The earliest responses of antigen-stimulated T cells consist of changes in the expression of various surface molecules, including cytokine receptors, as well as the secretion of cytokines. These are followed by proliferation of the antigen-specific cells, driven in part by the secreted cytokines, and then by differentiation of the activated cells into effector and memory cells. In the remainder of this chapter, we will describe these steps, their underlying mechanisms, and their functional consequences.
Changes in Surface Molecules During T-Cell Activation
After activation by antigen recognition and costimulation, there are characteristic changes in the expression of various surface molecules in T cells (Fig. 1). Many of the molecules that are expressed in activated T cells are also involved in the functional responses of the cells. These include the following:
• CD69. Within a few hours, T cells increase their expression of CD69. This protein binds to and reduces surface expression of the sphingosine-1 phosphate receptor 1 (S1PR1), which we described as a receptor that mediates egress of T cells from secondary lymphoid organs. The con sequence of decreased S1PR1 expression is that activated T cells are retained in lymphoid organs long enough to receive the signals that initiate their proliferation and differentiation into effector and memory cells. After that occurs, CD69 expression decreases, the activated T cells reexpress high levels of S1PR1, and therefore the effector and memory cells can exit the lymphoid organs.
• CD25 (IL-2Rα). The expression of this component of the receptor for the growth factor interleukin-2 (IL-2) enables activated T cells to respond to this cytokine. This process is described later.
• CD40 ligand (CD40L, CD154). Within 24 to 48 hours after antigen recognition, CD4+ T cells express high levels of the ligand for CD40. The expression of CD40L enables these activated T cells to mediate their key effector functions, which are to help macrophages and B cells. In addition, as discussed earlier, CD40L on the T cells activates DCs to become better APCs, thus providing a positive feedback mechanism for amplifying T-cell responses.
• Adhesion molecules and chemokine receptors. During activation, T cells reduce the expression of molecules that bring them to secondary lymphoid organs (such as L-selectin [CD62L] and the chemokine receptor CCR7) and increase the expression of molecules that are involved in their migration to peripheral sites of infection and tissue injury (such as the integrins LFA-1 and VLA-4, the ligands for E- and P-selectins, and various chemokine receptors). Activation also increases the expression of CD44, a receptor for the extracellular matrix molecule hyaluronan. Binding of CD44 to its ligand helps retain effector T cells in the tissues at sites of infection and tissue damage.
Fig1. Changes in surface molecules after T-cell activation. (A) The approximate kinetics of expression of selected molecules during activation of T cells by antigens and costimulators are shown. The illustrative examples include a transcription factor (c-FOS), a cytokine (IL-2), and surface proteins. These proteins are typically expressed at low levels in naive T cells and are induced by activating signals. CTLA-4 (cytotoxic T-lymphocyte antigen–4) and programmed cell death protein–1 are induced hours or 1–2 days after initial activation. The kinetics are estimates and will vary with the nature of the antigen, its dose and persistence, and the type of adjuvant. (B) The major functions of selected surface molecules are shown and described in the text. CD40L, CD40 ligand; IL-2R, interleukin-2 receptor; TCR, T-cell receptor.
Metabolic Changes During T-Cell Activation
When cells are activated, they need to increase their metabolic activity to cope with the increased demands of the cellular response. In the immune system, this phenomenon has been best studied in T cells. Upon activation by antigen and costimulators, T cells increase the transport of glucose and change their energy production from mitochondrial oxidative phosphorylation to glycolysis, even in the presence of abundant oxygen, a phenomenon known as aerobic glycolysis or the Warburg effect (Fig. 2). This was first described in tumor cells but is now recognized as an important mechanism used by many proliferating cell types. Although glycolysis generates less ATP (the molecule cells use to store and release energy) than oxidative phosphorylation, glycolysis does not use substrates other than glucose, such as amino acids and lipids, and thus preserves essential building blocks needed for the synthesis of new macromolecules and for cell division. Aerobic glycolysis in lymphocytes may be important not just for cellular proliferation but also for the differentiation of T cells into effector cells and for the production of effector cytokines.
Fig2. Metabolic changes during T-cell activation. In resting T cells, the major pathway of energy generation is mitochondrial oxidative phosphorylation. Upon activation, there is a switch to aerobic glycolysis, which generates less energy but preserves and produces the building blocks for cellular organelle biosynthesis, which is required for cell proliferation and functional responses. ATP, Adenosine triphosphate.
Cytokines in T-Cell Activation
Numerous cytokines play critical roles in adaptive immune responses. CD4+ helper T cells make the largest amount and variety of these cytokines, but some are also produced by CD8+ T cells and APCs. Cytokines secreted by DCs and other APCs are especially important for the differentiation of naive T cells into different types of effector cells. Various cytokines are involved in the proliferation and differentiation of antigen stimulated T cells and in the effector functions of these cells. Most of these cytokines act on nearby cells (paracrine action) or the cells that produce them (autocrine action). Cytokines that activate T cells work in concert with antigens and costimulation and are sometimes referred to as “signal 3.”
Here we discuss IL-2, a cytokine involved in early steps of T-cell responses.
Interleukin-2 Secretion and Interleukin-2 Receptor Expression
IL-2 is a growth, survival, and differentiation factor for T lymphocytes that plays a major role in the proliferation of anti gen-stimulated T cells and in the maintenance of functional regulatory T cells. IL-2 functions as an autocrine and paracrine cytokine.
IL-2 is produced mainly by CD4+ T lymphocytes activated by antigen and costimulators. Activation of these cells stimulates transcription of the IL2 gene and synthesis and secretion of the protein. IL-2 production is rapid and transient, starting within 1 to 2 hours after antigen recognition, peaking at about 8 to 12 hours, and declining by 24 hours. Secreted IL-2 is a 14- to 17-kD globular glycoprotein containing four α helices. It is the prototype of the four-α-helical cytokine family that interacts with type I cytokine receptors.
The high-affinity IL-2 receptor (IL-2R) is transiently expressed on activation of naive and effector T cells; regulatory T cells always express this receptor. The IL-2R consists of three noncovalently associated proteins: IL-2Rα (CD25), IL-2/15Rβ (CD122), and γc (CD132, also called IL-2Rγ) (Fig. 3). Of the three chains, only IL-2Rα is unique to the IL-2R. The β chain is also part of the IL-15 receptor, and the γ chain is shared with a number of cytokine receptors, including those for IL-4, IL-7, IL-9, IL-15, and IL-21, and is therefore called the common γ chain (γc). Both the β and γc chains engage JAK (Janus kinase)-STAT (signal transducers and activators of transcription) signaling pathways.
Fig3. The interleukin-2 receptor (IL-2R). (A) The structure of IL-2 bound to its trimeric receptor. (B) Different combinations of IL-2R chains are expressed on different cell types and bind IL-2 with different affinities. The cellular response to IL-2 is determined by which chains of the receptor are expressed. (The IL-2Rα chain is not expressed in isolation but is shown to indicate its binding affinity.) NK, Natural killer; Tregs, regulatory T cells. (A, Courtesy Drs. Kevin Jude and Chris Garcia, Stanford University. B, Modified from Spolski R, Li P, Leonard WJ. Biology and regulation of IL-2: from molecular mechanisms to human therapy. Nat Rev Immunol. 2018;18:648–659.)
IL-2Rβγc complexes are expressed at low levels on resting T cells (and on NK cells) and bind IL-2 with a Kd (equilibrium dissociation constant, a measure of binding affinity) of approximately 10−9 M. Expression of IL-2Rα and, to a lesser extent, of IL-2Rβ is increased on activation of naive CD4+ and CD8+ T cells (Fig. 4). The α chain associates with the βγc complex to form the complete IL-2R, the IL-2Rαβγc complex, which can bind IL-2 more tightly, with a Kd of approximately 10−11 M. Growth stimulation of activated T cells occurs at a similarly low IL-2 concentration. Because both IL-2 secretion and IL-2Rα production occur in response to antigen stimulation, the antigen-activated T cells are the ones that proliferate preferentially in response to the cytokine, compared with bystander cells that have not recognized the antigen. IL-2, produced in response to antigen stimulation, is itself a stimulus for induction of IL-2Rα, providing a feedback mechanism by which T-cell responses amplify themselves. Activated T cells express IL-2Rα transiently, long enough to respond to the growth factor and proliferate. CD4+ regulatory T cells constitutively express the complete IL-2R complex and require IL-2 for their maintenance, as discussed later and in Chapter 15. Chronic T-cell stimulation leads to shedding of the extracellular domain of IL-2Rα, and an increased level of shed IL-2Rα ectodomain in the serum is a marker of strong antigenic stimulation (e.g., acute rejection of a transplanted organ) and lymphoid malignancies.
Fig4. Regulation of interleukin-2 receptor (IL-2R) expression. Resting (naive) T lymphocytes express the IL-2Rβγc complex, which has a moderate affinity for IL-2. Activation of the T cells by antigen, costimulators, and IL-2 itself leads to expression of the IL-2Rα chain (also called CD25) and increased levels of the high-affinity IL-2Rαβγc complex. Regulatory T cells constitutively express the high-affinity IL-2Rαβγc com plex. APC, Antigen-presenting cell.
Functions of Interleukin-2
The biology of IL-2 is fascinating because it plays critical roles in both promoting and controlling T-cell responses and functions (Fig. 5).
• IL-2 stimulates the survival, proliferation, and differentiation of antigen-activated T cells. IL-2 promotes survival of cells by inducing the anti-apoptotic protein BCL-2. It stimulates cell cycle progression through activation of mTOR (mammalian target of rapamycin, also called molecular or mechanistic target of rapamycin), which induces the synthesis of cyclins and relieves a block in cell cycle progression through degradation of the cell cycle inhibitor p27. In addition, IL-2 increases production of effector cytokines, such as IFN-γ and IL-4, by T cells.
• IL-2 is required for the survival and function of regulatory T cells, which suppress immune responses against self and other antigens. These cells constitutively express the complete IL-2 receptor, including the α chain CD25, and are more sensitive to IL-2 than are activated and effector T cells. when we describe the properties and functions of regulatory T cells. An interesting feature of this function of IL-2 is that regulatory T cells do not produce the cytokine, so they depend on IL-2 made by other T cells responding to antigens (see Fig. 5B)
Fig5. Biologic actions of interleukin-2 (IL-2). (A) IL-2 stimulates the survival, proliferation, and differentiation of T lymphocytes, acting as an autocrine growth factor, leading to the generation of effector and memory cells. (B) IL-2 also promotes the survival of regulatory T cells and maintains their functional capability, and thus controls immune responses (e.g., against self antigens). TCR, T-cell receptor.
There are many ongoing attempts to produce versions of IL-2 that selectively activate effector cells by binding only to the IL-2βγ receptor (to stimulate immune responses in cancer patients) or act on regulatory T cells by binding to the IL-2Rα chain (to suppress immune responses in patients with autoimmune diseases).
Clonal Expansion of T Cells
T-cell proliferation in response to antigen recognition is mediated by a combination of signals from the antigen receptor, costimulators, and autocrine growth factors, primarily IL-2. The expansion of antigen-specific clones that results from this proliferation converts the small pool of naive antigen-specific lymphocytes into the large number of cells required to eliminate the antigen. Before antigen exposure, the frequency of naive T cells specific for any antigen is 1 in 105 to 106 lymphocytes or fewer in secondary lymphoid organs. After microbial antigen exposure, the frequency of CD8+ T cells specific for that microbe may increase to as many as 1 in 3 CD8+ T lymphocytes, representing a greater than 50,000-fold expansion of antigen-specific CD8+ T cells, and the number of specific CD4+ cells increases up to 1 in 100 CD4+ lymphocytes, or a 1000-fold expansion (Fig. 6). Studies in mice first showed this tremendous expansion of the antigen-specific population in some acute viral infections, and remarkably it occurred within as little as 1 week after infection. Equally remarkable was the finding that during this massive expansion of antigen-specific clones, bystander T cells not specific for the virus did not proliferate. The expansion of T cells specific for the Epstein-Barr virus and human immunodeficiency virus (HIV) in acutely infected humans is also on this order of magnitude.
Fig6. Clonal expansion of T cells. The numbers of CD4+ and CD8+ T cells specific for microbial anti gens and the expansion and decline of the cells during immune responses are illustrated. The numbers are approximations based on studies of T-cell responses in inbred mice. The relative numbers of viable infectious organisms in an individual over time is indicated by the tan-shaded curve (not related to the number of T cells).
Differentiation of Activated T Cells Into Effector
Cells Many of the progeny of the antigen-stimulated T cells differentiate into effector cells. effector cells of the CD4+ lineage express surface molecules and secrete cytokines that activate other cells (B lymphocytes, macrophages, and DCs). Whereas naive CD4+ T cells produce mostly IL-2 on activation, effector CD4+ T cells are capable of producing a large number and variety of cytokines that have diverse bio logic activities. Effector CD8+ cells are cytotoxic and kill infected cells. Because there are important differences in effector cells of the CD4+ and CD8+ lineages.
Development and Properties of Memory T Cells
T cell–mediated immune responses to an antigen usually result in the generation of memory T cells specific for that antigen, which may persist for years, even a lifetime. Memory cells provide effective defense against pathogens that are prevalent in the environment and may be repeatedly encountered. Despite the importance of immunologic memory, many fundamental questions about the generation and maintenance of memory cells have still not been answered.
The mechanisms that determine whether an individual anti gen-stimulated T cell will become a short-lived effector cell or enter the long-lived memory cell pool are not fully established, and there are three major competing models. Memory cells may develop from effector cells along a linear pathway, meaning that as effector T cells contract, a small population of memory pre cursor cells remains from which memory cells are generated (Fig. 7A). An alternative model proposes that some naive cells differentiate through multiple independent stages into memory T cells following activation. Additional stimulation may induce some of the memory cells to differentiate into terminal effector T cells that die rather than contribute to durable memory. This model implies unidirectional differentiation (Fig. 7B). Lastly, it has been proposed that effector and memory populations follow divergent differentiation and are two alternative fates of lymphocytes activated by antigen and other stimuli (Fig. 7C). The signals that drive the development of memory cells are also not fully understood. These signals may include the strength of TCR stimulation, the level of costimulation, the cytokine environment, and others. No single transcription factor determines whether an antigen-stimulated T cell will become a terminal effector cell or a memory cell; rather, this choice may be controlled by quantitative differences in numerous transcription factors and epigenetic reprogramming.
Fig7. Development of memory T cells. (A) Following activation, T cells develop into effectors, some of which survive as memory cells. (B) Activated T cells differentiate into self-renewing stem cell-like populations that can give rise to memory and effector cells. (C) Whereas memory T cells are maintained, effector cells die by apoptosis after performing their functions. Tcm , central memory T cells; Teff , effector T cells; Tem , effector memory T cells; Tscm , stem cell-like memory T cells.
Properties of Memory T Cells
The defining properties of memory cells are their ability to survive for prolonged periods after an antigen is eliminated and to mount larger and more rapid responses to antigens than do naive cells. Several features of memory cells account for these properties.
• Memory cells express increased levels of antiapoptotic proteins, which may be responsible for their prolonged survival. Whereas naive T cells live for weeks or months and are replaced by mature cells that develop in the thymus, memory T cells may survive for years. Thus, as humans age in an environment in which they are constantly exposed and responding to infectious agents, the proportion of memory cells induced by these microbes compared with naive cells progressively increases. In individuals older than 50 years of age, half or more of circulating T cells may be memory cells. The anti-apoptotic proteins that promote memory cell survival include BCL-2 and BCL-XL , which block apoptosis induced by a deficiency of survival signals. The presence of these proteins allows memory cells to survive even after antigen is eliminated and innate immune responses have subsided, when the stimuli for effector T-cell survival and proliferation are no longer present.
• Memory cells respond more rapidly to antigen stimulation than do naive cells specific for the same antigen. This is one reason why secondary responses to antigen exposure are more rapid than primary responses. A possible explanation for this accelerated response is that the gene loci for cytokines and other effector molecules are fixed in an accessible chromatin state in memory cells, in part because of changes in methylation and acetylation of histones. These epigenetically modified genes are poised to respond rapidly to antigen recognition.
• The number of memory T cells specific for any antigen is greater than the number of naive cells specific for the same antigen. As we discussed earlier, proliferation leads to a large clonal expansion in all adaptive immune responses, and the memory cells that remain from the expanded clone are typically 10- to 100-fold more numerous than the pool of naïve cells before antigen encounter. The increased population size is one reason that antigen challenge in a previously immunized individual induces a larger response than the first immunization in a naive individual.
• Memory cells are able to migrate to peripheral tissues and respond to antigens at these sites. naive T cells migrate preferentially to secondary lymphoid organs where they respond to antigens for the first time, but memory cells can migrate to virtually any tissue. These differences are related to differences in the expression of adhesion molecules and chemokine receptors. Some memory cells are resident in peripheral tissues and may contribute to recall responses to antigens in these tissues. In addition, memory T cells are less dependent on costimulation than are naive cells, allowing memory cells to respond to antigens presented by a wide range of APCs in peripheral tissues; in contrast, as we have discussed earlier, naive T cells are dependent on antigen presentation by mature DCs in secondary lymphoid organs.
• Memory cells undergo slow proliferation, and this ability to self-renew may contribute to the long-life span of the memory pool. The cycling of these cells may be driven by cytokines. Because of the capacity for self-renewal, memory cells have been likened to stem cells. Although they survive for long periods, memory cells are functionally inactive and have to be restimulated by antigen to become functional effector cells.
• The maintenance of memory cells is dependent on cytokines but does not require antigen recognition. The most important cytokine for the maintenance of memory CD4+ and CD8+ T cells is IL-7, which also plays a key role in early lymphocyte development (see Chapter 8) and in the survival of naive T cells. Predictably, high expression of the IL-7 receptor (CD127) is characteristic of memory T cells. Memory CD8+ T cells also depend on the related cytokine IL-15 for their survival. IL-7 and IL-15 induce the expression of anti-apoptotic proteins and stimulate low-level proliferation, both of which maintain populations of memory T cells for long periods. The ability of memory cells to survive without antigen recognition has been best demonstrated by experiments in mice in which antigen receptors are genetically deleted after mature lymphocytes have developed. In these mice the number of naive lymphocytes drops rapidly, but memory cells are maintained.
The most reliable phenotypic markers for memory T cells appear to be elevated surface expression of the IL-7 receptor and a protein of unknown function called CD27 and the absence of markers of naive and recently activated T cells. In humans, most naive T cells express the 200-kD isoform of the surface molecule CD45 called CD45RA (for “restricted A”), and most memory T cells express a 180-kD isoform of CD45 called CD45RO.
Both CD4+ and CD8+ memory T cells are heterogeneous and can be subdivided into subsets based on their homing properties and functions. However, memory T-cell subsets are best understood for CD8+ T cells and four distinct subsets of CD8+ memory T cells are now recognized.
• Central memory T cells (TCM ) express the chemokine receptor CCR7 and the adhesion molecule L-selectin and home mainly to lymph nodes. They have a limited capacity to perform effector functions when they encounter antigen, but they undergo brisk proliferative responses and generate many effector cells on antigen challenge. Thus, they provide a pool of memory cells that can respond to antigen challenge and develop into effector cells.
• Effector memory T cells (TEM ), on the other hand, do not express CCR7 or L-selectin, and they home to peripheral sites, especially mucosal tissues. On stimulation by an anti gen, TEM cells rapidly produce effector cytokines such as IFN-γ or become cytotoxic, but they do not proliferate much. This effector subset, therefore, is poised for a rapid response to exposure to a microbe, but complete eradication of infection may also require large numbers of effectors generated from the pool of central memory T cells. A subset of TEM cells in humans expresses the CD45RA isoform, which is characteristic of naive T cells. This population is called TEMRA cells (T effector memory RA+); whether it has unique functional properties is not known.
• Tissue-resident memory T cells (TRM ) are present in various nonlymphoid tissues, do not circulate in the blood, and may provide rapid defense against microbes in the tissues. Most of these cells express high levels of CD69, the molecule that reduces surface expression of S1PR1. As a result, these cells do not respond to the high concentrations of S1P in the lymph and blood, facilitating their retention in tissues.
• Stem-like memory T cells (TSCM ) in secondary lymphoid organs express high levels of the TCF-1 transcription factor, and account for the self-renewal and maintenance of central and effector memory T-cell pools, but it is unclear if they are required for the maintenance of the TRM pool.
Memory T cells are also heterogeneous in terms of cytokine profiles. For example, some CD4+ memory T cells may be derived from activated T cells that are not committed to the Th1, Th2, or Th17 phenotype, and when reactivated by exposure to antigen and cytokines, they can differentiate into any of these subsets. Other memory T cells may be derived from differentiated Th1, Th2, or Th17 populations and retain their respective cytokine profiles on reactivation.
