Chapter 8:  T Cell-Mediated Immunity

8-1. T-cell responses are initiated in peripheral lymphoid organs by activated antigen-presenting cells

Adaptive immune responses are not initiated at the site where a pathogen first establishes a focus of infection. They occur in the organized peripheral lymphoid tissues through which naive T cells are continually migrating. Pathogens or their products are transported to lymphoid tissue in the lymph that drains the infected tissue, or, more rarely, by the blood. Pathogens infecting mucosal surfaces accumulate in lymphoid tissues such as the Peyer's patches of the gut or the tonsils; those that enter the blood are trapped in the spleen; while those infecting peripheral sites are trapped in the lymph nodes directly downstream of the site of infection (see Section 1-3). All these lymphoid organs contain cells specialized for capturing antigen and presenting it to T cells. The most important of these are the dendritic cells, which capture antigen at the site of infection and then migrate to the downstream lymph node.

The delivery of antigen from a site of infection to downstream lymphoid tissue and its subsequent presentation to naive T cells is actively aided by the innate immune response to infection. As discussed in Chapter 2, this is rapidly triggered at the site of infection by nonclonotypic receptors that recognize molecular patterns that are associated with pathogens but not host cells. One of the induced responses of innate immunity is an inflammatory reaction that increases the entry of plasma into the infected tissues and the consequent drainage of tissue fluids into the lymph. Another is the induced maturation of tissue dendritic cells that have been taking up particulate and soluble antigens at the site of infection (Fig. 8.2). These cells are activated through receptors that signal the presence of pathogen components bound by dendritic cell receptors, or by cytokines produced during the inflammatory response. The dendritic cells respond by migrating to the lymph node and expressing the co-stimulatory molecules that are required, in addition to antigen, for the activation of naive T cells. Macrophages, which are phagocytic cells found in the tissues and scattered throughout lymphoid tissue, and B cells, which bind pathogen components, may be similarly induced through nonspecific receptors to express co-stimulatory molecules and act as antigen-presenting cells. Thus the innate immune response to infection hastens the transport of antigens to the local lymphoid tissue, and enables those cells that have taken up antigen to present it effectively to the naive T cells that migrate through this tissue.

The distribution of dendritic cells, macrophages, and B cells in a lymph node is shown in Fig. 8.3. Dendritic cells are present mainly in the T-cell areas. These cells are named after their fingerlike processes, which form a network of branches among the T cells. By the time they arrive in the lymph nodes, dendritic cells have lost their ability to capture new antigen. They are, however, able to present the antigens they ingested at the site of infection and in their mature, activated form they are the most potent antigenpresenting cells for naive T cells.

Macrophages are found in many areas of the lymph node, especially in the marginal sinus, where the afferent lymph enters the lymphoid tissue, and in the medullary cords, where the efferent lymph collects before flowing into the blood. Here they can actively ingest microbes and particulate antigens and so prevent them from entering the blood. As most pathogens are particulate, macrophages in the T-cell areas may stimulate immune responses to many sources of infection.

Finally, the B cells, which recirculate through the lymphoid tissues and concentrate in the lymphoid follicles, are particularly efficient at taking up soluble antigens such as bacterial toxins by the specific binding of antigen to the B-cell surface immunoglobulin. The antigen:receptor complex is internalized by receptor-mediated phagocytosis, and degraded fragments of the antigen can return to the B-cell surface complexed with MHC class II molecules. Antigen-specific B cells can thus activate naive CD4 T cells if the B cells are also induced to express co-stimulatory molecules. B cells are, however, very inefficient at initiating adaptive immune responses. This is because only those with the appropriate receptor specificity can internalize and present a particular antigen at high frequency, and these will be very scarce. Thus, the probability of their encountering a naive T cell specific for the same antigen is very low.

The antigen-presenting function of dendritic cells, macrophages, and B cells will be discussed in more detail in Sections 8-5 to 8-7. Only these three cell types express the specialized co-stimulatory molecules required to activate naive T cells; furthermore, all of these cell types express these molecules only when suitably activated in the context of a response to infection. Dendritic cells can take up, process, and present a wide variety of pathogens and antigens and appear to be the most important activators of naive T cells, whereas macrophages and B cells specialize in processing and presenting antigens from ingested pathogens and soluble antigens, respectively, and are also the targets of subsequent actions of armed effector CD4 T cells.

8-2. Naive T cells sample the MHC:peptide complexes on the surface of antigen-presenting cells as they migrate through peripheral lymphoid tissue

Naive T cells enter lymphoid tissue by crossing the walls of specialized venules known as high endothelial venules (HEV). They circulate continuously from the bloodstream to the lymphoid organs and back to the blood, making contact with many thousands of antigen-presenting cells in the lymphoid tissues every day. These contacts allow the sampling of MHC:peptide complexes on the surface of these antigen-presenting cells, which is important for two reasons. One is that it appears to reinforce the process of positive selection for self MHC recognition that occurred during T-cell development. As we discussed in Chapter 7, T-cell receptors are selected for their ability to interact with self MHC:self peptide complexes during T-cell development. In this way, a repertoire of mature T cells is selected that can be activated by nonself peptides bound to the same MHC molecules. Recent experiments show that T-cell survival in the periphery also depends on contact with self MHC:self peptide ligands (see Section 7-32), and that the signals required for survival are delivered effectively through interactions with MHC:peptide complexes on dendritic cells. Thus, as naive T cells migrate through peripheral lymphoid tissue, they receive survival signals through their interactions with dendritic cells. At the same time, the sampling of MHC:peptide ligands ensures that each T cell has a high probability of encountering antigens derived from pathogens at any site of infection. This is crucial for the initiation of an adaptive immune response, as only one naive T cell in 10⁴-10⁶ is likely to be specific for a particular antigen, and adaptive immunity depends on the activation and expansion of such rare antigen-specific T cells (Fig. 8.4). The T cells that do not encounter their antigen eventually reach the medulla of the lymph node, from where they are carried by the efferent lymphatics back to the blood to continue recirculating through other lymphoid organs. Naive T cells that recognize their antigen on the surface of a dendritic cell cease to migrate, and embark on the steps that generate armed effector cells. The generation of effector cells from a naive T cell takes several days. At the end of this period, the armed effector T cells leave the lymphoid organ and reenter the bloodstream to migrate to sites of infection.

8-3. Lymphocyte migration, activation, and effector function depend on cell-cell interactions mediated by cell-adhesion molecules

The migration of naive T cells through the lymph nodes, and their initial interactions with antigen-presenting cells, depend on cells binding to each other through interactions that are not antigen-specific. Similar interactions eventually guide the effector T cells into the peripheral tissues and play an important part in their interaction with target cells. Binding of T cells to other cells is controlled by an array of adhesion molecules on the surface of the T lymphocyte that recognize a complementary array of adhesion molecules on the surface of the interacting cell. The main classes of adhesion molecule involved in lymphocyte interactions are the selectins, the integrins, members of the immunoglobulin superfamily, and some mucinlike molecules. We have already encountered members of the first three classes in the recruitment of neutrophils and monocytes to sites of infection during an innate immune response (see Section 2-22). Most adhesion molecules play fairly broad roles in the generation of immune responses. Many that are involved in lymphocyte migration and the interactions of armed effector T cells with their targets are also involved in interactions between other leukocytes. Adhesion molecules are important in getting lymphocytes together in adaptive immune responses that involve T-cell-B-cell interactions, and we will describe these in Chapter 10, where we present an integrated view of the immune response.

The selectins (Fig. 8.5) are particularly important for leukocyte homing to particular tissues, and can be expressed either on leukocytes (L-selectin, CD62L) or on vascular endothelium (P-selectin, CD62P, and E-selectin, CD62E). L-Selectin is expressed on naive T cells and guides their exit from the blood into peripheral lymphoid tissues. P-Selectin and E-selectin are expressed on the vascular endothelium at sites of infection and serve to recruit effector cells into the tissues at these sites (see Sections 2-21 and 2-22). Selectins are cell-surface molecules with a common core structure, distinguished from each other by the presence of different lectinlike domains in their extracellular portion (see Fig. 2.34). The lectin domains bind to particular sugar groups, and each selectin binds to a cell-surface carbohydrate. L-Selectin binds to the carbohydrate moiety, sulfated sialyl-Lewis^x, of mucinlike molecules called vascular addressins, which are expressed on the surface of vascular endothelial cells. Two of these addressins, CD34 and GlyCAM-1, are expressed as sulfated sialyl-Lewis^x molecules on high endothelial venules in lymph nodes. A third, MAdCAM-1, is expressed on endothelium in mucosa, and guides lymphocyte entry into mucosal lymphoid tissue such as that of the gut.

The interaction between L-selectin and the vascular addressins is responsible for the specific homing of naive T cells to lymphoid organs but does not, on its own, enable the cell to cross the endothelial barrier into the lymphoid tissue. For this, proteins from two other families—the integrins and the immunoglobulin superfamily—are required. These proteins also play a critical part in the subsequent interactions of lymphocytes with antigen-presenting cells and later with their target cells.

The integrins comprise a large family of cell-surface proteins that mediate adhesion between cells, and between cells and the extracellular matrix, in normal development as well as in immune and inflammatory responses. Integrins bind tightly to their ligands after receiving signals that induce a change in conformation. For example, as we saw in Chapter 2, signaling by chemokines activates integrins on leukocytes to bind tightly to the vascular surface during migration of leukocytes into sites of inflammation. Chemokines similarly activate T-cell integrins during the migration of T lymphocytes into lymphoid organs and in the migration of activated T lymphocytes to sites of infection.

The migration of naive T cells into lymphoid tissues is mediated by the chemokine SLC (secondary lymphoid tissue chemokine). This is expressed by the high vascular endothelium, stromal cells, and dendritic cells in lymphoid tissue, and binds to the CCR7 chemokine receptor on naive T cells. This interaction, by a mechanism as yet unknown, is able to increase the affinity of integrin binding, arresting the T cell's progress through the blood and enabling it to enter the lymphoid tissue. Similar interactions with chemokines produced at sites of inflammation direct effector T-cell migration into the tissues; this will be discussed in more detail when we describe the functions of effector T cells in Chapter 10. Chemokines are not the only molecules able to signal the upregulation of integrin affinity; later in this chapter we will see how signaling through the T-cell receptor also triggers T-cell integrins to adhere tightly to their ligands on the antigen-presenting cell.

The integrins were introduced in Chapter 2, so we will just review their most important characteristics here. An integrin molecule consists of a large α chain that pairs noncovalently with a smaller β chain. There are several subfamilies of integrins, broadly defined by their common β chains. We will be concerned chiefly with the leukocyte integrins, which have a common β₂ chain with distinct α chains (Fig. 8.6). All T cells express a β₂ integrin known as lymphocyte function-associated antigen-1 (LFA-1). This leukocyte integrin is also found on macrophages and neutrophils, and is involved in their recruitment to sites of infection (see Sections 2-21 and 2-22). LFA-1 plays a similar role in the migration of both naive and effector T cells out of the blood. In addition, it is thought to be the most important adhesion molecule for T-lymphocyte activation, because antibodies to LFA-1 effectively inhibit the activation of both naive and armed effector T cells

Surprisingly, T-cell responses can be normal in patients lacking the β₂ integrin chain and hence all integrins that contain β₂, such as LFA-1. This is probably because T cells also express other adhesion molecules, including CD2 and β₁ integrins, which may be able to compensate for the absence of LFA-1. Expression of the β₁ integrins increases significantly at a late stage in T-cell activation, and they are thus often called VLAs, for very late activation antigens; we will see in Chapter 10 that they play an important part in directing armed effector T cells to their inflamed target tissues.

Many cell-surface adhesion molecules are members of the immunoglobulin superfamily, which also includes the antigen receptors of T and B cells, the co-receptors CD4, CD8, and CD19, and the invariant domains of MHC molecules. At least five adhesion molecules of the immunoglobulin superfamily are especially important in T-cell activation (Fig. 8.7). Three very similar intercellular adhesion molecules (ICAMs)—ICAM-1, ICAM-2, and ICAM-3—all bind to the T-cell integrin LFA-1. ICAM-1 and ICAM-2 are expressed on endothelium as well as on antigen-presenting cells; binding to these molecules enables lymphocytes to migrate through blood vessel walls. ICAM-3 is expressed only on leukocytes and is thought to play an important part in adhesion between T cells and antigen-presenting cells, particularly dendritic cells. In addition to binding LFA-1, ICAM-3 binds with high affinity to a recently discovered lectin called DC-SIGN, which is found only on dendritic cells. Another interaction involving immunoglobulin superfamily molecules is mediated by LFA-3 on the antigen-presenting cell binding to CD2 on the T cell; this interaction synergizes with that between LFA-1 and ICAM-1 and ICAM-2.

8-4. The initial interaction of T cells with antigen-presenting cells is mediated by cell-adhesion molecules

As they migrate through the cortical region of the lymph node, naive T cells bind transiently to each antigen-presenting cell they encounter. Antigenpresenting cells, and dendritic cells in particular, bind naive T cells very efficiently through interactions between LFA-1, CD2, and ICAM-3 on the T cell, and ICAM-1, ICAM-2, LFA-3, and DC-SIGN on the antigen-presenting cell (Fig. 8.8). The binding of ICAM-3 to DC-SIGN is unique to the interaction between dendritic cells and T cells, while the other molecules synergize in the binding of lymphocytes to all three types of antigen-presenting cell. Perhaps because of this synergy, the precise role of each adhesion molecule has been difficult to distinguish. People lacking LFA-1 can have normal T-cell responses, and this also seems to be the case for genetically engineered mice lacking CD2. It would not be surprising if there were enough redundancy in the molecules mediating T-cell adhesive interactions to enable immune responses to occur in the absence of any one of them; such molecular redundancy has been observed in other complex biological processes.

The transient binding of naive T cells to antigen-presenting cells is crucial in providing time for T cells to sample large numbers of MHC molecules on each antigen-presenting cell for the presence of specific peptide. In those rare cases in which a naive T cell recognizes its peptide:MHC ligand, signaling through the T-cell receptor induces a conformational change in LFA-1, which greatly increases its affinity for ICAM-1 and ICAM-2. This conformational change is the same as that induced by signaling through chemokine receptors during the migration of leukocytes to sites of infection (see Section 2-20), although its mechanism is not known. The change in LFA-1 stabilizes the association between the antigen-specific T cell and the antigen-presenting cell (Fig. 8.9). The association can persist for several days, during which time the naive T cell proliferates and its progeny, which also adhere to the antigen-presenting cell, differentiate into armed effector T cells.

Most T-cell encounters with antigen-presenting cells do not, however, result in recognition of an antigen. In these encounters, the T cells must be able to separate efficiently from the antigen-presenting cells so that they can continue to migrate through the lymph node, eventually leaving via the efferent lymphatic vessels to reenter the blood and continue circulating. Dissociation, like stable binding, may also involve signaling between the T cell and the antigen-presenting cells, but little is known of its mechanism.

8-5. Both specific ligand and co-stimulatory signals provided by an antigen-presenting cell are required for the clonal expansion of naive T cells

We saw in Chapter 3 that armed effector T cells are triggered when their antigen-specific receptors and either the CD4 or CD8 co-receptors bind to peptide:MHC complexes. By contrast, ligation of the T-cell receptor and co-receptor does not, on its own, stimulate naive T cells to proliferate and differentiate into armed effector T cells. The antigen-specific clonal expansion of naive T cells requires a second, or co-stimulatory, signal (Fig. 8.10), which must be delivered by the same antigen-presenting cell on which the T cell recognizes its antigen. CD8 T cells appear to require a stronger co-stimulatory signal than CD4 cells and, as we will see later, their clonal expansion is aided by CD4 cells interacting with the same antigen-presenting cell.

The best-characterized co-stimulatory molecules are the structurally related glycoproteins B7.1 (CD80) and B7.2 (CD86). We will call them the B7 molecules from here on, as functional differences between the two have yet to be defined. The B7 molecules are homodimeric members of the immunoglobulin superfamily that are found exclusively on the surfaces of cells that can stimulate T-cell proliferation. Their role in co-stimulation has been demonstrated by transfecting fibroblasts that express a T-cell ligand with genes encoding B7 molecules and showing that the fibroblasts could then stimulate the clonal expansion of naive T cells. The receptor for B7 molecules on the T cell is CD28, yet another member of the immunoglobulin superfamily (Fig. 8.11). Ligation of CD28 by B7 molecules or by anti-CD28 antibodies co-stimulates the clonal expansion of naive T cells, whereas anti-B7 antibodies, which inhibit the binding of B7 molecules to CD28, inhibit T-cell responses. Although other molecules have been reported to co-stimulate naive T cells, so far only the B7 molecules have been shown definitively to provide costimulatory signals for naive T cells in normal immune responses.

Once a naive T cell is activated, however, it expresses a number of proteins that contribute to sustaining or modifying the co-stimulatory signal that drives clonal expansion and differentiation. One such protein is CD40 ligand, so-called because it binds to CD40 on antigen-presenting cells. Binding of CD40 ligand by CD40 transmits activating signals to the T cell and also activates the antigen-presenting cell to express B7 molecules, thus stimulating further T-cell proliferation. CD40 and CD40 ligand belong to the TNF family of receptors and ligands and, as we will describe later in this chapter, have a central role in the effector function of fully differentiated T cells. Their earlier role in sustaining the development of a T-cell response is demonstrated by mice lacking CD40 ligand; when these mice are immunized, the clonal expansion of responding T cells is curtailed at an early stage. Another pair of TNF family molecules that appear to contribute to co-stimulation of T cells are the T-cell molecule 4-1BB (CD137) and its ligand 4-1BBL, which is expressed on activated dendritic cells, macrophages, and B cells. As with CD40L and CD40, the effects of this receptor-ligand interaction are bidirectional, with both the T cell and the antigen-presenting cell receiving activating signals; this process is sometimes referred to as the T-cell/antigen-presenting cell dialogue.

CD28-related proteins are also induced on activated T cells and serve to modify the co-stimulatory signal as the T-cell response develops. One is CTLA-4 (CD152), an additional receptor for B7 molecules. CTLA-4 closely resembles CD28 in sequence, and the two proteins are encoded by closely linked genes. However, CTLA-4 binds B7 molecules about 20 times more avidly than does CD28 and delivers an inhibitory signal to the activated T cell (Fig. 8.12). This makes the activated progeny of a naive T cell less sensitive to stimulation by the antigen-presenting cell and limits the amount of an autocrine T-cell growth factor, interleukin-2 (IL-2), that is produced. Thus, binding of CTLA-4 to B7 molecules is essential for limiting the proliferative response of activated T cells to antigen and B7. This was confirmed by producing mice with a disrupted CTLA-4 gene; such mice develop a fatal disorder characterized by massive lymphocyte proliferation.

A third CD28-related protein is induced on activated T cells and can enhance T-cell responses; this inducible co-stimulator, or ICOS, binds a ligand known as LICOS, the ligand of ICOS, which is distinct from B7.1 and B7.2. LICOS is produced on activated dendritic cells, monocytes, and B cells, but its contribution to immune responses has not yet been clearly defined. Although it resembles CD28 in driving T-cell growth, it differs from CD28 in not inducing IL-2; instead it induces IL-10 (see Fig. 8.32).

Thus antigen-presenting cells engage in a co-stimulatory dialogue with T cells that recognize the antigens they display. This dialogue involves the delivery and receipt of signals through a number of different molecules, but appears to be initiated through the binding of B7 molecules to CD28 on a naive T cell. Antigen-presenting cells are activated to express B7 molecules on detecting the presence of infection through receptors of the innate immune system. The requirement for the simultaneous delivery of antigen-specific and co-stimulatory signals by one cell in the activation of naive T cells means that only such activated antigen-presenting cells, principally the dendritic cells that migrate into lymphoid tissue after being activated by binding and ingesting pathogens, can initiate T-cell responses. This is important, because not all potentially self-reactive T cells are deleted in the thymus; peptides derived from proteins made only in specialized cells in peripheral tissues might not be encountered during negative selection of thymocytes. Self-tolerance could be broken if naive autoreactive T cells could recognize self antigens on tissue cells and then be co-stimulated by an antigen-presenting cell, either locally or at a distant site. Thus, the requirement that the same cell presents both the specific antigen and the co-stimulatory signal is important in preventing destructive immune responses to self tissues. Indeed, antigen binding to the T-cell receptor in the absence of co-stimulation not only fails to activate the cell, it instead leads to a state called anergy, in which the T cell becomes refractory to activation by specific antigen even when the antigen is subsequently presented to it by a professional antigen-presenting cell (Fig. 8.13).

Now that we have discussed the molecular interactions that allow naive T cells to adhere transiently to antigen-presenting cells and scan their MHC:peptide complexes, and also the adhesion and co-stimulatory molecules that contribute to T-cell activation once a specific antigen is encountered, we will look more closely at the properties of the three types of antigen-presenting cell. Dendritic cells, macrophages, and B cells differ in their selectivity of antigen uptake, their antigen-processing properties, and their co-stimulatory and migratory behavior, and thus have distinctive functions in initiating T-cell responses.

8-6. Dendritic cells specialize in taking up antigen and activating naive T cells

The only known function of dendritic cells is to present antigen to T cells, and the mature dendritic cells found in lymphoid tissues are by far the most potent stimulators of naive T cells. This ability is not shared, however, by the immature dendritic cells found under most surface epithelia and in most solid organs such as the heart and kidneys. Dendritic cells arise from myeloid progenitors within the bone marrow, and emerge from the bone marrow to migrate in the blood to peripheral tissues. In these tissues, they have an immature phenotype that is associated with low levels of MHC proteins, and they lack co-stimulatory B7 molecules (Fig 8.14, top panel). They are not yet equipped to stimulate naive T cells. However, they share with their close relatives the macrophages, the ability to recognize and ingest pathogens through receptors that recognize features common to microbial surfaces, and they are very active in taking up antigens by phagocytosis using receptors such as DEC 205. Other extracellular antigens are taken up nonspecifically by macropinocytosis, in which large volumes of surrounding fluid are engulfed.

Typical of immature dendritic cells are the Langerhans' cells of the skin. These are actively phagocytic and contain large granules, known as Birbeck granules, which may be a type of phagosome. An infection triggers the migration of Langerhans' cells to the regional lymph nodes (Figs 8.15 and 8.2). Here, they rapidly lose the ability to take up and process antigen, but synthesize new MHC molecules that present peptides of pathogens at a high level. On arriving in the regional lymph node, they also express B7 molecules, which can co-stimulate naive T cells, and also large numbers of adhesion molecules, which enable them to interact with antigen-specific T cells. In this way the Langerhans' cells capture antigens from invading pathogens and differentiate into mature dendritic cells that are uniquely fitted for presenting these antigens and activating naive T cells.

Immature dendritic cells persist in the peripheral tissues for variable lengths of time. When an infection occurs, they are stimulated to migrate via the lymphatics to the local lymphoid tissues, where they have a completely different phenotype. The dendritic cells in lymphoid tissue are no longer able to engulf antigens by phagocytosis or by macropinocytosis. However, they now express very high levels of long-lived MHC class I and MHC class II molecules; this enables them to stably present peptides from proteins acquired from the infecting pathogens. They also express very high levels of adhesion molecules, including DC-SIGN, as well as high levels of B7 molecules (Fig. 8.14, center panel). They also secrete a chemokine that specifically attracts naive T cells; this chemokine, called DC-CK, is expressed only in dendritic cells in lymphoid tissues. These properties help to explain dendritic cells' ability to stimulate strong naive T-cell responses.

Although activated mature dendritic cells will also present some self peptides, the T-cell receptor repertoire has been purged in the thymus of receptors that recognize self peptides presented by dendritic cells (see Chapter 7), and thus T-cell responses against ubiquitous self antigens are avoided. In addition, tissue dendritic cells reaching the end of their life-span without having been activated by infection also travel via the lymphatics to local lymphoid tissue. Because they do not express the appropriate costimulatory molecules, these cells induce tolerance to any self antigens derived from peripheral tissues that they display.

The signals that activate tissue dendritic cells to migrate and mature after taking up antigen are clearly of key importance in determining whether an adaptive immune response will be initiated. These signals can be generated through direct interactions with pathogens or by cytokine stimulation, but in both cases they are thought to be a consequence of the recognition of invading pathogens by nonclonotypic receptors of the innate immune system. The best-understood example is the response to gram-negative bacteria, whose cell walls contain lipopolysaccharide (LPS). Receptors that recognize LPS are found on dendritic cells and macrophages, and these associate with the Toll-like signaling receptor TLR-4, which then activates the transcription factor NFκB (see Sections 2-17 and 6-15). Signaling through this pathway induces the expression of B7 molecules, and of cytokines such as TNF-α, which stimulate the migration of tissue dendritic cells. Thus an immature tissue dendritic cell that binds and internalizes a gram-negative bacterium is induced to migrate to local lymphoid tissue and present bacterium-derived peptide antigens to naive T cells. Other members of the TLR family are expressed on tissue dendritic cells, and are thought to be involved in detecting and signaling the presence of other classes of pathogen. Other types of receptor that can bind pathogens, such as receptors for complement, or phagocytic receptors such as the mannose receptor, are also expressed on dendritic cells and may contribute to their activation.

Pathogens that have evolved to escape recognition by phagocytic receptors are taken up by tissue dendritic cells through the process of macropinocytosis, and can then be presented to T cells. This is thought to occur after intracellular degradation of the pathogen to reveal components that trigger activation of the dendritic cell. Bacterial DNA containing unmethylated CpG dinucleotide motifs induces the rapid activation of dendritic cells. This probably occurs after recognition of the DNA by an intracellular receptor called TLR-9. Exposure to bacterial DNA activates NFκB and mitogen-activated protein kinase (MAP kinase) signaling pathways, leading to the production of cytokines such as IL-6, IL-12, IL-18, and interferon (IFN)-α and IFN-γ. In turn, these induce and augment the expression of co-stimulatory molecules. Bacterial heat-shock proteins are another internal bacterial constituent that can activate the antigen-presenting function of dendritic cells. Some viruses are thought to be recognized inside the dendritic cell, as a consequence of the production of double-stranded RNA in the course of their replication. As discussed in Section 2-25, viral infection also induces the production of IFN-α by infected cells. IFN-α is one of the cytokines that can activate dendritic cells to express co-stimulatory molecules.

Dendritic cells are likely to be particularly important in stimulating T-cell responses to viruses, which fail to induce co-stimulatory activity in other types of antigen-presenting cell. Viruses may infect dendritic cells by binding to any of several molecules on the cell surface, or after being engulfed but not destroyed by immature dendritic cells. Such viruses synthesize their proteins using the dendritic cell's own protein synthesis machinery, leading to surface expression of viral peptides by MHC class I molecules just as in other types of infected cell. Viral peptides will also be presented on both MHC class I and MHC class II molecules as a result of uptake of viral particles by phagocytic receptors such as the mannose receptor, which can recognize many viruses, or through macropinocytosis. The mechanism by which peptides generated by degradation of viral proteins in the endosomal pathway can be presented by MHC class I molecules is not known, nor, in fact, whether there is only one such mechanism. Nevertheless, it is clear that extracellular proteins taken up by dendritic cells can give rise to peptides presented by MHC class I molecules. In this way, viruses that are not able to infect dendritic cells are still able to stimulate effective immune responses. Thus, any virus-infected cell is able to activate naive CD8 T cells, generating cytotoxic CD8 effector T cells that can kill infected cells, and also to activate CD4 T cells that can stimulate the production of antibodies.

Dendritic cells are believed to present antigens from fungal as well as viral and bacterial pathogens. Indeed, they are thought to initiate immune responses to a wide range of pathogens, and to be able to distinguish between different classes of pathogen. This is reflected in the synthesis of different effector molecules by the activated dendritic cells, which in turn influence the differentiation of the responding T cells into different subclasses, which is discussed further in Section 10-5. In addition to pathogen-associated antigens, dendritic cells are thought to present protein antigens from environmental sources that trigger allergic reactions upon inhalation (see Chapter 12), and alloantigens deriving from a transplanted organ, which form the basis for graft rejection (see Chapter 13). In principle, any nonself antigen will be immunogenic if it is taken up and presented by a dendritic cell that is activated to migrate to nearby lymphoid tissues and mature. The normal physiology of dendritic cells is to migrate, and this is increased by stimuli that activate the linings of the lymphatics, like transplantation, which is why dendritic cells are so potent at stimulating allograft reactions.

8-7. Macrophages are scavenger cells that can be induced by pathogens to present foreign antigens to naive T cells

As we learned in Chapter 2, many of the microorganisms that enter the body are engulfed and destroyed by phagocytes, which provide an innate, antigen-nonspecific first line of defense against infection. Microorganisms that are destroyed by phagocytes without additional help from T cells do not cause disease and do not require an adaptive immune response. Pathogens, by definition, have developed mechanisms to avoid elimination by innate immunity, and the targeting and removal of such pathogens is the function of the adaptive immune response. Mononuclear phagocytes or macrophages that have bound and ingested microorganisms but have failed to destroy them, contribute to the adaptive immune response by acting as antigenpresenting cells. As we will see later in this chapter and in Chapter 10, the adaptive immune response is in turn able to stimulate the microbicidal and phagocytic capacities of these cells.

Resting macrophages have few or no MHC class II molecules on their surface, and do not express B7 molecules. The expression of both MHC class II and B7 molecules is induced by the ingestion of microorganisms and recognition of their foreign molecular patterns. Macrophages, like tissue dendritic cells, have a variety of receptors that recognize microbial surface components, including the mannose receptor, the scavenger receptor, complement receptors, and several Toll-like receptors (see Chapter 2). These receptors function in the innate immune defense mediated by macrophages; they are involved in the uptake of microorganisms by phagocytosis and in signaling for the secretion of pro-inflammatory cytokines that recruit and activate more phagocytes. In addition, they can play the same role as tissue dendritic cells, and allow the macrophage to function as an antigen-presenting cell. Once bound, microorganisms are engulfed and degraded in the endosomes and lysosomes, generating peptides that can be presented by MHC class II molecules. At the same time, the receptors recognizing these microorganisms transmit a signal that leads to expression of MHC class II molecules and B7 molecules.

Thus the induction of co-stimulatory activity by common microbial constituents occurs in both dendritic cells and macrophages. This is believed to allow the immune system to distinguish antigens borne by infectious agents from antigens associated with innocuous proteins, including self proteins. Indeed, many foreign proteins do not induce an immune response when injected on their own, presumably because they fail to induce costimulatory activity in antigen-presenting cells. When such protein antigens are mixed with bacteria, however, they become immunogenic, because the bacteria induce the essential co-stimulatory activity in cells that ingest the protein (Fig. 8.16). Bacteria used in this way are known as adjuvants (see Appendix I, Section A-4). We will see in Chapter 13 how self tissue proteins mixed with bacterial adjuvants can induce autoimmune diseases, illustrating the crucial importance of the regulation of co-stimulatory activity in discrimination of self from nonself.

As macrophages continuously scavenge dead or dying cells, which are rich sources of self antigens, it is particularly important that they should not activate T cells in the absence of microbial infection. The Kupffer cells of the liver sinusoids and the macrophages of the splenic red pulp, in particular, remove large numbers of dying cells from the blood daily. Kupffer cells express little MHC class II and no TLR-4, the Toll-like receptor on human cells that signals the presence of LPS. Thus, although they generate large amounts of self peptides in their endosomes and lysosomes, these macrophages are not likely to elicit an autoimmune response.

8-8. B cells are highly efficient at presenting antigens that bind to their surface immunoglobulin

Macrophages cannot take up soluble antigens efficiently, whereas immature dendritic cells can take up large amounts of antigen from extracellular fluid by macropinocytosis. B cells, by contrast, are uniquely adapted to bind specific soluble molecules through their cell-surface immunoglobulin. B cells internalize the antigens bound by their surface immunoglobulin receptors and then display peptide fragments of antigen as peptide:MHC class II complexes. Because this mechanism of antigen uptake is highly efficient, and B cells constitutively express high levels of MHC class II molecules, high levels of specific peptide:self MHC class II complexes are generated at the B-cell surface (Fig. 8.17). This pathway of antigen presentation allows B cells to be targeted by antigen-specific CD4 T cells, which drive their differentiation, as we will see in Chapter 9. In circumstances in which the presenting B cells are induced to express co-stimulatory activity, it also allows B cells to activate naive T cells.

B cells do not constitutively express co-stimulatory activity but, as with dendritic cells and macrophages, they can be induced by various microbial constituents to express B7.1 and especially B7.2. Indeed, B7.1 was first identified as a molecule expressed on B cells activated by microbial lipopolysaccharide. These observations help explain why it is essential to co-inject bacterial adjuvants in order to produce an immune response to soluble proteins such as ovalbumin, hen egg-white lysozyme, and cytochrome c, which may require B cells as antigen-presenting cells.

The requirement for induced co-stimulatory activity also helps explain why, although B cells present soluble proteins efficiently, they are unlikely to initiate responses to soluble self proteins in the absence of infection. In the absence of co-stimulation, antigen not only fails to activate naive T cells but causes them to become anergic, or nonresponsive (see Fig. 8.13). This provides an additional safeguard to the mechanisms discussed in Chapter 7 whereby potentially self-reactive T and B cells are eliminated or inactivated as they develop in the thymus and bone marrow.

Although much of what we know about the immune system in general, and about T-cell responses in particular, has been learned from the study of immune responses to soluble protein immunogens presented by B cells, it is not clear how important B cells are in priming naive T cells in natural immune responses. Soluble protein antigens are not abundant during natural infections; most natural antigens, such as bacteria and viruses, are particulate, whereas soluble bacterial toxins act by binding to cell surfaces and so are present only at low concentrations in solution. Some natural immunogens enter the body as soluble molecules; examples are insect toxins, anticoagulants injected by blood-sucking insects, snake venoms, and many allergens. However, tissue dendritic cells could also be responsible for activating naive T cells that recognize these antigens. Although tissue dendritic cells could not concentrate these antigens in the same way as antigen-specific B cells, they may be more likely to encounter a naive T cell with the appropriate antigen specificity than the limited number of B cells able to bind and concentrate a particular antigen. The chances of a B cell encountering a T cell that can recognize the peptide antigens it displays is greatly increased once a naive T cell has been detained in lymphoid tissue by finding its antigen on the surface of a dendritic cell.

T-cell responses can thus be primed by three distinct types of antigen- presenting cell. Dendritic cells are optimally equipped to present a wide variety of antigens to naive T cells, while macrophages stimulate T-cell responses to the pathogens they take up but are unable to eliminate, and B cells specialize in presenting fragments of the antigen to which their surface immunoglobulin binds (Fig. 8.18). In each of these cell types, as we saw in Chapter 2, the expression of co-stimulatory activity is controlled so as to provoke responses against pathogens while avoiding immunization against self.

8-9. Activated T cells synthesize the T-cell growth factor interleukin-2 and its receptor

Naive T cells can live for many years without dividing. These small resting cells have condensed chromatin and a scanty cytoplasm and synthesize little RNA or protein. On activation, they must reenter the cell cycle and divide rapidly to produce the large numbers of progeny that will differentiate into armed effector T cells. Their proliferation and differentiation are driven by a cytokine called interleukin-2 (IL-2), which is produced by the activated T cell itself.

The initial encounter with specific antigen in the presence of the required co-stimulatory signal triggers entry of the T cell into the G₁ phase of the cell cycle; at the same time, it also induces the synthesis of IL-2 along with the α chain of the IL-2 receptor. The IL-2 receptor has three chains: α, β, and γ (Fig. 8.19). Resting T cells express a form of this receptor composed of β and γ chains which binds IL-2 with moderate affinity, allowing resting T cells to respond to very high concentrations of IL-2. Association of the α chain with the β and γ chains creates a receptor with a much higher affinity for IL-2, allowing the cell to respond to very low concentrations of IL-2. Binding of IL-2 to the high-affinity receptor then triggers progression through the rest of the cell cycle. T cells activated in this way can divide two to three times a day for several days, allowing one cell to give rise to a clone composed of thousands of progeny that all bear the same receptor for antigen (Fig. 8.20). IL-2 also promotes the differentiation of these cells into armed effector T cells.

8-10. The co-stimulatory signal is necessary for the synthesis and secretion of IL-2

The production of IL-2 determines whether a T cell will proliferate and become an armed effector cell, and the most important function of the co-stimulatory signal is to promote the synthesis of IL-2. Antigen recognition by the T-cell receptor ultimately induces the synthesis of several transcription factors (see Chapter 6). One of these factors, NFAT (nuclear factor of activated T cells), binds to the promoter region of the IL-2 gene and is needed to activate its transcription. IL-2 gene transcription on its own, however, does not lead to the production of IL-2, which additionally requires CD28 ligation by B7. One effect of signaling through CD28 is thought to be the stabilization of IL-2 mRNA. Cytokine mRNAs are very short-lived because of an ‘instability’ sequence in their 3′ untranslated region. This instability prevents sustained cytokine production and release, and enables cytokine activity to be tightly regulated. The stabilization of IL-2 mRNA increases IL-2 synthesis by 20- to 30-fold. A second effect of CD28 ligation is to activate transcription factors (AP-1 and NFκB) that increase transcription of IL-2 mRNA by about threefold. These two effects together increase IL-2 protein production by as much as 100-fold. When a T cell recognizes specific antigen in the absence of co-stimulation through its CD28 molecule, little IL-2 is produced and the T cell does not proliferate.

The central importance of IL-2 in initiating adaptive immune responses is well illustrated by the drugs that are most commonly used to suppress undesirable immune responses such as transplant rejection. The immunosuppressive drugs cyclosporin A and FK506 (tacrolimus) inhibit IL-2 production by disrupting signaling through the T-cell receptor, whereas rapamycin (sirolimus) inhibits signaling through the IL-2 receptor. Cyclosporin A and rapamycin act synergistically to inhibit immune responses by preventing the IL-2-driven clonal expansion of T cells. The mode of action of these drugs will be considered in detail in Chapter 14.

8-11. Antigen recognition in the absence of co-stimulation leads to T-cell tolerance

Antigen recognition in the absence of co-stimulation inactivates naive T cells, inducing a state known as anergy. The most important change in anergic T cells is their inability to produce IL-2. This prevents them from proliferating and differentiating into effector cells when they encounter antigen, even if the antigen is subsequently presented by antigen-presenting cells. This helps to ensure the tolerance of T cells to self tissue antigens. Although anergy has only been demonstrated formally in vitro, there is sufficiently compelling evidence from studies in vivo showing peripheral tolerance to various antigens to assume that it happens in this setting as well.

As we saw in Section 7-24, any protein synthesized by all cells will be presented by antigen-presenting cells in the thymus and will cause clonal deletion of the T cells reactive to these ubiquitous self proteins. However, many proteins have specialized functions and are made only by the cells of certain tissues. Because MHC class I molecules present only peptides derived from proteins synthesized within the cell, such tissue-specific peptides will not be displayed on the MHC molecules of thymic cells, and T cells recognizing them are unlikely to be deleted in the thymus. An important factor in avoiding autoimmune responses to such tissue-specific proteins is the absence of co-stimulatory activity on tissue cells. Naive T cells recognizing self peptides on tissue cells are not activated; instead they may be induced to enter a state of anergy (Fig. 8.21).

Although the deletion of potentially autoreactive T cells is readily understood as a simple way to maintain self tolerance, the retention of anergic T cells specific for tissue antigens is less easy to understand. It would seem more economical and efficient to eliminate such cells; indeed, binding of the T-cell receptor on peripheral T cells in the absence of co-stimulators can lead to programmed cell death as well as to anergy. Nevertheless, some T cells persist in an anergic state in vivo. One possible explanation for this is that such anergic T cells have a role in preventing responses by naive, nonanergic T cells to foreign antigens that mimic self peptide:self MHC complexes. The persisting anergic T cells could recognize and bind to such peptide:MHC complexes on antigen-presenting cells without responding, and thus could compete with naive, potentially autoreactive cells of the same specificity. In this way, anergic T cells could serve to prevent the accidental activation of autoreactive T cells by infectious agents, thus actively contributing to tolerance.

8-12. Proliferating T cells differentiate into armed effector T cells that do not require co-stimulation to act

Late in the proliferative phase of the T-cell response induced by IL-2, after 4–5 days of rapid growth, activated T cells differentiate into armed effector T cells that can synthesize all the effector molecules required for their specialized functions as helper or cytotoxic T cells. In addition, all classes of armed effector T cells have undergone changes that distinguish them from naive T cells. One of the most critical is in their activation requirements: once a T cell has differentiated into an armed effector cell, encounter with its specific antigen results in immune attack without the need for co-stimulation (Fig. 8.22).

This applies to all classes of armed effector T cells. Its importance is particularly easy to understand in the case of cytotoxic CD8 T cells, which must be able to act on any cell infected with a virus, whether or not the infected cell can express co-stimulatory molecules. However, it is also important for the effector function of CD4 cells, as armed effector CD4 T cells must be able to activate B cells and macrophages that have taken up antigen, even if, as is often the case, they have too little co-stimulatory activity to activate a naive CD4 T cell.

Changes are also seen in the cell-adhesion molecules expressed by armed effector T cells. They express higher levels of LFA-1 and CD2, but lose cell-surface L-selectin and thus cease to recirculate through lymph nodes. Instead, they express the integrin VLA-4, which allows them to bind to vascular endothelium at sites of inflammation. This allows the armed effector T cells to enter sites of infection and put their armory of effector proteins to good use. These changes in the T-cell surface are summarized in Fig. 8.23.

8-13. The differentiation of CD4 T cells into T _H 1 or T _H 2 cells determines whether humoral or cell-mediated immunity will predominate.

Naive CD8 T cells emerging from the thymus are already predestined to become cytotoxic cells, even though they are not yet expressing any of the differentiated functions of armed effector cells. The case of CD4 T cells, however, is more complex. Naive CD4 T cells can differentiate upon activation into either T_H1 or T_H2 cells, which differ in the cytokines they produce and thus in their function. The decision on which fate the progeny of a naive CD4 T cell will follow is made during the clonal expansion that takes place after the first encounter with antigen (Fig. 8.24).

The factors that determine whether a proliferating CD4 T cell will differentiate into a T_H1 or a T_H2 cell are not fully understood. The cytokines elicited by infectious agents (principally IFN-γ, IL-12, and IL-4), the co-stimulators used to drive the response, and the nature of the peptide:MHC ligand all have an effect. In particular, because the decision to differentiate into T_H1 versus T_H2 cells occurs early in the immune response, the cytokines produced in response to pathogens by cells of the innate immune system play an important part in shaping the subsequent adaptive response; we will learn more about this in Chapter 10.

The consequences of inducing T_H1 versus T_H2 cells are profound: the selective production of T_H1 cells leads to cell-mediated immunity, whereas the production of predominantly T_H2 cells provides humoral immunity. A striking example of the difference this can make to the outcome of infection is seen in leprosy, a disease caused by infection with Mycobacterium leprae. M. leprae, like M. tuberculosis, grows in macrophage vesicles, and effective host defense requires macrophage activation by T_H1 cells. In patients with tuberculoid leprosy, in which T_H1 cells are preferentially induced, few live bacteria are found, little antibody is produced, and, although skin and peripheral nerves are damaged by the inflammatory responses associated with macrophage activation, the disease progresses slowly and the patient usually survives. However, when T_H2 cells are preferentially induced, the main response is humoral, the antibodies produced cannot reach the intracellular bacteria, and the patients develop lepromatous leprosy, in which M. leprae grows abundantly in macrophages, causing gross tissue destruction that is eventually fatal.

8-14. Naive CD8 T cells can be activated in different ways to become armed cytotoxic effector cells

Naive CD8 T cells differentiate into cytotoxic cells, and perhaps because the effector actions of these cells are so destructive, naive CD8 T cells require more co-stimulatory activity to drive them to become armed effector cells than do naive CD4 T cells. This requirement can be met in two ways. The simplest is activation by dendritic cells, which have high intrinsic costimulatory activity. These cells can directly stimulate CD8 T cells to synthesize the IL-2 that drives their own proliferation and differentiation (Fig. 8.25). This has been exploited to generate cytotoxic T-cell responses against tumors, as we will see in Chapter 14.

Cytotoxic T-cell responses to some viruses and tissue grafts, however, seem to require the presence of CD4 T cells during the priming of the naive CD8 T cell. In these responses, both the naive CD8 T cell and the CD4 T cell must recognize related antigens on the surface of the same antigen-presenting cell. In this case, it is thought that the actions of the CD4 T cell may be needed to compensate for inadequate co-stimulation of naive CD8 T cells by the antigen-presenting cell. This compensatory effect is currently thought to occur by the recruitment of an armed effector CD4 T cell that activates the antigenpresenting cell to express higher levels of co-stimulatory activity. We have seen that this is one of the actions of the CD40 ligand, which is expressed once T cells have been activated. Binding of CD40 ligand on the CD4 T cell to CD40 on the antigen-presenting cell induces B7 expression and enables the antigen-presenting cell to co-stimulate the CD8 T cell directly (Fig. 8.26).

Summary

The crucial first step in adaptive immunity is the activation of naive antigen-specific T cells by antigen-presenting cells. This occurs in the lymphoid tissues and organs through which naive T cells are constantly passing. The most distinctive feature of antigen-presenting cells is the expression of co-stimulatory molecules, of which the B7.1 and B7.2 molecules are the best characterized. Naive T cells will respond to antigen only when one cell presents both specific antigen to the T-cell receptor and a B7 molecule to CD28, the receptor for B7 on the T cell. The three cell types that can serve as antigen-presenting cells are dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in eliciting immune responses. Tissue dendritic cells take up antigens by phagocytosis and macropinocytosis and are stimulated by infection to migrate to the local lymphoid tissue, where they differentiate into mature dendritic cells expressing co-stimulatory activity. They serve as the most potent activators of naive T-cell responses. Macrophages efficiently ingest particulate antigens such as bacteria and are induced by infectious agents to express MHC class II molecules and costimulatory activity. The unique ability of B cells to bind and internalize soluble protein antigens via their receptors may be important in activating T cells to this class of antigen, provided that co-stimulatory molecules are also induced on the B cell. In all three types of antigen-presenting cell, the expression of co-stimulatory molecules is activated in response to signals from receptors that also function in innate immunity to signal the presence of infectious agents (see Chapter 2).

The activation of T cells by antigen-presenting cells leads to their proliferation and the differentiation of their progeny into armed effector T cells. This depends on the production of cytokines, in particular the T-cell growth factor IL-2, which binds to a high-affinity receptor on the activated T cell. T cells whose antigen receptors are ligated in the absence of co-stimulatory signals fail to make IL-2 and instead become anergic or die. This dual requirement for both receptor ligation and co-stimulation helps to prevent naive T cells from responding to self antigens on tissue cells, which lack co-stimulatory activity. Proliferating T cells develop into armed effector T cells, the critical event in most adaptive immune responses. Once an expanded clone of T cells achieves effector function, its armed effector T-cell progeny can act on any target cell that displays antigen on its surface. Effector T cells can mediate a variety of functions. Their most important functions are the killing of infected cells by CD8 cytotoxic T cells and the activation of macrophages by T_H1 cells, which together make up cell-mediated immunity, and the activation of B cells by both T_H2 and T_H1 cells to produce different classes of antibody, thus driving the humoral immune response.

8-15. Effector T-cell interactions with target cells are initiated by antigen-nonspecific cell-adhesion molecules

Once an effector T cell has completed its differentiation in the lymphoid tissue it must find target cells that are displaying the MHC:peptide complex that it recognizes. Some T_H2 cells encounter their B-cell targets without leaving the lymphoid tissue, as we discuss further in Chapter 9. However, most of the armed effector T cells emigrate from their site of activation in lymphoid tissues and enter the blood via the thoracic duct. Because of the cell-surface changes that have occurred during differentiation, they can now migrate into tissues, particularly at sites of infection. They are guided to these sites by changes in the adhesion molecules expressed on the endothelium of the local blood vessels as a result of infection, and by local chemotactic factors, as we will see in Chapter 10.

The initial binding of an effector T cell to its target, like that of a naive T cell to an antigen-presenting cell, is an antigen-nonspecific interaction mediated by LFA-1 and CD2. The level of LFA-1 and of CD2 is twofold to fourfold higher on armed effector T cells than on naive T cells, and so armed effector T cells can bind efficiently to target cells that have lower levels of ICAMs and LFA-3 on their surface than do the professional antigen-presenting cells. This interaction is normally transient unless recognition of antigen on the target cell through the T-cell receptor triggers an increase in the affinity of the T-cell's LFA-1 for its ligands on the target cell. The T cell binds more tightly to its target and remains bound for long enough to release its effector molecules. Armed CD4 effector T cells, which activate macrophages or induce B cells to secrete antibody, must maintain contact with their targets for relatively long periods. Cytotoxic T cells, by contrast, can be observed under the microscope attaching to and dissociating from successive targets relatively rapidly as they kill them (Fig. 8.28). Killing of the target, or some local change in the T cell, then allows the effector T cell to detach and address new targets. How armed CD4 effector T cells disengage from their antigen-negative targets is not known, although current evidence suggests that CD4 binding directly to MHC class II molecules on target cells that are not displaying specific antigen, signals the cell to detach.

8-16. Binding of the T-cell receptor complex directs the release of effector molecules and focuses them on the target cell

When binding to peptide:MHC complexes, the T-cell receptor molecules and their cross-linked co-receptors cluster at the site of cell-cell contact. Clustering of the T-cell receptors then signals a reorientation of the cytoskeleton that polarizes the effector cell so as to focus the release of effector molecules at the site of contact with the target cell, as illustrated for a cytotoxic T cell in Fig. 8.29. Polarization of the cell starts with the local reorganization of the cortical actin cytoskeleton at the site of contact; this in turn leads to the reorientation of the microtubule-organizing center (MTOC), the center from which the microtubule cytoskeleton is produced, and of the Golgi apparatus (GA), through which most proteins destined for secretion travel. In the cytotoxic T cell, the cytoskeletal reorientation focuses exocytosis of the preformed lytic granules at the site of contact with its target cell.

The polarization of a T cell also focuses the secretion of soluble effector molecules whose synthesis is induced de novo by ligation of the T-cell receptor. For example, the secreted cytokine IL-4, which is the principal effector molecule of T_H2 cells, is confined and concentrated at the site of contact with the target cell (see Fig. 9.6). It has been shown that the enhanced binding of LFA-1 to ICAM-1 creates a molecular seal surrounding the clustered T-cell receptors, CD4 co-receptors, and CD28 molecules (Fig. 8.30).

Thus, the antigen-specific T-cell receptor controls the delivery of effector signals in three ways: it induces the stable binding of effector cells to their specific target cells to create a tightly held, narrow space in which effector molecules can be concentrated; it focuses their delivery at the site of contact by inducing a reorientation of the secretory apparatus of the effector cell; and it triggers their synthesis and/or release. All these receptor-coordinated mechanisms contribute to the selective action of effector molecules on the target cell bearing specific antigen. In this way, effector T-cell activity is highly selective for those target cells that display antigen, although the effector molecules themselves are not antigen-specific.

8-17. The effector functions of T cells are determined by the array of effector molecules they produce

The effector molecules produced by armed effector T cells fall into two broad classes: cytotoxins, which are stored in specialized lytic granules and released by cytotoxic CD8 T cells, and cytokines and related membrane-associated proteins, which are synthesized de novo by all effector T cells. The cytotoxins are the principal effector molecules of cytotoxic T cells and will be discussed further in Section 8-22. Their release, in particular, must be tightly regulated as they are not specific: they can penetrate the lipid bilayer and trigger an intrinsic death program in any cell. By contrast, cytokines and membrane-associated proteins act by binding to specific receptors on the target cell. Cytokines and membrane-associated proteins are the principal mediators of CD4 T-cell effector actions, and the main effector actions of CD4 cells are therefore directed at specialized cells that express receptors for these proteins.

The effector actions and main effector molecules of all three functional classes of effector T cell are summarized in Fig. 8.31. The cytokines are a diverse group of proteins and we will briefly review them before discussing the T-cell cytokines and their contributions to the effector actions of cytotoxic CD8 T cells, T_H1 cells, and T_H2 cells. As we will see, soluble cytokines and membrane-associated molecules often act in combination to mediate the effects of T cells on their target cells.

The membrane-associated effector molecules, which we will discuss further in Section 8-20, are all structurally related to tumor necrosis factor (TNF), and their receptors on target cells are members of the TNF receptor (TNFR) family. All three classes of effector T cell express one or more members of the TNF family upon recognizing their specific antigen on the target cell. The membrane-bound TNF family member CD40 ligand is of particular importance for CD4 T-cell effector function; it is induced on T_H1 and T_H2 cells, and delivers activating signals to B cells and macrophages through the TNFR protein CD40. TNF-α is made by T_H1 cells, some T_H2 cells, and by cytotoxic T cells in soluble and membrane-associated forms, and can also deliver activating signals to macrophages. Some members of the TNF family can stimulate death by apoptosis. Thus Fas ligand (CD95L), the principal membraneassociated TNF-related molecule expressed by cytotoxic T cells, can trigger death by apoptosis in target cells bearing the receptor protein Fas (CD95); some T_H1 cells also express Fas ligand and can kill Fas-bearing cells with which they interact. Death by this mechanism appears to be important for removing activated Fas-bearing lymphocytes; if it fails, a lymphoproliferative disease associated with severe autoimmunity results.

8-18. Cytokines can act locally or at a distance

Cytokines are small soluble proteins secreted by one cell that can alter the behavior or properties of the cell itself or of another cell. They are released by many cells in addition to those of the immune system. We have already discussed the cytokines released by phagocytic cells in Chapter 2, where we dealt with the inflammatory reactions that play an important part in innate immunity; here we are concerned mainly with the cytokines that mediate the effector functions of T cells. Cytokines produced by lymphocytes are often called lymphokines, but this nomenclature can be confusing because some lymphokines are also secreted by nonlymphoid cells; we will therefore use the generic term ‘cytokine’ for all of them. Most cytokines produced by T cells are given the name interleukin (IL) followed by a number: we have encountered several interleukins already in this chapter. Cytokines of immunological interest are listed in Appendix III.

Most cytokines have a multitude of different biological effects when tested at high concentration in biological assays in vitro but targeted disruption of genes for cytokines and cytokine receptors in knockout mice (see Appendix I, Section A-47) has helped to clarify their physiological roles. The major actions of the cytokines produced by effector T cells are given in Fig. 8.32. As the effect of a cytokine varies depending on the target cell, the actions are listed according to the major target cell types—B cells, T cells, macrophages, hematopoietic cells, and tissue cells.

The main cytokine released by CD8 effector T cells is IFN-γ, which can block viral replication or even lead to the elimination of virus from infected cells without killing them. T_H1 cells and T_H2 cells release different, but overlapping, sets of cytokines, which define their distinct actions in immunity. T_H2 cells secrete IL-4 and IL-5, which activate B cells, and IL-10, which inhibits macrophage activation. T_H1 cells secrete IFN-γ, which is the main macrophage-activating cytokine, and lymphotoxin (LT-α or TNF-β), which activates macrophages, inhibits B cells and is directly cytotoxic for some cells. The T_H0 cells from which both of these functional classes derive (see Fig. 8.24) also secrete cytokines, including IL-2, IL-4, and IFN-γ, and may therefore have a distinctive effector function.

We have already discussed in Section 8-16 how the T-cell receptor can orchestrate the polarized release of these cytokines so that they are concentrated at the site of contact with the target cell. Furthermore, most of the soluble cytokines have local actions that synergize with those of the membrane-bound effector molecules. The effect of all these molecules is therefore combinatorial, and as the membrane-bound effectors can only bind to receptors on an interacting cell, this is another mechanism by which selective effects of cytokines are focused on the target cell. The effects of some cytokines are further confined to target cells by tight regulation of their synthesis: as we will see later, the synthesis of cytokines such as IL-2, IL-4, and IFN-γ is controlled, so that secretion from T cells does not continue after the interaction with a target cell ends.

Some cytokines, however, have more distant effects. IL-3 and GM-CSF (see Fig. 8.32), for example, which are released by both types of CD4 effector T cell, act on bone marrow cells to stimulate the production of macrophages and granulocytes, both of which are important nonspecific effector cells in both humoral and cell-mediated immunity. IL-3 and GM-CSF also stimulate the production of dendritic cells from bone marrow precursors. IL-5, produced by T_H2 cells, can increase the production of eosinophils, which contribute to the late phase of allergic reactions, in which there is a predominant activation of T_H2 cells (see Chapter 12). Whether a cytokine effect is local or more distant is likely to reflect the amounts released, the degree to which this release is focused on the target cell, and the stability of the cytokine in vivo but, for most of the cytokines, in particular those with more distant effects, these factors are not yet known.

8-19. Cytokines and their receptors fall into distinct families of structurally related proteins

Cytokines can be grouped by structure into families—the hematopoietins, the interferons, and the TNF family (Fig. 8.33)—and their receptors can likewise be grouped (Fig. 8.34). We have already encountered members of all of these families in Chapter 2, and have given an overview of the chemokine family there (see Section 2-20). We will focus here on the receptors for the hematopoietins, the TNF family, and IFN-γ on account of their role in T-cell effector function. Members of the TNF family act as trimers, most of which are membrane-bound and so are quite distinct in their properties from the other cytokines. Nevertheless, they share some important properties with the soluble T-cell cytokines, as they are also synthesized de novo upon antigen recognition by T cells, and affect the behavior of the target cell.

Many of the soluble cytokines made by effector T cells are members of the hematopoietin family. These cytokines and their receptors can be further divided into subfamilies characterized by functional similarities and genetic linkage. For instance, IL-3, IL-4, IL-5, IL-13, and GM-CSF are related structurally, their genes are closely linked in the genome, and all are major cytokines produced by T_H2 cells. In addition, they bind to closely related receptors, which form the family of class I cytokine receptors. The IL-3, IL-5, and GM-CSF receptors share a common β chain. Another subgroup of class I cytokine receptors is defined by their use of the γ chain of the IL-2 receptor; this is shared by receptors for the cytokines IL-2, IL-4, IL-7, IL-9, and IL-15 and is now called the common γ chain (γ_c). More distantly related, the receptor for IFN-γ is a member of a small family of cytokine receptors with some similarities to the hematopoietin receptor family. These so-called class II cytokine receptors include the receptor for IFN-α and IFN-β, and the IL-10 receptor. Overall, the structural, functional, and genetic relations between the cytokines and their receptors suggest that they may have diversified in parallel during the evolution of increasingly specialized effector functions.

These specific functional effects depend on intracellular signaling events that are triggered by the cytokines binding to their specific receptors. The hematopoietin and interferon receptors all signal through a similar pathway, which is described in Chapter 6. The key signaling molecules of this pathway are members of the Janus family of cytoplasmic tyrosine kinases (JAKs) and their targets the signal transducing activators of transcription (STATs), which enter the nucleus to activate specific genes. As the JAKs and STATs are present as families of related molecules, different members may be activated to achieve different effects.

8-20. The TNF family of cytokines are trimeric proteins that are often associated with the cell surface

TNF-α is made by T cells in soluble and membrane-associated forms, both of which are made up of three identical protein chains (a homotrimer, see Fig. 8.33). TNF-β (LT-α) can be produced as a secreted homotrimer, but is usually linked to the cell surface by forming heterotrimers with a third, membrane-associated, member of this family called LT-β. The receptors for these molecules, TNFR-I and TNFR-II, form homotrimers when bound to either TNF-α or LT. The trimeric structure is characteristic of all members of the TNF family, and the ligand-induced trimerization of their receptors seems to be the critical event in initiating signaling.

Most effector T cells express members of the TNF protein family as cell-surface molecules. The most important TNF-family proteins in T-cell effector function are TNF-α, TNF-β, Fas ligand, and CD40 ligand, the latter two always being cell-surface associated. These molecules all bind receptors that are members of the TNFR family; TNFR-I and II can each interact with either TNF-α or TNF-β, whereas Fas ligand and CD40 ligand bind respectively to the transmembrane protein Fas and to the transmembrane protein CD40 on target cells

Fas is expressed on many cells, especially on activated lymphocytes. Activation of Fas by the Fas ligand has profound consequences for the cell as Fas contains a ‘death domain’ in its cytoplasmic tail, which can initiate an activation cascade of cellular proteases called caspases that leads to apoptotic cell death (see Fig. 6.23). Fas is important in maintaining lymphocyte homeostasis, as can be seen from the effects of mutations in the Fas or Fas ligand genes. Mice and humans with a mutant form of Fas develop a lymphoproliferative disease associated with severe autoimmunity. A mutation in the gene encoding the Fas ligand in another mouse strain creates a nearly identical phenotype. These mutant phenotypes represent the best-characterized examples of generalized autoimmunity caused by single-gene defects. Other TNFR family members, including TNFR-I, are also associated with death domains and can also induce programmed cell death. Thus, TNF-α and TNF-β can induce programmed cell death by binding to TNFR-I.

The cytoplasmic tail of CD40 lacks a death domain; instead, it appears to be linked to proteins called TRAFs (TNF-receptor-associated factors), about which little is known. CD40 is involved in macrophage and B-cell activation; the ligation of CD40 on B cells promotes growth and isotype switching, whereas CD40 ligation on macrophages induces them to secrete TNF-α and to become receptive to much lower concentrations of IFN-γ. Deficiency in CD40 ligand expression is associated with immunodeficiency, as we will learn in Chapters 9 and 11.

Summary

Interactions between armed effector T cells and their targets are initiated by transient nonspecific adhesion between the cells. T-cell effector functions are elicited only when peptide:MHC complexes on the surface of the target cell are recognized by the receptor on an armed effector T cell. This recognition event triggers the armed effector T cell to adhere more strongly to the antigen-bearing target cell and to release its effector molecules directly at the target cell, leading to the activation or death of the target. The consequences of antigen recognition by an armed effector T cell are determined largely by the set of effector molecules it produces on binding a specific target cell. CD8 cytotoxic T cells store preformed cytotoxins in specialized lytic granules whose release can be tightly focused at the site of contact with the infected target cell. Cytokines, and one or more members of the TNF family of membrane-associated effector proteins, are synthesized de novo by all three types of effector T cell. T_H2 cells express B-cell activating effector molecules, whereas T_H1 cells express effector molecules that activate macrophages. CD8 T cells express membrane-associated Fas ligand that induces programmed cell death in cells bearing Fas; they also release IFN-γ. Membrane-associated effector molecules can deliver signals only to an interacting cell bearing the appropriate receptor, whereas soluble cytokines can act on cytokine receptors expressed locally on the target cell, or on hematopoietic cells at a distance. The actions of cytokines and membrane-associated effector molecules through their specific receptors, together with the effects of cytotoxins released by CD8 cells, account for most of the effector functions of T cells.

8-21. Cytotoxic T cells can induce target cells to undergo programmed cell death

Cells can die in either of two ways. Physical or chemical injury, such as the deprivation of oxygen that occurs in heart muscle during a heart attack, or membrane damage with antibody and complement, leads to cell disintegration or necrosis. The dead or necrotic tissue is taken up and degraded by phagocytic cells, which eventually clear the damaged tissue and heal the wound. The other form of cell death is known as programmed cell death or apoptosis. Apoptosis is a normal cellular response that is crucial in the tissue remodeling that occurs during development and metamorphosis in all multicellular animals. As we saw in Chapter 7, most thymocytes die an apoptotic death when they fail positive selection or, much less often, are negatively selected as a result of recognizing self antigens. Early changes seen in apoptosis are nuclear blebbing, alteration in cell morphology, and, eventually, fragmentation of the DNA. The cell then destroys itself from within, shrinking by shedding membrane-bound vesicles, and degrading itself until little is left. A hallmark of this type of cell death is the fragmentation of nuclear DNA into 200-base-pair (bp) pieces through the activation of endogenous nucleases that cleave the DNA between nucleosomes, each of which contains about 200 bp of DNA.

Cytotoxic T cells kill their targets by programming them to undergo apoptosis (Fig. 8.35). When cytotoxic T cells are mixed with target cells and rapidly brought into contact by centrifugation, they can program antigen-specific target cells to die within 5 minutes, although death may take hours to become fully evident. The short period required by cytotoxic T cells to program their targets to die reflects the release of preformed effector molecules, which activate an endogenous apoptotic pathway within the target cell.

As well as killing the host cell, the apoptotic mechanism may also act directly on cytosolic pathogens. For example, the nucleases that are activated in apoptosis to destroy cellular DNA can also degrade viral DNA. This prevents the assembly of virions and thus the release of infectious virus, which could otherwise infect nearby cells. Other enzymes activated in the course of apoptosis may destroy nonviral cytosolic pathogens. Apoptosis is therefore preferable to necrosis as a means of killing infected cells; in necrosis, intact pathogens are released from the dead cell and these can continue to infect healthy cells, or can parasitize the macrophages that ingest them.

8-22. Cytotoxic effector proteins that trigger apoptosis are contained in the granules of CD8 cytotoxic T cells

The principal mechanism through which cytotoxic T cells act is by the calcium-dependent release of specialized lytic granules upon recognition of antigen on the surface of a target cell. These granules are modified lysosomes that contain at least two distinct classes of cytotoxic effector protein that are expressed selectively in cytotoxic T cells (Fig. 8.36). Such proteins are stored in the lytic granules in an active form, but conditions within the granules prevent them from functioning until after their release. One of these cytotoxic proteins, known as perforin, polymerizes to form transmembrane pores in target cell membranes. The other class of cytotoxic proteins comprises at least three proteases called granzymes, which belong to the same family of enzymes—the serine proteases—as the digestive enzymes trypsin and chymotrypsin. Granules that store perforin and granzymes can be seen in armed CD8 cytotoxic effector cells in tissue lesions.

When purified granules from cytotoxic T cells are added to target cells in vitro, they lyse the cells by creating pores in the lipid bilayer. The pores consist of polymers of perforin, which is a major constituent of these granules. On release from the granule, perforin forms a cylindrical structure that is lipophilic on the outside and hydrophilic down a hollow center with an inner diameter of 16 nm (Fig. 8.37). It is not known whether this structure is first formed and then inserted into the lipid bilayer of the target cell membrane, or whether it is formed in the bilayer itself. The pore that is formed allows water and salts to pass rapidly into the cell. With the integrity of the cell membrane destroyed, the cells die rapidly. Large numbers of purified granules can kill target cells in vitro without inducing fragmentation of cellular DNA, but this lytic mechanism of cell killing probably occurs only at artificially high levels of perforin that do not reflect the physiological activity of cytotoxic T cells.

Both perforin and granzymes are required for effective cell killing. The separate roles of perforin and granzymes have been investigated in a cell system that relies upon similarities between the lytic granules of T cells and the granules of mast cells. Release of mast cell granules occurs on cross-linking of the Fcε receptor (see Chapter 9), just as release of lytic granules from CD8 T cells occurs on cross-linking of the T-cell receptor. The mechanism of signaling for granule release is thought to be the same or similar in both cases, as both the Fcε receptor and the T-cell receptor have ITAM motifs in their cytoplasmic domains, and cross-linking leads to tyrosine phosphorylation of the ITAMs (see Chapter 6).

When a mast-cell line is transfected with the gene for perforin or for granzyme, the gene products are stored in mast cell granules, and when the cell is activated through its Fcε receptor, these granules are released. When transfected with the gene for perforin alone, mast cells can kill other cells, but large numbers of the transfected cells are needed as the killing is not very efficient. By contrast, mast cells transfected with the gene for granzyme B alone are unable to kill other cells. However, when perforin-transfected mast cells are also transfected with the gene encoding granzyme B, the cells or their purified granules become as effective at killing targets as granules from cytotoxic cells, and granules from both types of cell induce DNA fragmentation. This suggests that perforin makes pores through which the granzymes can move into the target cell.

The granzymes are proteases, so although they have a role in triggering apoptosis in the target cell, they cannot act directly to fragment the DNA. Rather, they must activate an enzyme, or more probably an enzyme cascade, in the target cell. Granzyme B can cleave the ubiquitous cellular enzyme CPP-32, which is believed to have a key role in programmed cell death in all cells. CPP-32 is a caspase and activates a nuclease, called caspase-activated deoxyribonuclease or CAD, by cleaving an inhibitory protein (ICAD) that binds to and inactivates CAD. This enzyme is believed to be the final effector of DNA degradation in apoptosis.

Cells undergoing programmed cell death are rapidly ingested by nearby phagocytic cells. The phagocytes recognize some change in the cell membrane, most probably the exposure of phosphatidylserine, which is normally found only in the inner leaflet of the membrane. The ingested cell is then completely broken down and digested by the phagocyte without the induction of co-stimulatory proteins. Thus, apoptosis is normally an immunologically ‘quiet’ process; that is, apoptotic cells do not normally contribute to or stimulate immune responses.

The importance of perforin in this process is well illustrated in mice that have had their perforin gene knocked out. Such mice are severely defective in their ability to mount a cytotoxic T-cell response to many but not all viruses, whereas mice that are defective in the granzyme B gene have a less profound defect, probably because there are several genes coding for granzymes.

8-23. Activated CD8 T cells and some CD4 effector T cells express Fas ligand, which can also activate apoptosis

The release of granule contents accounts for most of the cytotoxic activity of CD8 effector T cells, as shown by the loss of most killing activity in perforin knockout mice. This granule-mediated killing is strictly calcium-dependent, yet some cytotoxic actions of CD8 T cells survive calcium depletion. Moreover, some CD4 T cells can also kill other cells, yet do not contain granules and make neither perforin nor granzymes. These observations imply that there must be a second perforin-independent mechanism of cytotoxicity. This mechanism involves the binding of Fas in the target cell membrane by the Fas ligand, which is present in the membranes of activated cytotoxic T cells and T_H1 cells. Ligation of Fas leads to activation of caspases, which induce apoptosis in the target cell (see Fig. 6.23). As discussed in Section 8-20, the lymphoproliferative and autoimmune disorders seen in mice and humans with mutations in genes for either Fas or Fas ligand imply that this pathway of killing is very important in regulating peripheral immune responses. Fas is expressed on activated lymphocytes and Fas-Fas ligand interactions are important in terminating lymphocyte growth after the removal of the initiating pathogen.

8-24. Cytotoxic T cells are selective and serial killers of targets expressing specific antigen

When cytotoxic T cells are offered a mixture of equal amounts of two target cells, one bearing specific antigen and the other not, they kill only the target cell bearing the specific antigen. The ‘innocent bystander’ cells and the cytotoxic T cells themselves are not killed, despite the fact that cloned cytotoxic T cells can be recognized and killed by other cytotoxic T cells just like any tissue cell. At first sight this may seem surprising, because the effector molecules released by cytotoxic T cells lack any specificity for antigen. The explanation probably lies in the highly polar release of the effector molecules. As we saw in Fig. 8.29, cytotoxic T cells orient their Golgi apparatus and microtubule-organizing center to focus secretion on the point of contact with a target cell. Granule movement toward the point of contact is shown in Fig. 8.38. Cytotoxic T cells attached to several different target cells reorient their secretory apparatus toward each cell in turn and kill them one by one, strongly suggesting that the mechanism whereby cytotoxic mediators are released allows attack at only one point of contact at any one time. The narrowly focused action of cytotoxic CD8 T cells allows them to kill single infected cells in a tissue without creating widespread tissue damage (Fig. 8.39) and is of critical importance in tissues where cell regeneration does not occur, as in neurons of the central nervous system, or is very limited, as in the pancreatic islets.

Cytotoxic T cells can kill their targets rapidly because they store preformed cytotoxic proteins in forms that are inactive in the environment of the lytic granule. Cytotoxic proteins are synthesized and loaded into the lytic granules during the first encounter of a naive cytotoxic precursor T cell with its specific antigen. Ligation of the T-cell receptor similarly induces de novo synthesis of perforin and granzymes in armed effector CD8 T cells, so that the supply of lytic granules is replenished. This makes it possible for a single CD8 T cell to kill many targets in succession.

8-25. Cytotoxic T cells also act by releasing cytokines

Although the secretion of perforin and granzymes is the main way by which cytotoxic CD8 T cells eliminate infection, with the expression of Fas ligand playing a lesser role, most cytotoxic CD8 T cells also release the cytokines IFN-γ, TNF-α, and TNF-β, which contribute to host defense in several ways. IFN-γ directly inhibits viral replication, and also induces the increased expression of MHC class I and other molecules involved in peptide loading of the newly synthesized MHC class I proteins in infected cells. This increases the chance that infected cells will be recognized as target cells for cytotoxic attack. IFN-γ also activates macrophages, recruiting them to sites of infection both as effector cells and as antigen-presenting cells. The activation of macrophages by IFN-γ is a critical component of the host immune response to intracellular protozoan pathogens such as Toxoplasma gondii. IFN-γ also has a secondary role in decreasing the tryptophan concentration within responsive cells and thus can kill intracellular parasites, effectively by starvation. TNF-α or TNF-β can synergize with IFN-γ in macrophage activation, and in killing some target cells through their interaction with TNFR-I. Thus, armed effector cytotoxic CD8 T cells act in a variety of ways to limit the spread of cytosolic pathogens. The relative importance of each of these mechanisms remains to be determined.

Summary

Armed effector cytotoxic CD8 T cells are essential in host defense against pathogens that live in the cytosol, the commonest of which are viruses. These cytotoxic T cells can kill any cell harboring such pathogens by recognizing foreign peptides that are transported to the cell surface bound to MHC class I molecules. Cytotoxic CD8 T cells carry out their killing function by releasing two types of preformed cytotoxic protein: the granzymes, which seem able to induce apoptosis in any type of target cell, and the pore-forming protein perforin, which punches holes in the target-cell membrane through which the granzymes can enter. These properties allow the cytotoxic T cell to attack and destroy virtually any cell that is infected with a cytosolic pathogen. A membrane-bound molecule, the Fas ligand, expressed by CD8 and some CD4 T cells, is also capable of inducing apoptosis by binding to Fas expressed by some target cells. Cytotoxic CD8 T cells also produce IFN-γ, which is an inhibitor of viral replication and is an important inducer of MHC class I expression and macrophage activation. Cytotoxic T cells kill infected targets with great precision, sparing adjacent normal cells. This precision is critical in minimizing tissue damage while allowing the eradication of infected cells.

8-26. Armed T_H1 cells have a central role in macrophage activation

A number of important pathogens live within macrophages, whereas many others are ingested by macrophages from the extracellular fluid. In many cases, the macrophage is able to destroy such pathogens without the need for T-cell activation, as we have seen in Chapter 2, but in several clinically important infections CD4 T cells are needed to provide activating signals for macrophages. The induction of antimicrobial mechanisms in macrophages is known as macrophage activation and is the principal effector action of T_H1 cells. Among the extracellular pathogens that are killed when macrophages are activated is Pneumocystis carinii, which, because of a deficiency of CD4 T cells, is a common cause of death in people with AIDS. Macrophage activation can be measured by the ability of activated macrophages to damage a broad spectrum of microbes as well as certain tumor cells. This ability to act on extracellular targets extends to healthy self cells, which means that macrophages must normally be maintained in a nonactivated state.

Macrophages require two signals for activation. One of these is provided by IFN-γ; the other can be provided by a variety of means, and is needed to sensitize the macrophage to respond to IFN-γ. Armed effector T_H1 cells can deliver both signals. IFN-γ is the most characteristic cytokine produced by armed T_H1 cells on interacting with their specific target cells, whereas the CD40 ligand expressed by the T_H1 cell delivers the sensitizing signal by contacting CD40 on the macrophage (Fig. 8.40). CD8 T cells are also an important source of IFN-γ and can activate macrophages presenting antigens derived from cytosolic proteins; mice lacking MHC class I molecules, and which thus have no CD8 T cells, show increased susceptibility to some parasitic infections. Macrophages can be made more sensitive to IFN-γ by very small amounts of bacterial lipopolysaccharide, and this latter pathway may be particularly important when CD8 T cells are the primary source of the IFN-γ. It is also possible that membrane-associated TNF-α or TNF-β can substitute for CD40 ligand in macrophage activation. These cell-associated molecules apparently stimulate the macrophage to secrete TNF-α, and antibody to TNF-α can inhibit macrophage activation. T_H2 cells are inefficient macrophage activators because they produce IL-10, a cytokine that can deactivate macrophages, and they do not produce IFN-γ. They do express CD40 ligand, however, and can deliver the contact-dependent signal required to activate macrophages to respond to IFN-γ.

8-27. The production of cytokines and membrane-associated molecules by armed CD4 T _H 1 cells requires new RNA and protein synthesis.

Within minutes of the recognition of specific antigen by armed effector cytotoxic CD8 T cells, directed exocytosis of preformed perforins and granzymes programs the target cell to die via apoptosis. In contrast, when armed T_H1 cells encounter their specific ligand, they must synthesize de novo the cytokines and cell-surface molecules that mediate their effects. This process requires hours rather than minutes, so T_H1 cells must adhere to their target cells for far longer than cytotoxic T cells.

Recognition of its target by a T_H1 cell rapidly induces transcription of cytokine genes and new protein synthesis begins within an hour of receptor triggering. The newly synthesized cytokines are then delivered directly through micro-vesicles of the constitutive secretory pathway to the site of contact between the T-cell membrane and the macrophage. It is thought that the newly synthesized cell-surface CD40 ligand is also expressed in this polarized fashion. This means that, although all macrophages have receptors for IFN-γ, the macrophage actually displaying antigen to the armed T_H1 cell is far more likely to become activated by it than are neighboring uninfected macrophages.

8-28. Activation of macrophages by armed T _H 1 cells promotes microbial killing and must be tightly regulated to avoid tissue damage.

T_H1 cells activate infected macrophages through cell contact and the focal secretion of IFN-γ. This generates a series of biochemical responses that converts the macrophage into a potent antimicrobial effector cell (Fig. 8.41). Activated macrophages fuse their lysosomes more efficiently to phagosomes, exposing intracellular or recently ingested extracellular microbes to a variety of microbicidal lysosomal enzymes. Activated macrophages also make oxygen radicals and nitric oxide (NO), both of which have potent antimicrobial activity, as well as synthesizing antimicrobial peptides and proteases that can be released to attack extracellular parasites.

Additional changes in the activated macrophage help to amplify the immune response. The number of MHC class II molecules, B7 molecules, CD40, and TNF receptors on the macrophage surface increases, making the cell both more effective at presenting antigen to fresh T cells, which may thereby be recruited as effector cells, and more responsive to CD40 ligand and to TNF-α. TNF-α synergizes with IFN-γ in macrophage activation, particularly in the induction of the reactive nitrogen metabolite NO, which has broad anti-microbial activity. The NO is produced by the enzyme inducible NO synthase (iNOS), and mice that have had the gene for iNOS knocked out are highly susceptible to infection with several intracellular pathogens. Activated macrophages secrete IL-12, which directs the differentiation of activated naive CD4 T cells into T_H1 effector cells, as we will learn in Chapter 10. These and many other surface and secreted molecules of activated macrophages are instrumental in the effector actions of macrophages in cell-mediated responses, and they are also important effectors in humoral immune responses, which we will discuss in Chapter 9, and in recruiting other immune cells to sites of infection, a function to which we will return in Chapter 10.

Because activated macrophages are extremely effective in destroying pathogens, one may ask why macrophages are not simply maintained in a state of constant activation. Besides the fact that macrophages consume huge quantities of energy to maintain the activated state, macrophage activation in vivo is usually associated with localized tissue destruction that apparently results from the release of antimicrobial mediators such as oxygen radicals, NO, and proteases, which are also toxic to host cells. The ability of activated macrophages to release toxic mediators is important in host defense because it enables them to attack large extracellular pathogens that they cannot ingest, such as parasitic worms. This can only be achieved, however, at the expense of tissue damage. Tight regulation of the activity of macrophages by T_H1 cells thus allows the specific and effective deployment of this potent means of host defense while minimizing local tissue damage and energy consumption.

Macrophage activation is contained by mechanisms that control IFN-γ synthesis by activated effector T cells. This seems to be achieved by regulating the half-life of the mRNA encoding IFN-γ. IFN-γ mRNA, like that encoding a variety of other cytokines, contains a sequence (AUUUA)_n in its 3′ untranslated region that greatly reduces its half-life, and this serves to limit the period of cytokine production. Activation of the T cell appears to induce the production of a new protein that promotes cytokine mRNA degradation: treatment of activated effector T cells with the protein synthesis inhibitor cycloheximide greatly increases the level of cytokine mRNA. The rapid destruction of cytokine mRNA, together with the focal delivery of IFN-γ at the point of contact between the activated T_H1 cell and its macrophage target, thus limits the action of the effector T cell to the infected macrophage. We will see in Chapter 9, when we consider the activation of B cells by T_H2 cells, that the same mechanisms direct and limit T-cell help to the specific antigen-binding B cell. In addition, macrophage activation itself is markedly inhibited by cytokines such as transforming growth factor-β (TGF-β), IL-4, IL-10, and IL-13. Because several of these inhibitory cytokines are produced by T_H2 cells, the induction of CD4 T cells belonging to the T_H2 subset represents an important pathway for controlling the effector functions of activated macrophages.

8-29. T _H 1 cells coordinate the host response to intracellular pathogens.

The activation of macrophages by armed T_H1 cells expressing CD40 ligand and secreting IFN-γ is central to the host response to pathogens that proliferate in macrophage vesicles. In mice in which the IFN-γ gene or the CD40 ligand gene has been destroyed by targeted gene disruption, production of anti-microbial agents by macrophages is impaired, and the animals succumb to sublethal doses of Mycobacterium species and Leishmania species. Macrophage activation is also critical in controlling vaccinia virus. Mice lacking TNF receptors also show increased susceptibility to these pathogens. However, although IFN-γ and CD40 ligand are probably the most important effector molecules synthesized by T_H1 cells, the immune response to pathogens that proliferate in macrophage vesicles is complex, and other cytokines secreted by T_H1 cells have a crucial role in coordinating these responses (Fig. 8.42). For example, macrophages that are chronically infected with intracellular bacteria may lose the ability to become activated. Such cells could provide a reservoir of infection that is shielded from immune attack. Activated T_H1 cells can express Fas ligand and thus kill a limited range of target cells that express Fas, including macrophages, thereby destroying these infected cells.

Whereas some intravesicular bacteria pose a hazard by incapacitating chronically infected macrophages, others, including some mycobacteria and Listeria monocytogenes, can escape from cell vesicles and enter the cytoplasm, where they are not susceptible to macrophage activation. Their presence can, however, be detected by cytotoxic CD8 T cells, which can release them by killing the cell. The pathogens released when macrophages are killed either by T_H1 cells or by cytotoxic CD8 T cells can be taken up by freshly recruited macrophages still capable of activation to antimicrobial activity.

Another very important function of T_H1 cells is the recruitment of phagocytic cells to sites of infection. T_H1 cells recruit macrophages by two mechanisms. First, they make the hematopoietic growth factors IL-3 and GM-CSF, which stimulate the production of new phagocytic cells in the bone marrow. Second, TNF-α and TNF-β, which are secreted by T_H1 cells at sites of infection, change the surface properties of endothelial cells so that phagocytes adhere to them, while chemokines such as macrophage chemotactic protein (MCP-1), produced by T_H1 cells in the inflammatory response, serve to direct the migration of these phagocytic cells through the vascular endothelium to the site of the infection (see Section 10-8).

When microbes effectively resist the microbicidal effects of activated macrophages, chronic infection with inflammation can develop. Often, this has a characteristic pattern, consisting of a central area of macrophages surrounded by activated lymphocytes. This pathological pattern is called a granuloma (Fig. 8.43). Giant cells consisting of fused macrophages usually form the center of these granulomas. This serves to ‘wall-off’ pathogens that resist destruction. T_H2 cells seem to participate in granulomas along with T_H1 cells, perhaps by regulating their activity and preventing widespread tissue damage. In tuberculosis, the center of the large granulomas can become isolated and the cells there die, probably from a combination of lack of oxygen and the cytotoxic effects of activated macrophages. As the dead tissue in the center resembles cheese, this process is called caseation necrosis. Thus, the activation of T_H1 cells can cause significant pathology. Their nonactivation, however, leads to the more serious consequence of death from disseminated infection, which is now seen frequently in patients with AIDS and concomitant mycobacterial infection.

Summary

CD4 T cells that can activate macrophages have a critical role in host defense against those intracellular and extracellular pathogens that resist killing after being engulfed by macrophages. Macrophages are activated by membrane-bound signals delivered by activated T_H1 cells as well as by the potent macrophage-activating cytokine IFN-γ, which is secreted by activated T cells. Once activated, the macrophage can kill intracellular and ingested bacteria. Activated macrophages can also cause local tissue damage, which explains why this activity must be strictly regulated by antigen-specific T cells. T_H1 cells produce a range of cytokines and surface molecules that not only activate infected macrophages but can also kill chronically infected senescent macrophages, stimulate the production of new macrophages in bone marrow, and recruit fresh macrophages to sites of infection. Thus, T_H1 cells have a central role in controlling and coordinating host defense against certain intracellular pathogens. It is likely that the absence of this function explains the preponderance of infections with intracellular pathogens in adult AIDS patients.