Chapter 60: Adaptive Immunity: T-Cell–Mediated Immunity

Innate immunity (see Chapter 58) and antibodies (see Chapter 61) are important mechanisms for preventing infections from taking hold, but in many infectious diseases, it is primarily the T cells that orchestrate resistance and recovery. Furthermore, T cells are important in the immune system’s surveillance for cancer, and they are responsible for most autoimmune diseases and rejection of organ transplants. The strongest evidence for the importance of T cells comes from the increase in infections and cancers that occurs when T-cell function is reduced by immunosuppressive drugs, by acquired diseases such as human immunodeficiency virus (HIV), or in congenital (primary) immunodeficiency syndromes.

The constituents of the T-cell–mediated immune system include several cell types: (1) macrophages and dendritic cells (DCs), which phagocytize microbes and present antigens to T cells (see Chapter 58); (2) effector/helper CD4-positive T cells, which use antigen receptors to recognize antigen and make cytokines that enhance or suppress immune functions; (3) cytotoxic CD8-positive T cells, which use antigen receptors to detect and kill infected cells; and (4) natural killer (NK) cells, which detect and kill infected cells using innate receptors.

The major defining feature of cell-mediated immunity, covered in detail in this chapter, is that it is critically dependent on cytokines produced by these cells. Although the interactions between various cells are complex, the result is relatively simple: opportunistic microbes only cause disease when T-cell–mediated immunity is compromised.

As discussed in previous chapters, lymphocyte precursors develop into mature B cells and T cells in the bone marrow and thymus, respectively, and these are therefore called primary lymphoid organs (see Chapter 59). The result is an enormous diversity of adaptive immune cell “clones,” and each clone has a unique and specific antigen receptor, which is either a B-cell receptor (BCR) or a T-cell receptor (TCR). At this stage, a lymphocyte is considered mature, because it has a functional antigen receptor, but naïve, because it has not yet encountered a foreign antigen that can strongly bind to its TCR or BCR. Note that only a few lymphocyte clones might be specific for any given antigen.

How do lymphocyte clones survey our entire barrier, bloodstream, and organs for microbial antigens? Secondary lymphoid organs concentrate and filter antigenic material so that immune cells can sample it and remove it if necessary. After lymphocytes complete their maturation (see Chapter 59), they exit to circulate through the secondary lymphoid organs via blood and lymphatic vessels (Figure 60–1).

Lymphatics are a specialized circulatory system parallel to the blood system, with one-way valves that keep the lymph circulating in one direction. The lymphatic vessels drain all of your body’s tissues, concentrating and filtering foreign material through draining lymph nodes. The initial phase of T-cell activation occurs in the secondary lymphoid organs when the antigen receptors of T cells and/or B cells recognize antigens. The initial activation, or priming, of naïve T cells depends on antigen-presenting cells (APCs), which are usually DCs.

Lymphoid Tissue Architecture

Secondary lymphoid organs can be divided into zones based on different functions and cellular makeup, and chemokines direct the migration of cells to their appropriate zones. The follicle is an area mostly made up of B cells, with an adjacent or surrounding T-cell zone. DCs that pick up antigens first encounter naïve T cells in the T-cell zone.

DCs process foreign proteins that are either extracellular (which they take up into vesicles) or cytoplasmic. They break these proteins down into small peptides and then load them onto major histocompatibility complex (MHC) proteins. The peptide–MHC complex is transported to the surface of the DC, where the peptide is presented to the TCRs of circulating T cells in the nearby T-cell zone of the secondary lymphoid tissue.

How do these DCs and T cells meet one another in the T-cell zone? T cells circulate freely through the bloodstream and lymphatics, using its chemokine receptor CCR7 to migrate toward chemokines produced by structural fibroblast cells in the T-cell zones of the secondary lymphoid tissue (Figure 60–2). After a DC ingests a microbe, the accompanying pathogen-associated molecular patterns (PAMPs) stimulate the cell to express the same chemokine receptor, CCR7, and therefore, the chemokine gradient also attracts the DC to the T-cell zone.

Note that some DCs and macrophages wait in the outer layers of the secondary lymphoid tissue to ingest freely circulating antigens. This is particularly important in the secondary lymphoid organs that receive antigen directly. These include the spleen, which filters the blood, so that damaged or infected red blood cells and bloodborne pathogens can be cleared and processed for antigen presentation. This also occurs in the mucosa-associated lymphoid tissues (MALT), which similarly survey and receive antigen directly from the contiguous mucosal epithelial barrier. In lymph nodes, there is an additional route of entry, namely the afferent lymphatics that drain all tissues. Antigens can either travel through these lymphatics to be ingested and processed when they arrive in a draining lymph node, or else the DCs in the peripheral tissue can take up the antigens and carry them through the afferent lymphatics to the T-cell zone using CCR7 for navigation.

T-Cell Receptor Signaling

T cells recognize only polypeptide antigens, in the form of short peptide chains. The specific polypeptide that binds to a TCR is called its cognate antigen (or cognate peptide). Furthermore, they recognize those polypeptides only when they are presented in association with MHC proteins. (As described in Chapter 59, there are rare innate-like T cells that recognize antigens presented by nonclassical MHC antigen presentation proteins, but these are minor exceptions!)

Remember the “rule of eight”: CD4-positive T cells recognize antigen in association with class II MHC proteins (4 × 2 = 8), whereas CD8-positive T cells recognize antigen in association with class I MHC proteins (8 × 1 = 8). This is called MHC restriction because each type of T cell is “restricted” to recognize antigen only when presented by the appropriate class of MHC protein. As described in Chapter 59, MHC restriction is a feature of thymic positive selection and is mediated by specific binding sites on the TCR as well as on the CD4 and CD8 proteins that bind to specific regions on the MHC proteins. Furthermore, the specific class I and class II genes you inherit from your parents ensure that your T cells are selected to only recognize antigen presented by your APCs (see Chapter 62).

When the TCR interacts with the peptide–MHC complex, the CD4 or CD8 protein on the surface of the T cell also interacts with the class II or class I MHC protein on the APC. This binding is reinforced by other protein interactions (e.g., lymphocyte function-associated antigen 1 [LFA-1] binding to intracellular adhesion molecule 1 [ICAM-1]), stabilizing the contact between the T cell and the APC. The initial activation of naïve T cells is called priming, and it occurs when the TCR recognizes a peptide–MHC complex presented by a DC in the T-cell zone of a secondary lymphoid organ. A series of cell–cell interactions provide two signals that prime the naïve T cell (Figure 60–3).

Signal 1 is the interaction of the TCR with its cognate peptide complexed with the MHC protein. When the peptide–MHC protein complex on the DC is strongly bound by the TCR, a signal is transmitted by the CD3 protein complex through several pathways that eventually lead to a large influx of calcium into the cell. (Stimulation of the TCR activates a series of phosphokinases, which then activate phospholipase C, which cleaves phosphoinositide to produce inositol triphosphate, which opens the calcium channels. The structure of the TCR is presented in Chapter 59. Further details of the signal transduction pathway are beyond the scope of this book.) Calcium activates calcineurin, a serine phosphatase. Calcineurin moves to the nucleus and is involved in the activation of the genes for interleukin-2 (IL-2) and the high-affinity IL-2 receptor. (Calcineurin function is blocked by cyclosporine, one of the most effective drugs used to prevent rejection of organ transplants [see Chapter 62].)

Co-stimulation (Signal 2)

The process of activating naïve T cells does not occur as a simple “on–off” switch. As mentioned earlier, two signals are required in the initial activation of naïve T cells, and the interactions that provide signal 2 are called co-stimulation. The best example of co-stimulation is the B7 protein on a DC that interacts with the CD28 protein on the T cell (see Figure 60–3A). Resting APCs express low levels of B7 proteins, but they increase these levels upon stimulation of their pattern recognition receptors by microbial products, such as PAMPs, or adjuvants, which are nonantigenic ingredients in vaccines (see Chapter 58).

The requirement for co-stimulation is important because it prevents inadvertent T-cell activation by benign antigens. For example, if the TCR recognizes a cognate antigen on an adjacent APC but the co-stimulatory signal is absent, the T cell adopts a state of unresponsiveness called anergy (see Figure 60–3B). As described earlier, foreign antigens from pathogens generally contain PAMPs that stimulate the pattern recognition receptors of APCs, whereas “self”-antigens do not. Co-stimulation (signal 2) tells the T cell that its cognate antigen is being presented in an inflammatory context.

Note that all of the above pathways, from TCR recognition to IL-2 stimulating proliferation, are important for activation of both CD4-positive and CD8-positive T cells. However, a unique characteristic of CD8-positive cells is that they require additional “help” in the form of cytokines from CD4-positive cells to become fully functional effector cells (see Figure 57–1). As discussed below, CD8-positive cells can be extremely lethal to host cells. Because they recognize peptide presented by class I MHC proteins, which are expressed by all nucleated cells, the additional requirement that CD8-positive cells get “help” is added insurance that the CD8-positive cells are not activated inadvertently. (The central role of CD4-positive cells in orchestrating so many different components of immune responses, as described below, explains why HIV-associated deficiency of CD4-positive cells predisposes to so such a variety of severe opportunistic infections.)

The end result of TCR stimulation is the activation of the T cell to produce various cytokines (e.g., IL-2), as well as to express the high-affinity IL-2 receptor. IL-2, also known as T-cell growth factor, stimulates the T cells to multiply, resulting in “clonal proliferation” of a population of antigen-specific T cells. As they proliferate, different progeny cells of this clonal population take on one of a number of essential functions. Some of these cells remain in the secondary lymphoid organ, while others exit via the blood or efferent lymphatics and migrate to inflamed tissues where the same TCR–peptide–MHC pathways restimulate them to exert their effector functions. Figure 60–4 is an overview of the priming of naïve CD4-positive and CD8-positive T cells in a lymph node draining a site of infection.

T-cell functions can be divided into four main categories: CD4-positive cells become (1) effector/helper (Teff or Th) cells, which leave the lymphoid organ and coordinate immune responses in inflamed tissue; (2) follicular helper (Tfh) cells, which move into the B-cell follicle of the lymphoid organ and help the B cells; and (3) regulatory T (Treg) cells, which suppress inflammation. CD8-positive cells become (4) cytotoxic T cells (or cytotoxic T lymphocytes, usually abbreviated CTL), which kill virus-infected cells and tumor cells. Remember, all of these T cells require cell–cell interactions and TCR–peptide–MHC recognition, both for their initial priming and later for their effector functions. Also, after an infection is cleared, each of these T-cell types can contribute clones to the pool of memory T cells that patrol the body and respond rapidly to reinfection. Figure 60–5 is an overview of the “help” provided by the various subsets of CD4-positive effector/helper (Th-1, Th-2, and Th-17) and follicular helper T (Tfh) cells.

Effector/Helper T Cells

CD4-positive T lymphocytes perform a variety of functions that help an immune response by enhancing the functions of other cells. T effector/helper (Th) cells leave the lymph node, migrate to inflamed tissues in the body, and produce cytokines. Different infectious pathogens must be handled by the immune system in different ways. In order to provide a targeted immune defense against a specific organism, Th cells can produce various cytokines that have various effects.

However, this means that the Th cells must be programed to produce the appropriate cytokines for the appropriate organisms. This occurs through a process of further differentiation: the signals that Th cells encounter at the time of first antigen recognition commit them to become one of several specialist cell types, or Th subsets. In inflamed tissue, the Th subsets interact with APCs, and when they sense their unique antigen, they respond in a way that is defined by their subset membership.

Table 60–1 lists the major innate cytokines that influence Th subset differentiation. Analogous to a multipotent stem cell, the original naïve cell clone has the potential to become any of the subsets. But as the progeny cells divide, their transcriptional programs are reinforced through a process of epigenetic modification. The earliest cues that start the process of differentiation are only partially known, but after only a few cell divisions, clones of the original cell can be identified that have specialist capabilities, defined by their signature transcription factors and cytokines.

Most of our understanding of Th cell subsets comes from studies in which Th cell clones can be transferred among genetically identical mice. Despite exposures to new infectious and inflammatory stimuli, these Th cells and their progeny continue to have the same signature cytokines of their original subset. For example, all effector/helper Th cell subsets express the gene PRDM1 (encoding the protein BLIMP-1) and have the ability to make copious IL-2, migrate from the secondary lymphoid organ to the site of infection, and express further cytokines upon re-stimulation of their TCR.

1. Th-1 cells are primarily responsible for “classical” activation of macrophages, leading to enhanced phagocytosis, phagolysosome free radical production, and granuloma formation (Figure 60–6 and Table 60–2). Th-1 cells arise after antigens arrive in the T-cell zone of the secondary lymphoid tissue. DCs present peptide fragments complexed with class II MHC proteins to nearby naïve CD4-positive T cells. Activation and clonal proliferation of antigen-specific Th cells occur as a result of TCR stimulation, co-stimulatory signals, and IL-2 production from the T cells.

   In certain infections, the DCs produce IL-12 at the time of Th cell activation, leading to the differentiation of these CD4-positive cells into Th-1 cells. Th-1 cells express the signature transcription factor TBX21 and produce the cytokine gamma interferon (IFN-γ). The activated Th-1 cell clones then move out of the lymphoid tissue, into the arterial circulation, and into the inflamed tissue by means of inflammation-induced extravasation (see Figure 58–4). There, they come in contact with macrophages, and after recognizing the same peptide presented with class II MHC by these macrophages, the Th-1 cells make more IFN-γ as well as tumor necrosis factor (TNF). These cytokines activate the macrophages to be more effective killers of phagocytized intracellular organisms and help the macrophages form large granulomas to wall off microbes that are hard to kill.

   Th-1 cells and macrophages play a role in host defense against many bacteria, fungi, and viruses, as well as against tumors, but individuals with deficiencies in IL-12 or IFN-γ are particularly susceptible to mycobacterial infections, such as tuberculosis. In addition to their role in controlling phagocytized pathogens, overactive Th-1 cells are associated with autoimmune and inflammatory diseases, including Crohn’s disease, psoriasis, and rheumatoid arthritis.

2. The Th-17 cell subset is closely related to the Th-1 cell subset but is generated by high levels of IL-1, IL-6, and IL-23 at the time of initial activation by DCs (see Figure 60–6 and Table 60–2). Th-17 cells express the signature transcription factors RORC and STAT3, which are reinforced by autocrine signaling from the cytokine IL-21. Th-17 cells also produce the cytokines IL-17 (the source of their name), which stimulates phagocytes and mucosal epithelial cells to increase the production of IL-1, IL-6, and neutrophil-attracting chemokines. Th-17 cells also make IL-22, which stimulates mucosal epithelial cells to increase the production of antimicrobial defensins and tight junction proteins. Together, the cytokines from Th-17 cells and the neutrophils they recruit defend the barrier tissues against bacterial and fungal infections.

   Patients with genetic mutations causing IL-17 deficiency have particular susceptibility to mucocutaneous infections from the yeast Candida albicans. In addition, loss of Th-17 cells in HIV disease is associated with chronic translocation of small numbers of bacteria from the intestinal lumen across the bowel wall and into the portal circulation. Like Th-1 cells, overactive Th-17 cells are associated with autoimmune and inflammatory diseases.

3. The Th-2 cell subset is most commonly associated with infection by certain helminth worms, such as Schistosoma and Strongyloides, which have a tissue-invasive stage of their life cycle (Figure 60–7 and Table 60–2). The signature Th-2 transcription factor is GATA3, and the signature Th-2 cytokines are IL-4 and IL-13, two cytokines that share the same receptor and therefore have similar effects. These cytokines increase the production of mucus by goblet cells at barrier surfaces, cause smooth muscle cells to be hypercontractile, and cause “alternative” activation of macrophages, leading to collagen deposition often seen in wound healing. IL-4 also signals in autocrine fashion to reinforce the Th-2 transcriptional program (analogous to IL-21 for Th-17 cells). Th-2 cells also make IL-5, which is the specific factor that recruits and maintains eosinophils, and IL-9, which activates mast cells.

   When dysregulated, Th-2 cells cause allergic disease, such as atopic dermatitis, allergic asthma, and eosinophilic gastrointestinal disease. As shown in Figure 60–7, another important part of the Th2 immune response is IgE, which mast cells and eosinophils use to detect antigen. Tfh cells are likely the main source of IL-4 that helps B cells mature to become IgE-producing plasma cells.

Follicular Helper T Cells

Tfh cells differentiate from naïve CD4-positive T cells like the other activated T cells, but rather than disperse from the lymphoid tissue to other sites, they migrate into the B-cell follicles (see Figure 60–7 and Table 60–2). The positioning of Tfh cells within the lymphoid tissue is absolutely critical to their function, as it dictates which cells they will encounter. They find the follicle by downregulating the chemokine receptor CCR7 and upregulating the chemokine receptor CXCR5, which senses chemokines produced by the stromal cells of the follicle. The earliest signals that start the Tfh cell transcriptional program are unknown, but as they proliferate and migrate to the follicle, Tfh cells begin to express the transcription factor BCL6 and suppress expression of PRDM1 (the gene encoding BLIMP-1). In addition, Tfh cells produce IL-21, which, in an autocrine fashion, increases their own BCL-6 and CXCR5 levels.

Tfh cells help B cells primarily through the production of cytokines such as IL-4 and IL-21 and also by expression of CD40 ligand (CD40L) on the Tfh cell surface, which interacts with CD40 on the surface of the B cell. (For more details on how Tfh cells promote B-cell activities, see Chapter 61.) Because of their central role in B-cell function, Tfh cells are particularly important for antibody responses, including responses to vaccines. A mutation in the gene encoding CD40L causes hyper-IgM syndrome, in which B cells are unable to “class switch” from IgM to the more mature immunoglobulin isotypes, as discussed in Chapter 68. Inappropriate Tfh activation can lead to self-reactive antibodies in autoimmune disease.

Regulatory T Cells

Normal immune responses can become pathologic if they are unrestrained. This can cause tissue damage, either from excessive inflammation at the site of an infection or from inappropriate activation of self-reactive (i.e., autoimmune) adaptive cells. The subset of CD4-positive cells called suppressor or regulatory T cells (Tregs) is responsible for limiting immune responses and maintaining tolerance of self-antigens and harmless commensal antigens. Some Tregs are programmed to be suppressive cells in the thymus, whereas others are programmed at the time of priming in secondary lymphoid organs.

The Treg cell subset expresses the signature transcription factor FOXP3. Patients with mutations in this gene have absent Tregs, enlarged secondary lymphoid organs, and severe autoimmune disease in numerous tissues. There are multiple mechanisms by which Tregs probably suppress immunity, although the most well-described mechanisms involve inhibiting Th and CTL activation. These mechanisms are described in greater detail in Chapter 66. There is significant interest in targeting these pathways, because boosting Treg function could be beneficial for transplant rejection and autoimmune disease, and inhibiting Treg function could be beneficial for cancer immunotherapy and chronic infections.

Table 60–2 lists the major cytokines produced by the various Th cell subsets and their main target cells.

Cytotoxic T Cells

CD8-positive cytotoxic (or cytolytic) T lymphocytes (CTLs) are particularly effective at killing virus-infected cells. Mature naïve CD8-positive T cells arise in the thymus and recognize non–self-peptide antigens complexed with class I MHC proteins (see Chapter 59). Because all nucleated cells express class I MHC, all nucleated cells bearing the cognate peptide for that CTL are a potential APC. To prevent inadvertent activation of self-reactive CTLs, there is an additional requirement that, during their activation, CTLs receive IL-2 produced by nearby CD4-positive T cells also undergoing activation.

For example, if a virus (e.g., influenza virus) infects and lyses a respiratory epithelial cell, virus particles (virions) are released to be phagocytized by DCs (Figure 60–8). The DCs transport these particles into the secondary lymphoid tissues, and viral peptide antigens appear on the surface of the DC in association with MHC proteins. Viral “peptide A” (red circle) is presented with class II MHC and recognized by the TCR of a CD4-positive T cell. In addition, through the process of “cross-presentation” (see Chapter 58), viral “peptide B” (gray circle) is presented with class I MHC and recognized by the TCR of a CD8-positive T cell (cell with green nucleus).

The CD4-positive cells help the DC–CD8-positive cell interaction in two ways: (1) When activated, the “A”-specific CD4-positive T cell expresses high surface levels of CD40L, which interacts with CD40 on the surface of the DC. This signals to the DC to further increase its expression of co-stimulatory molecules, ensuring activation of the “B”-specific CD8-positive cell. (Note that this mechanism of CD40L “licensing” of DCs is similar to the CD40L that Tfh cells provide to B cells, mentioned earlier and described in Chapter 61.) (2) The “A”-specific CD4-positive T cell also secretes IL-2, which directly signals the “B”-specific CD8-positive T-cell clone to proliferate. These new CTLs are virus-specific killers, able to recognize and kill any cell that displays viral peptide “B” on its surface.

The main function of CTLs is to secrete perforins and proteases into infected cells. Perforins form a channel through the membrane, which allows the cell contents to leak out. It also allows the proteases to enter the cell cytosol and degrade cellular proteins. One of these proteases, granzyme B, cleaves procaspases into their active form, initiating apoptosis. Another mechanism by which CTLs kill target cells is the Fas-Fas ligand (FasL) interaction. Fas is a protein displayed on the surface of many cells. FasL is induced on the surface of the cytotoxic cell when its TCR recognizes its cognate antigen on the surface of a target cell. When Fas and FasL interact, the caspases that initiate apoptosis in the target cell are activated. After killing the virus-infected cell, the CTL itself is not damaged and can continue to kill other cells infected with the same virus.

Like Th-1 cells, CTLs express the transcription factor TBX21 and produce the cytokine IFN-γ. CTLs are especially important as an immune defense against viruses and some intracellular bacteria, such as Listeria monocytogenes. This is because these intracellular pathogens reside within host cells and use the cell machinery to divide and spread. These pathogens spend little time outside of host cells, meaning they are not susceptible to antibody and complement, so the best way to defeat them is for CTLs to kill the host cells, allowing phagocytes to engulf the remains. (Cytotoxic T cells have no effect on free virus, only on virus-infected cells.) In some cases, the killing effect of CTLs is actually pathogenic: the severe liver damage caused by hepatitis viruses is not the result of viral cytotoxicity, but rather the result of a robust CTL response that kills virus-infected hepatocytes.

CTLs are also important in the surveillance of the body for cancer: when malignant cells accumulate somatic mutations, they begin to generate novel (non-”self”) proteins, and CTLs that recognize and are activated by DCs presenting these peptide “neoantigens” can infiltrate the tumor and kill the malignant cells expressing those proteins. Transplanted cells from an allograft can similarly be recognized as non-”self” based on the presence of different human leukocyte antigen (HLA) polymorphisms and are therefore targets of CTLs (see Chapter 62).

Memory T Cells

Memory T cells, as the name implies, endow our host defenses with the ability to respond rapidly and vigorously for many years after the initial exposure to a microbe or other foreign material. The primary immune response occurs after the initial exposure to the antigen, when the naïve T cells are first primed. The specific T-cell clones primed during the primary response proliferate to large numbers, outnumbering many of the other T-cell clones in the circulation. For example, it is estimated that during infectious mononucleosis caused by Epstein–Barr virus (EBV), 40% of all circulating CD8-positive T cells are specific for EBV lytic phase proteins. After the infection has resolved, many of the antigen-specific T cells die by apoptosis, and the remaining few persist as memory cells.

Memory cells live for many years and have the capacity to reproduce themselves over many cell generations. On subsequent exposure to the antigen, these few T-cell clones rapidly proliferate again as part of a secondary immune response, generating many more specific T cells. This secondary response to a specific antigen is stronger and faster because: (1) the starting pool of memory cells is greater than the starting pool of that clone during the primary response, so it takes less time to reexpand this population; (2) compared with naïve cells, memory cells have a lower threshold of activation, meaning smaller amounts of antigen and co-stimulation are required; and (3) activated memory cells produce greater amounts of cytokines than do naïve T cells at the time of initial priming. (See Chapter 61 for a further discussion of primary and secondary antibody responses.)

Certain proteins, particularly staphylococcal enterotoxins and toxic shock syndrome toxin, act as “superantigens” (Figure 60–9). They do this by binding to the MHC proteins and the TCRs on the surfaces of adjacent APCs and T cells, respectively, forcing the signaling molecules together. As a result, the T cell receives a strong TCR signal regardless of the peptide that is displayed in complex with the MHC molecule. Superantigens are “super” not because they activate each individual T cell more strongly, but rather because they activate a vastly larger number of the available T cells, in many cases bypassing the need for co-stimulation. For example, staphylococcal toxic shock syndrome toxin-1 (TSST-1) binds class II MHC proteins directly to the variable portion of the β chain of the TCR, specifically Vβ2. This causes unrestrained activation of any CD4-positive T cells that use this Vβ in their TCR, regardless of that TCR’s antigen specificity and regardless of the peptide complexed with the MHC protein.

Because a large percentage of human T cells use Vβ2 (up to 30%), if all of these T cells are activated, it causes massive amounts of IL-2 released from the T cells and IL-1 and TNF from macrophages. These cytokines account for many of the findings seen in toxin-mediated staphylococcal diseases, such as toxic shock syndrome. Certain viral proteins (e.g., those of mouse mammary tumor virus [a retrovirus]) also possess superantigen activity. (Although not all superantigens bind Vβ2, they all cause pathology by activating an excessive number of T cells irrespective of those cells’ TCR specificities.)

Evaluation of “immunocompetence” often depends on demonstrating intact T-cell–mediated immune responses to commonly present “harmless” antigens (i.e., a T-cell–mediated hypersensitivity reaction) or, more specifically, on laboratory assessments of T-cell numbers and function.

Enumeration of T Cells & Subpopulations

The number of each type of immune cell can be precisely counted by use of a flow cytometer (see Chapter 64). Cells are labeled with antibodies tagged with fluorescent dyes. These dyes have properties that give them specific light excitation and emission wavelengths. The various antibodies specifically bind to proteins on the surface of cells. Single cells are passed through the beam of a laser, exciting the fluorescent dyes, and the number of cells that emit light of a particular color is registered. Specific antibodies directed against T-cell markers permit the enumeration of total T cells and the percentage that are CD4-positive, CD8-positive, regulatory, etc. The normal number of CD4-positive cells in adults is between 500 and 1500 cells/μL, whereas in patients with advanced HIV/AIDS, this number drops to less than 200 cells/μL. (In Chapter 59, we described the polymerase chain reaction [PCR] assay for T-cell receptor excision circles [TRECs], which is an inexpensive newborn screening test to identify T-cell deficiencies.)

In Vivo Tests for T-Cell Competence (Skin Tests)

Skin Tests for Preexisting (Memory) T-Cell–Mediated Hypersensitivity

Most normal persons respond with inflammatory reactions to skin test antigens of Candida and other benign environmental antigens because of immune memory of past exposure to these antigens. After pricking the skin with a small quantity of these protein antigens, normal memory T-cell responses take 2 to 3 days to develop, causing an area of skin induration and redness. Absence of this immune response suggests impairment of T-cell–mediated immunity. This is also the principle behind skin testing for latent tuberculosis infection.

Skin Tests for Newly Developed T-Cell–Mediated Hypersensitivity

Most normal persons readily develop reactivity to simple chemicals (e.g., dinitrochlorobenzene [DNCB]) applied to their skin in lipid solvents. When the same chemical is applied to the same area 7 to 14 days later, the host’s newly primed T cells generate a skin reaction. Immunocompromised persons with inadequate development of T-cell–mediated responses fail to generate a reaction on DNCB rechallenge.

In Vitro Tests for T-Cell Proliferation & Function

As described earlier, flow cytometry can be used to count specific cell populations from a pool of the patient’s cells. It can also be used to identify cell proliferation by determining the percentage of cells that incorporate alkyne-modified nucleotides added to a cell culture. Uptake and incorporation of these nucleotides only occur in dividing cells.

Similarly, flow cytometry can be used to assay cell activation by determining the level of certain surface markers or measuring the production of certain cytokines. These tests can be used in conjunction with a variety of stimuli to test either antigen-specific T-cell responses, as in the case of IFN-γ release assays (IGRAs) used to diagnose latent tuberculosis, or nonspecific T-cell responses, as in the case of assays that use “mitogens” such as phytohemagglutinin or concanavalin A, which are plant extracts that bypass the TCR to stimulate T cells.

Finally, CTL cytotoxic function can be assayed by culturing CD8-positive cells with MHC-matched cells displaying foreign (e.g., viral) peptides. Some fraction of the CD8-positive cells should recognize these foreign peptides, and if they function normally, they should increase their expression of activation markers and cell-killing effector proteins and should cause widespread death of the target cells in the culture.