by the combination of TGF-β and IL-4 and regulated by the transcription factors PU.1, STAT6, IRF4, and GATA-3.90 They primarily produce IL-9, IL-10, and IL-21.91 Functionally, Th9 cells appear to be effector rather than regulatory, and they have been implicated in the development of allergic reactions, particularly in the lungs.92 Recent data indicates that Th9 cells in vivo might be implicated in tumor immunity in melanoma.93,94
Following a successful immune response to antigen including the exposure to and recognition of antigen, expansion of T-cell subsets and exertion of effector function, both naïve CD4+ and CD8+ T-cells can develop into long-lived memory T cells that provide enhanced protection from re-exposure to the same or a related pathogen.95,96 Memory T-cell populations maintain the ability to both survive independently of cognate antigen97 and self-renew in response to external homeostatic signals such as IL-15 and IL-7, hence maintaining themselves at stable levels for many years.98 In addition, they have less-stringent requirements for activation and an enhanced capacity for lymphokine production upon rechallenge with the same antigen, and require a lower level of costimulatory factors.
Memory CD4+ or CD8+ T lymphocytes can also be distinguished from naïve cells based on their surface phenotypes of CD45 isoforms, rate of cycling, and migration. CD45, also known as leukocyte common antigen or T200, consists of a family of membrane glycoproteins, ranging from 180 to 220 kDa, that are expressed on all leukocytes.99 Each member is the product of a single complex gene on chromosome 1 that contains 34 exons. Exons 3 through 7 may be spliced differently at the RNA transcript level to generate several distinct messenger RNA and protein products. The deduced amino acid sequences of these protein products have extracellular domains ranging from 391 to 552 amino acids, a transmembrane region, and a highly conserved cytoplasmic domain of 705 amino acids. This large cytoplasmic domain contains an intrinsic tyrosine phosphatase activity that is important in the regulation of various activation pathways involving tyrosine kinase activity, such as those involved in signal transduction via the TCR for antigen.100 Different isoforms of CD45, designated as CD45R, have distinct expression patterns during lymphocyte ontogeny and activation. Therefore, cell subsets can be readily characterized by flow cytometry approaches using specific monoclonal antibodies. Naïve CD4+ T cells express CD45RA, whereas memory CD4+ T cells and CD8+ T cells express CD45RO. CD45RB can also be useful for distinguishing memory T cells. Within the CD4+ memory T-cell population, for example, there is an increase of helper activity associated with the shift from a CD45RBbright to a CD45RBdim phenotype.101
In addition, the distinct expression of chemokine receptors (CCRs) or homing molecules can be used to characterize T-cell subsets: relative to naïve T cells, memory T cells express lower levels of L-selectin (CD62L) and higher levels of CD29 and CD44.102 More recent studies demonstrate that memory cells can be further subdivided into CD44+CD62L+CCR7+ central memory T cells (TCM cells) and CD44+CD62L−CCR7− effector memory T cells (TEM cells).103 Because of their constitutive expression of CCR7 and CD62L, TCM cells home to secondary lymphoid organs, where they have little or no immediate effector function, but show greater sensitivity to antigenic stimulation in comparison to naïve T cells, are less dependent on costimulation, and upregulate CD40L to a greater extent. However, upon recognition of their cognate antigen, they have a high proliferative potential and can rapidly differentiate into large numbers of effector cells. TEM cells, in contrast, have higher migratory potential and display immediate effector function. Therefore, TCM cells are predominantly found in the CD4 lineage and are enriched in lymph nodes and tonsils, whereas TEM cells are more frequent in the CD8 compartment in lung, liver, and intestines. Accordingly, CD8+ TEM cells carry large amounts of perforin, and both CD4+ and CD8+ TEM cells can produce IFN-γ, IL-4, and IL-5 within hours after following antigenic stimulation.
It has long been controversial whether memory T cells arise during the contraction phase and develop directly from effector cells, or whether they diverge early during an immune response, and arise in parallel with short-lived effector cells. Recent studies, however, have provided evidence for an early delineation of the effector versus memory T-cell fates regulated through specific transcription factors and cytokines, such as IL-7 receptor α-chain (IL-7R) expression, IL-2, IL-12, and T-BET, EOMES, and BLIMP-1.104,105