Chapter 9:  The Humoral Immune Response

9-1. The humoral immune response is initiated when B cells that bind antigen are signaled by helper T cells or by certain microbial antigens alone

It is a general rule in adaptive immunity that naive antigen-specific lymphocytes are difficult to activate by antigen alone. Naive T cells require a co-stimulatory signal from professional antigen-presenting cells; naive B cells require accessory signals that can come either from an armed helper T cell or, in some cases, directly from microbial constituents.

Antibody responses to protein antigens require antigen-specific T-cell help. B cells can receive help from armed helper T cells when antigen bound by surface immunoglobulin is internalized and returned to the cell surface as peptides bound to MHC class II molecules. Armed helper T cells that recognize the peptide:MHC complex then deliver activating signals to the B cell. Thus, protein antigens binding to B cells both provide a specific signal to the B cell by cross-linking its antigen receptors and allow the B cell to attract antigenspecific T-cell help. These antigens are unable to induce antibody responses in animals or humans who lack T cells, and they are therefore known as thymus-dependent or TD antigens (Fig. 9.2, top two panels).

The B-cell co-receptor complex of CD19:CD21:CD81 (see Section 6-8) can greatly enhance B-cell responsiveness to antigen. CD21 (also known as complement receptor 2, CR2) is a receptor for the complement fragment C3d (see Section 2-11). When mice are immunized with hen egg lysozyme coupled to three linked molecules of the complement fragment C3dg, the modified lysozyme induces antibody without added adjuvant at doses up to 10,000 times smaller than unmodified hen egg lysozyme. Whether binding of CD21 enhances B-cell responsiveness by increasing B-cell signaling, by inducing co-stimulatory molecules on the B cell, or by increasing the receptormediated uptake of antigen, is not yet known. As we will see later in this chapter, antibodies already bound to antigens can activate the complement system, thus coating the antigen with C3d and producing a more potent antigen, which in turn leads to more efficient B-cell activation and antibody production.

Although armed peptide-specific helper T cells are required for B-cell responses to protein antigens, many microbial constituents, such as bacterial polysaccharides, can induce antibody production in the absence of helper T cells. These microbial antigens are known as thymus-independent or TI antigens because they induce antibody responses in individuals who have no T lymphocytes. The second signal required to activate antibody production to TI antigens is either provided directly by recognition of a common microbial constituent (see Fig. 9.2, bottom panel) or by a nonthymus-derived accessory cell in conjunction with massive cross-linking of B-cell receptors, which would occur when a B cell binds repeating epitopes on the bacterial cell. Thymus-independent antibody responses provide some protection against extracellular bacteria, and we will return to them later.

9-2. Armed helper T cells activate B cells that recognize the same antigen

T-cell dependent antibody responses require the activation of B cells by helper T cells that respond to the same antigen; this is called linked recognition. This means that before B cells can be induced to make antibody to an infecting pathogen, a CD4 T cell specific for peptides from this pathogen must first be activated to produce the appropriate armed helper T cells. This presumably occurs by interaction with an antigen-presenting dendritic cell (see Section 8-1). Although the epitope recognized by the armed helper T cell must therefore be linked to that recognized by the B cell, the two cells need not recognize identical epitopes. Indeed, we saw in Chapter 5 that T cells can recognize internal peptides that are quite distinct from the surface epitopes on the same protein recognized by B cells. For more complex natural antigens, such as viruses, the T cell and the B cell might not even recognize the same protein. It is, however, crucial that the peptide recognized by the T cell be a physical part of the antigen recognized by the B cell, which can thus produce the appropriate peptide after internalization of the antigen bound to its B-cell receptors.

For example, by recognizing an epitope on a viral protein coat, a B cell can internalize a complete virus particle. After internalization, the virus particle is degraded and peptides from internal viral proteins as well as coat proteins can be displayed by MHC class II molecules on the B-cell surface. Helper T cells that have been primed earlier in an infection by macrophages or dendritic cells presenting these internal peptides can then activate the B cell to make antibodies that recognize the coat protein (Fig. 9.3).

The specific activation of the B cell by a T cell sensitized to the same antigen or pathogen depends on the ability of the antigen-specific B cell to concentrate the appropriate peptide on its surface MHC class II molecules. B cells that bind a particular antigen are up to 10,000 times more efficient at displaying peptide fragments of that antigen on their MHC class II molecules than are B cells that do not bind the antigen. Armed helper T cells will thus help only those B cells whose receptors bind an antigen containing the peptide they recognize.

The requirement for linked recognition has important consequences for the regulation and manipulation of the humoral immune response. One is that linked recognition helps ensure self tolerance, as will be described in Chapter 13. An important application of linked recognition is in the design of vaccines, such as that used to immunize infants against Haemophilus influenzae type B. This bacterial pathogen can infect the lining of the brain, called the meninges, causing meningitis and, in severe cases, neurological damage or death. Protective immunity to this pathogen is mediated by antibodies against its capsular polysaccharide. Although adults make very effective thymus-independent responses to these polysaccharide antigens, such responses are weak in the immature immune system of the infant. To make an effective vaccine for use in infants, therefore, the polysaccharide is linked chemically to tetanus toxoid, a foreign protein against which infants are routinely and successfully vaccinated (see Chapter 14). B cells that bind the polysaccharide component of the vaccine can be activated by helper T cells specific for peptides of the linked toxoid (Fig. 9.4).

Linked recognition was originally discovered through studies of the production of antibodies to haptens (see Appendix I, Section A-1). Haptens are small chemical groups that cannot elicit antibody responses on their own because they cannot cross-link B-cell receptors and they cannot recruit T-cell help. When coupled at high density to a carrier protein, however, they become immunogenic, because the protein will carry multiple hapten groups that can now cross-link B-cell receptors. In addition, T-cell dependent responses are possible because T cells can be primed to peptides derived from the protein. Coupling of a hapten to a protein is responsible for the allergic responses shown by many people to the antibiotic penicillin, which reacts with host proteins to form a coupled hapten that can stimulate an antibody response, as we will learn in Chapter 12.

9-3. Antigenic peptides bound to self MHC class II molecules trigger armed helper T cells to make membrane-bound and secreted molecules that can activate a B cell

Armed helper T cells activate B cells when they recognize the appropriate peptide:MHC class II complex on the B-cell surface (Fig. 9.5). As with armed T_H1 cells acting on macrophages, recognition of peptide:MHC class II complexes on B cells triggers armed helper T cells to synthesize both cellbound and secreted effector molecules that synergize in activating the B cell. One particularly important T-cell effector molecule is a membrane-bound molecule of the tumor necrosis factor (TNF) family known as CD40 ligand (CD40L, also known as CD154) because it binds to the B-cell surface molecule CD40. CD40 is a member of the TNF-receptor family of cytokine receptors (see Section 8-20) however, it does not contain a ‘death domain.’ It is involved in directing all phases of the B-cell response. Binding of CD40 by CD40L helps to drive the resting B cell into the cell cycle and is essential for B-cell responses to thymus-dependent antigens.

B cells are stimulated to proliferate in vitro when they are exposed to a mixture of artificially synthesized CD40L and the cytokine interleukin-4 (IL-4). IL-4 is also made by armed T_H2 cells when they recognize their specific ligand on the B-cell surface, and IL-4 and CD40L are thought to synergize in driving the clonal expansion that precedes antibody production in vivo. IL-4 is secreted in a polar fashion by the T_H2 cell and is directed at the site of contact with the B cell (Fig. 9.6) so that it acts selectively on the antigen-specific target B cell.The combination of B-cell receptor and CD40 ligation, along with IL-4 and other signals derived from direct T-cell contact, leads to B-cell proliferation. Some of these contact signals have recently been elucidated. They involve other TNF/TNF-receptor family members, including CD30 and CD30 ligand and BLyS (B lymphocyte stimulator) and its receptor on B cells, TACI. After several rounds of proliferation, B cells can further differentiate into antibody-secreting plasma cells. Two additional cytokines, IL-5 and IL-6, both secreted by helper T cells, contribute to these later stages of B-cell activation.

9-4. Isotype switching requires expression of CD40L by the helper T cell and is directed by cytokines

Antibodies are remarkable not only for the diversity of their antigen-binding sites but also for their versatility as effector molecules. The specificity of an antibody response is determined by the antigen-binding site, which consists of the two variable V domains, V_H and V_L; however, the effector action of the antibody is determined by the isotype of its heavy-chain C region (see Section 4-15). A given heavy-chain V domain can become associated with the C region of any isotype through the process of isotype switching (see Section 4-16). We will see later in this chapter how antibodies of each isotype contribute to the elimination of pathogens. The DNA rearrangements that underlie isotype switching and confer this functional diversity on the humoral immune response are directed by cytokines, especially those released by armed effector CD4 T cells.

All naive B cells express cell-surface IgM and IgD, yet IgM makes up less than 10% of the immunoglobulin found in plasma, where the most abundant isotype is IgG. Much of the antibody in plasma has therefore been produced by B cells that have undergone isotype switching. Little IgD antibody is produced at any time, so the early stages of the antibody response are dominated by IgM antibodies. Later, IgG and IgA are the predominant isotypes, with IgE contributing a small but biologically important part of the response. The overall predominance of IgG results, in part, from its longer lifetime in the plasma (see Fig. 4.16).

Isotype switching does not occur in individuals who lack functional CD40L, which is necessary for productive interactions between B cells and helper T cells; such individuals make only small amounts of IgM antibodies in response to thymus-dependent antigens and have abnormally high levels of IgM in their plasma. These IgM antibodies may be induced by thymusindependent antigens expressed by the pathogens that chronically infect these patients, who suffer from severe humoral immunodeficiency, as we will see in Chapter 11.

Most of what is known about the regulation of isotype switching by helper T cells has come from experiments in which mouse B cells are stimulated with bacterial lipopolysaccharide (LPS) and purified cytokines in vitro. These experiments show that different cytokines preferentially induce switching to different isotypes. Some of these cytokines are the same as those that drive B-cell proliferation in the initiation of a B-cell response. In the mouse, IL-4 preferentially induces switching to IgG1 and IgE, whereas transforming growth factor (TGF)-β induces switching to IgG2b and IgA. T_H2 cells make both of these cytokines as well as IL-5, which induces IgA secretion by cells that have already undergone switching. Although T_H1 cells are relatively poor initiators of antibody responses, they participate in isotype switching by releasing interferon (IFN)-γ, which preferentially induces switching to IgG2a and IgG3. The role of cytokines in directing B cells to make the different antibody isotypes is summarized in Fig. 9.7.

Cytokines induce isotype switching by stimulating the formation and splicing of mRNA transcribed from the switch recombination sites that lie 5′ to each heavy-chain C gene (see Fig. 4.20). When activated B cells are exposed to IL-4, for example, transcription from a site upstream of the switch regions of C_γ1 and C_ε can be detected a day or two before switching occurs (Fig. 9.8). Recent data suggest that the production of a spliced switch transcript has a role in directing switching, but the mechanism is not yet clear. Each of the cytokines that induces switching seems to induce transcription from the switch regions of two different heavy-chain C genes, promoting specific recombination to one or other of these genes only. Such a directed mechanism is supported by the observation that individual B cells frequently undergo switching to the same C gene on both chromosomes, even though the antibody heavy chain is only being expressed from one of the chromosomes. Thus, helper T cells regulate both the production of antibody by B cells and the isotype that determines the effector function of the antibody.

9-5. Antigen-binding B cells are trapped in the T-cell zone of secondary lymphoid tissues and are activated by encounter with armed helper T cells

One of the most puzzling features of the antibody response is how an antigenspecific B cell manages to encounter a helper T cell with an appropriate antigen specificity. This question arises because the frequency of naive lymphocytes specific for any given antigen is estimated to be between 1 in 10,000 and 1 in 1,000,000. Thus, the chance of an encounter between a T lymphocyte and a B lymphocyte that recognize the same antigen should be between 1 in 10⁸ and 1 in 10¹². Achieving such an encounter is a far more difficult challenge than getting effector T cells activated, because, in the latter case, only one of the two cells involved has specific receptors. Moreover, T cells and B cells mostly occupy quite distinct zones in peripheral lymphoid tissue (see Fig. 1.8). As in naive T-cell activation (see Chapter 8), the answer seems to lie in the antigen-specific trapping of migrating lymphocytes.

When an antigen is introduced into an animal, it is captured and processed by professional antigen-presenting cells, especially the dendritic cells that migrate from the tissues into the T-cell zones of local lymph nodes. Recirculating naive T cells pass by such cells continuously and those rare T cells whose receptors bind peptides derived from the antigen are trapped very efficiently. This trapping clearly involves the specific antigen receptor on the T cell, although it is stabilized by the activation of adhesion molecules and chemokines as we learned in Sections 8-3 and 8-4. Ingenious experiments using mice transgenic for rearranged immunoglobulin genes show that, in the presence of the appropriate antigen, B cells with antigen-specific receptors are also trapped in the T-cell zones of lymphoid tissue by a similar mechanism. On encountering antigen, migrating antigen-binding B cells are arrested by the activation of adhesion molecules and the engagement of chemokine receptors such as CCR7, a receptor for MIP-3β and SLC.

Trapping of B cells in the T-cell zones provides an elegant solution to the problem posed at the beginning of this section. T cells are themselves trapped and activated to helper status in the T-cell zones, and when B cells migrate into lymphoid tissue through high endothelial venules they first enter these same T-cell zones. Most of the B cells move quickly through the T-cell zone into the B-cell zone (the primary follicle), but those B cells that have bound antigen are trapped. Thus, antigen-binding B cells are selectively trapped in precisely the correct location to maximize the chance of encountering a helper T cell that can activate them. Interaction with armed helper T cells activates the B cell to establish a primary focus of clonal expansion (Fig. 9.9). Here, at the border between T-cell and B-cell zones, both types of lymphocyte will proliferate for several days to constitute the first phase of the primary humoral immune response.

After several days, the primary focus of proliferation begins to involute. Many of the lymphocytes comprising the focus undergo apoptosis. However, some of the proliferating B cells differentiate into antibodysynthesizing plasma cells and migrate to the red pulp of the spleen or the medullary cords of the lymph node. The differentiation of a B cell into a plasma cell is accompanied by many morphological changes that reflect its commitment to the production of large amounts of secreted antibody. The properties of resting B cells and plasma cells are compared in Fig. 9.10. Plasma cells have abundant cytoplasm dominated by multiple layers of rough endoplasmic reticulum (see Fig. 1.19). The nucleus shows a characteristic pattern of peripheral chromatin condensation, a prominent perinuclear Golgi apparatus is visible, and the cisternae of the endoplasmic reticulum are rich in immunoglobulin, which makes up 10–20% of all the protein synthesized. MHC class II molecules are not expressed, so plasma cells can no longer present antigen to helper T cells, although these T cells may still provide important signals for plasma cell differentiation and survival, like IL-6 and CD40L. Surface immunoglobulin is still expressed on plasma cells at low levels, and recent evidence suggests that the survival of plasma cells may be determined in part by their ability to continue to bind antigen. Plasma cells have a range of life-spans. Some survive for only days to a few weeks after their final differentiation, whereas others are very long-lived and account for the persistence of antibody responses.

9-6. The second phase of the primary B-cell immune response occurs when activated B cells migrate to follicles and proliferate to form germinal centers

There is another fate for some of the B cells and T cells that proliferate in the primary focus. Some of these cells migrate into a primary lymphoid follicle (Fig. 9.11) where they continue to proliferate and ultimately form a germinal center (Fig. 9.12). Germinal centers are composed mainly of proliferating B cells, but antigen-specific T cells make up about 10% of germinal center lymphocytes and provide indispensable help to the B cells. The germinal center is essentially an island of cell division that sets up amidst a sea of resting B cells in the primary follicles; germinal center B cells displace the resting B cells toward the periphery of the follicle, forming a mantle zone of resting cells around the center. Primary follicles contain resting B cells clustered around a dense network of processes extending from a specialized cell type, the follicular dendritic cell (FDC). Follicular dendritic cells attract both naive and activated B cells into the follicles by secreting the chemokine BLC (see Section 7-30).

The early events in the primary focus lead to the prompt secretion of specific antibody that serves as immediate protection to the infected individual. The germinal center reaction, on the other hand, provides for a more effective later response, should the pathogen establish a chronic infection or the host become reinfected. To this end, B cells undergo a number of important modifications in the germinal center These include somatic hypermutation (see Chapter 4), which alters the V regions of B cells, affinity maturation, which selects for survival of B cells with high affinity for the antigen, and isotype switching (see Sections 9-4 and 4-16), which allows these selected B cells to express a variety of effector functions in the form of antibodies of different isotypes. The selected B cells will either differentiate into memory B cells, the function of which will be described in Chapter 10, or into plasma cells, which will begin to secrete higher-affinity and isotype-switched antibody during the latter part of the primary immune response.

The germinal center is a site of intense cell proliferation, with B cells dividing every 6 to 8 hours. Initially, these rapidly proliferating B cells dramatically reduce their expression of surface immunoglobulin, particularly of IgD. These B cells are termed centroblasts. As time goes on, some B cells reduce their rate of division and begin to express higher levels of surface immunoglobulin. These are termed centrocytes. The centroblasts at first proliferate in the dark zone of the germinal center (see Fig. 9.12), so called because the proliferating cells are densely packed. With further development, B cells begin to fill the light zone of the germinal center, an area of the follicle that is more richly supplied with follicular dendritic cells and less densely packed with cells. It was thought originally that only the centroblasts in the dark zone proliferated, whereas centrocytes in the light zone did not divide. Indeed, this may be the case in chronic germinal centers found in inflamed tonsils that have been surgically removed. However, in newly forming germinal centers in mice, it is now apparent that proliferation can occur in both light and dark zones, and that proliferative cells in the dark zone can express moderate amounts of immunoglobulin on their surface. So the distinction between dark and light zones as areas of B-cell proliferation or quiescence does not strictly apply to primary germinal centers, at least in mice. Follicular dendritic cells, which originally were most prominent in the light zone, appear to react to germinal center formation and begin to extend more prominently throughout the germinal center as it develops. The result is that a mature germinal center at day 15 after immunization more resembles a light zone, with few of the classic dark zone characteristics. This view of germinal center evolution may help to explain how B cells with high affinity for immunizing antigen are selected, as we now discuss.

9-7. Germinal center B cells undergo V-region somatic hypermutation and cells with mutations that improve affinity for antigen are selected

The process of somatic hypermutation, as one of the four mechanisms that create immunoglobulin diversity, was described in Chapter 4. Here we describe the signals that initiate hypermutation and the biological consequences of mutation for those cells. Somatic hypermutation is normally restricted to B cells that are proliferating in germinal centers. This was first shown by FACS sorting of germinal center B cells (see Appendix I, Section A-22) and sequencing of the V genes of cell lines derived from them; later, it was shown more directly by sequencing the V genes that were amplified by PCR of DNA isolated from germinal center B cells that had been micro-dissected from histologic sections. However, in vitro studies have shown that B cells can be induced to undergo hypermutation outside of germinal centers when their B-cell receptors are cross-linked and they receive help, including cytokines and CD40L stimulation, from activated T cells. In fact, mice that lack germinal centers owing to a mutation in the lymphotoxin-α gene (see Section 7-30) still support B-cell hypermutation, although where this takes place is unknown.

Unlike the other mechanisms of immunoglobulin diversification (see Section 4-6), which generate B cells with radically differing B-cell receptors, somatic hypermutation has the potential to create a series of related B cells that differ subtly in their specificity and affinity for antigen. This is because somatic hypermutation generally involves individual point mutations that change only a single amino acid. Immunoglobulin V-region genes accumulate mutations at a rate of about one base pair change per 10³ base pairs per cell division. The mutation rates of all other somatic cell DNA are much lower: around one base pair change per 10¹⁰ base pairs per cell division. As each of the expressed heavy- and light-chain V-region genes is encoded by about 360 base pairs, and about three out of every four base changes results in an altered amino acid, every second B cell will acquire a mutation in its receptor at each division. These mutations also affect some DNA flanking the rearranged V gene but they generally do not extend into the C-region exons. Thus, random point mutations are somehow targeted to the rearranged V genes in a B cell.

The point mutations accumulate in a stepwise manner as B-cell clones expand in the germinal center. Generally, a B cell will not acquire more than one or two new mutations in each generation. Mutations can affect the ability of a B cell to bind antigen and thus will affect the fate of the B cell in the germinal center, as diagrammed in Fig. 9.13. Most mutations have a negative impact on the ability of the B-cell receptor to bind the original antigen. For example, some mutations will abolish receptor function altogether by introducing a stop codon that prevents proper translation; other deleterious mutations alter framework region amino acids that are essential for correct immunoglobulin folding; and still others alter amino acids in the complementarity-determining regions that are responsible for contacting antigen. These deleterious mutations are disastrous for the cells that harbor them; these cells are eliminated by apoptosis either because they can no longer make a B-cell receptor or because they cannot compete with sibling cells that bind antigen more strongly. Deleterious mutation is evidently a frequent event, as germinal centers are filled with apoptotic B cells that are quickly engulfed by macrophages, resulting in tingible body macrophages, which contain dark-staining nuclear debris in their cytoplasm and are a longrecognized histologic feature of germinal centers.

More rarely, mutations will improve the affinity of a B-cell receptor for antigen. Cells that harbor these mutations are efficiently selected and expanded. Whether this is due to prevention of cell death and/or enhancement of cell division is still unclear. In either case, it is clear that selection is incremental. After each round of mutation, B cells begin to express the new receptor, and it determines the cell's fate, whether favorable or unfavorable. If favorable, the cell undergoes another round of division and mutation and the expression and selection process is repeated. In this way, the affinity and specificity of positively selected B cells is continually refined during the germinal center response. The fact that both centroblasts and centrocytes proliferate and can express immunoglobulin explains how mutation and positive selection can take place simultaneously throughout the germinal center without the need for migration back and forth between the dark and light zones. Evidence of positive and negative selection is seen in the pattern of somatic hyper-mutations in V regions of B cells that have survived passage through the germinal center (see Section 4-9). The existence of negative selection is shown by the relative scarcity of amino acid replacements in the framework regions, reflecting the loss of cells that had mutated any one of the many residues that are critical for immunoglobulin V-region folding. Negative selection is an important force in the germinal center, most likely eliminating about one in every two cells. Were it not for substantial negative selection, B cells dividing three to four times per day in a single germinal center would quickly create enough progeny to overwhelm the entire organism; more than a billion cells could be created in 10 days in a single germinal center. Instead, a germinal center actually contains a few thousand B cells at its peak.

The mark of positive selection, on the other hand, is an accumulation of numerous amino acid replacements in the complementarity-determining regions (see Fig. 4.9). The consequence of these cycles of proliferation, mutation, and selection, which all happen within the germinal center, is that the average affinity of the population of responding B cells for its antigen increases over time, largely explaining the observed phenomenon of affinity maturation of the antibody response. The selection process can be quite stringent: although 50 to 100 B cells may seed the germinal center, most of these leave no progeny, and by the time the germinal center reaches maximum size, it is typically composed of the descendants of only one or a few B cells.

9-8. Ligation of the B-cell receptor and CD40, together with direct contact with T cells, are all required to sustain germinal center B cells

Germinal center B cells are inherently prone to die and, in order to survive, they must receive specific signals. It was originally discovered in vitro that germinal center B cells could be kept alive by simultaneously cross-linking their B-cell receptors and ligating their cell-surface CD40. In vivo, these signals are delivered by antigen and T cells, respectively. Additional signals are also required for survival, which are delivered by direct contact with T cells. The nature of these signals is still obscure, but one signaling system involving the TNF-family member BLyS (the T-cell signal) and TACI (its receptor on B cells) has recently been found to be essential for the maintenance of germinal centers.

The source of antigen in the germinal center has been the matter of some controversy. Antigen can be trapped and stored for long periods of time in the form of immune complexes on follicular dendritic cells (Figs 9.14 and 9.15) and it was therefore assumed that this was the antigen that sustained germinal center B-cell proliferation. While this may be true under certain circumstances, there is now evidence that antigen on follicular dendritic cells is not required to sustain a normal germinal center response. Indeed, the role of the antigen depot on these cells is unknown, although it could be to maintain long-lived plasma cells. Where does the antigen that sustains the germinal center come from? Under normal circumstances, it is most likely that live pathogens carried to the lymphoid tissues and multiplying there will continue to provide antigens until they are eliminated by the immune response, after which the germinal center decays. Immunizations with protein antigens are usually given in a form that slowly releases the antigen over time, which mimics the situation with live pathogens. Indeed, it is difficult to stimulate germinal center formation by immunization without either a live replicating pathogen or a sustained release of antigen in adjuvant (see Appendix I, Section A-4).

How the various signals that maintain the germinal center exert their effects on B cells is not completely understood. The combined signals from the B-cell receptor and CD40 seem to upregulate a protein called Bcl-X_L, a relative of Bcl-2, which promotes B-cell survival (see Chapter 6). There are doubtless many other signals yet to be discovered that promote B-cell differentiation.

9-9. Surviving germinal center B cells differentiate into either plasma cells or memory cells

The purpose of the germinal center reaction is to enhance the later part of the primary immune response. Some germinal center cells differentiate first into plasmablasts and then into plasma cells. Plasmablasts continue to divide rapidly but have begun to specialize to secrete antibody at a high rate; they are destined to become nondividing, terminally differentiated plasma cells and thus represent an intermediate stage of differentiation. These plasma cells will migrate to the bone marrow, where a subset of them will live for a long period of time. Plasma cells obtain signals from bone marrow stromal cells that are essential for their survival. These plasma cells provide a source of long-lasting high-affinity antibody.

Other germinal center cells differentiate into memory B cells. Memory B cells are long-lived descendents of cells that were once stimulated by antigen and had proliferated in the germinal center. These cells divide very slowly if at all; they express surface immunoglobulin, but do not secrete antibody at a high rate. Since the precursors of memory B cells once participated in a germinal center reaction, memory B cells inherit the genetic changes that occurred in germinal center cells, including somatic mutations and the gene rearrangements that result in isotype switch (see Sections 4-9 and 4-16). The signals that control which differentiation path a B cell takes, and even whether at any given point the B cell continues to divide instead of differentiating, are unclear.

It has been proposed that signals from follicular dendritic cells (FDCs) are important in stimulating a B cell to become a memory cell. However, memory cells can develop in mutant mice lacking FDCs, albeit with reduced efficiency, so there may be other sources of signals. Another possibility is that affinity for antigen controls B-cell differentiation, with high-affinity cells perhaps being preferentially stimulated to become memory cells while the lower-affinity cells are allowed to undergo further cycles of proliferation, mutation, and selection. This is just one of the mysteries of the germinal center that immunologists have yet to solve. Immunological memory is discussed in detail in Chapter 10.

9-10. B-cell responses to bacterial antigens with intrinsic ability to activate B cells do not require T-cell help

Although antibody responses to most protein antigens are dependent on helper T cells, humans and mice with T-cell deficiencies nevertheless make antibodies to many bacterial antigens. This is because the special properties of some bacterial polysaccharides, polymeric proteins, and lipopolysaccharides enable them to stimulate naive B cells in the absence of peptide-specific T-cell help. These antigens are known as thymus-independent antigens (TI antigens) because they stimulate strong antibody responses in athymic individuals. These nonprotein bacterial products cannot elicit classical T-cell responses, yet they induce antibody responses in normal individuals. However, B-cell responses to these TI antigens are influenced by the presence of T cells, perhaps indirectly through cytokines such as IL-5 since they are greatly diminished in animals that have no T cells at all.

Thymus-independent antigens fall into two classes that activate B cells by two different mechanisms. TI-1 antigens possess an intrinsic activity that can directly induce B-cell division. At high concentration, these molecules cause the proliferation and differentiation of most B cells regardless of their antigen specificity; this is known as polyclonal activation (Fig. 9.16, top two panels). TI-1 antigens are thus often called B-cell mitogens, a mitogen being a substance that induces cells to undergo mitosis. An example of a B-cell mitogen and TI-1 antigen is LPS, which binds to LPS-binding protein and CD14 (see Chapter 2), which then associate with the receptor TLR-4 on B cells. LPS activates B cells only at doses at least 100 times greater than those needed to activate dendritic cells. Thus, when B cells are exposed to concentrations of TI-1 antigens that are 10³-10⁵ times lower than those used for polyclonal activation, only those B cells whose B-cell receptors also specifically bind the TI-1 molecules become activated. At these low antigen concentrations, sufficient amounts of TI-1 for B-cell activation can only be concentrated on the B-cell surface with the aid of this specific binding (Fig. 9.16, bottom two panels). In the presence of large amounts of the TI-1 antigen, this concentrating effect is not required, and all B cells can be stimulated.

It is likely that, as with any pathogen antigen, concentrations of TI-1 antigens are low during the early stages of infections in vivo; thus, only antigen-specific B cells are likely to be activated and these will produce antibodies specific for the TI-1 antigen. Such responses have an important role in defense against several extracellular pathogens, as they arise earlier than thymus-dependent responses since they do not require prior priming and clonal expansion of helper T cells. However, TI-1 antigens are inefficient inducers of isotype switching, affinity maturation, or memory B cells, all of which require specific T-cell help.

9-11. B-cell responses to bacterial polysaccharides do not require peptide-specific T-cell help

The second class of thymus-independent antigens consist of molecules such as bacterial capsular polysaccharides that have highly repetitive structures. These thymus-independent antigens, called TI-2 antigens, contain no intrinsic B-cell-stimulating activity. Whereas TI-1 antigens can activate both immature and mature B cells, TI-2 antigens can activate only mature B cells; immature B cells, as we saw in Chapter 7, are inactivated by repetitive epitopes. This might be why infants do not make antibodies to polysaccharide antigens efficiently; most of their B cells are immature. Responses to several TI-2 antigens are prominent among B-1 cells (also known as CD5 B cells), which comprise an autonomously replicating subpopulation of B cells, and among marginal zone B cells, another unique subset of nonrecirculating B cells that line the border of the splenic white pulp (see Chapter 7). Although B-1 cells arise early in development, young children do not make a fully effective response to carbohydrate antigens until about 5 years of age. On the other hand, marginal zone B cells are rare at birth and accumulate with age; they may thus be responsible for most physiological TI-2 responses, which also increase with age.

TI-2 antigens most probably act by extensively cross-linking the B-cell receptors of mature B cells specific for the antigen (Fig. 9.17, left panels). Excessive receptor cross-linking, however, renders mature B cells unresponsive or anergic, just as it does immature B cells. Thus, epitope density seems to be critical in the activation of B cells by TI-2 antigens: at too low a density, receptor cross-linking is insufficient to activate the cell; at too high a density, the B cell becomes anergic.

Although responses to TI-2 antigens can occur in nude mice (which lack a thymus), depletion of all T cells by knocking out the TCRβ and TCRδ loci eliminates responses to TI-2 antigens. Moreover, responses to TI-2 antigens can be augmented in vivo by transferring small numbers of T cells to these T-cell deficient mice. How T cells contribute to TI-2 responses is not clear. One possibility is that T cells can recognize and become activated by TI-2 antigens through cell-surface molecules shared by all T cells (Fig. 9.17, right panels). Alternatively, the help might come from γ:δ T cells or from CD4 CD8 double-negative α:β T cells. The T-cell receptors on these cells recognize certain polysaccharides bound to unconventional MHC class I or class I-like molecules such as CD1. Such T cells can develop outside the thymus, principally in the gut.

B-cell responses to TI-2 antigens provide a prompt and specific response to an important class of pathogen. Many common extracellular bacterial pathogens are surrounded by a polysaccharide capsule that enables them to resist ingestion by phagocytes. The bacteria not only escape direct destruction by phagocytes but also avoid stimulating T-cell responses through the presentation of bacterial peptides by macrophages. Antibody that is produced rapidly in response to this polysaccharide capsule without the help of peptide-specific T cells can coat these bacteria, promoting their ingestion and destruction by phagocytes by mechanisms we will describe later in this chapter. The common encapsulated extracellular bacteria are often known as pyogenic bacteria, as they typically cause the formation of abundant pus, which consists chiefly of dead and dying neutrophils that have been recruited to the site of infection. Both IgM and IgG antibodies are induced by TI-2 antigens and are likely to be an important part of the humoral immune response in many bacterial infections. We mentioned earlier the importance of antibodies to the capsular polysaccharide of Haemophilus influenzae type B, a TI-2 antigen, in protective immunity to this bacterium. A further example of the importance of TI-2 responses can be seen in patients with an immunodeficiency disease known as the Wiskott-Aldrich syndrome. These patients can respond, although poorly, to protein antigens but fail to make antibody against polysaccharide antigens and are highly susceptible to infection with encapsulated bacteria. Thus, the TI responses are important components of the humoral immune response to nonprotein antigens that do not engage peptide-specific T-cell help; the distinguishing features of thymus-dependent, TI-1, and TI-2 antibody responses are summarized in Fig. 9.18.

Summary

B-cell activation by many antigens, especially monomeric proteins, requires both binding of the antigen by the B-cell surface immunoglobulin—the B-cell receptor—and interaction of the B cell with antigen-specific helper T cells. Helper T cells recognize peptide fragments derived from the antigen internalized by the B cell and displayed by the B cells as peptide:MHC class II complexes. Helper T cells stimulate the B cell through the binding of CD40L on the T cell to CD40 on the B cell, through interaction of other TNF-TNF-receptor family ligand pairs, and by the directed release of cytokines. The initial interaction occurs in the T-cell area of secondary lymphoid tissue, where both antigen-specific and helper T cells and antigen-specific B cells are trapped as a consequence of binding antigen; further interactions between T cells and B cells occur after migration into the B-cell zone or follicle, and formation of a germinal center. Helper T cells induce a phase of vigorous B-cell proliferation, and direct the differentiation of the clonally expanded progeny of the naive B cells into either antibody-secreting plasma cells or memory B cells. During the differentiation of activated B cells, the antibody isotype can change in response to cytokines released by helper T cells, and the antigen-binding properties of the antibody can change by somatic hypermutation of V-region genes. Somatic hypermutation and selection for high-affinity binding occur in the germinal centers. Helper T cells control these processes by selectively activating cells that have retained their specificity for the antigen and by inducing proliferation and differentiation into plasma cells and memory B cells. Some nonprotein antigens stimulate B cells in the absence of linked recognition by peptide-specific helper T cells. These thymus-independent antigens induce only limited isotype switching and do not induce memory B cells. However, responses to these antigens have a critical role in host defense against pathogens whose surface antigens cannot elicit peptide-specific T-cell responses.

9-12. Antibodies of different isotype operate in distinct places and have distinct effector functions

Pathogens most commonly enter the body across the epithelial barriers of the mucosa lining the respiratory, digestive, and urogenital tracts, or through damaged skin, and can then establish infections in the tissues. Less often, insects, wounds, or hypodermic needles introduce microorganisms directly into the blood. The body's mucosal surfaces, tissues, and blood are all protected by antibodies from such infections; these antibodies serve to neutralize the pathogen or promote its elimination before it can establish a significant infection. Antibodies of different isotypes are adapted to function in different compartments of the body. Because a given V region can become associated with any C region through isotype switching (see Section 4-16), the progeny of a single B cell can produce antibodies, all specific for the same eliciting antigen, that provide all of the protective functions appropriate for each body compartment.

The first antibodies to be produced in a humoral immune response are always IgM, because IgM can be expressed without isotype switching (see Figs 4.20 and 9.8). These early IgM antibodies are produced before B cells have undergone somatic hypermutation and therefore tend to be of low affinity. IgM molecules, however, form pentamers whose 10 antigen-binding sites can bind simultaneously to multivalent antigens such as bacterial capsular polysaccharides. This compensates for the relatively low affinity of the IgM monomers by multipoint binding that confers high overall avidity. As a result of the large size of the pentamers, IgM is mainly found in the blood and, to a lesser extent, the lymph. The pentameric structure of IgM makes it especially effective in activating the complement system, as we will see in the last part of this chapter. Infection of the bloodstream has serious consequences unless it is controlled quickly, and the rapid production of IgM and its efficient activation of the complement system are important in controlling such infections. Some IgM is also produced in secondary and subsequent responses, and after somatic hypermutation, although other isotypes dominate the later phases of the antibody response.

Antibodies of the other isotypes—IgG, IgA, and IgE—are smaller in size and diffuse easily out of the blood into the tissues. Although IgA can form dimers, as we saw in Chapter 4, IgG and IgE are always monomeric. The affinity of the individual antigen-binding sites for their antigen is therefore critical for the effectiveness of these antibodies, and most of the B cells expressing these isotypes have been selected for increased affinity of antigen-binding in germinal centers. IgG is the principal isotype in the blood and extracellular fluid, whereas IgA is the principal isotype in secretions, the most important being those of the mucus epithelium of the intestinal and respiratory tracts. Whereas IgG efficiently opsonizes pathogens for engulfment by phagocytes and activates the complement system, IgA is a less potent opsonin and a weak activator of complement. This distinction is not surprising, as IgG operates mainly in the body tissues, where accessory cells and molecules are available, whereas IgA operates mainly on epithelial surfaces where complement and phagocytes are not normally present, and therefore functions chiefly as a neutralizing antibody.

Finally, IgE antibody is present only at very low levels in blood or extracellular fluid, but is bound avidly by receptors on mast cells that are found just beneath the skin and mucosa, and along blood vessels in connective tissue. Antigen binding to this IgE triggers mast cells to release powerful chemical mediators that induce reactions, such as coughing, sneezing, and vomiting, that can expel infectious agents, as will be discussed below when we describe the receptors that bind immunoglobulin C regions and engage effector functions. The distribution and main functions of antibodies of the different isotypes are summarized in Fig. 9.19.

9-13. Transport proteins that bind to the Fc regions of antibodies carry particular isotypes across epithelial barriers

IgA-secreting plasma cells are found predominantly in the connective tissue called the lamina propria, which lies immediately below the basement membrane of many surface epithelia. From there, the IgA antibodies can be transported across the epithelium to its external surface, for example, to the lumen of the gut or the bronchi. IgA antibody synthesized in the lamina propria is secreted as a dimeric IgA molecule associated with a single J chain (see Fig. 4.23). This polymeric form of IgA binds specifically to the poly-Ig receptor, which is present on the basolateral surfaces of the overlying epithelial cells (Fig. 9.20). When the poly-Ig receptor has bound a molecule of dimeric IgA, the complex is internalized and carried through the cytoplasm of the epithelial cell in a transport vesicle to its luminal surface. This process is called transcytosis. At the apical or luminal surface of the epithelial cell, the poly-Ig receptor is cleaved enzymatically, releasing the extracellular portion of the receptor still attached to the Fc region of the dimeric IgA. This fragment of receptor, called the secretory component, may help to protect the IgA dimer from proteolytic cleavage. Some molecules of dimeric IgA diffuse from the lamina propria into the extracellular spaces of the tissues, draining into the bloodstream before being excreted into the gut via the bile. Therefore, it is not surprising that patients with obstructive jaundice, a condition in which bile is not excreted, show a marked increase in dimeric IgA in the plasma.

The principal sites of IgA synthesis and secretion are the gut, the respiratory epithelium, the lactating breast, and various other exocrine glands such as the salivary and tear glands. It is believed that the primary functional role of IgA antibodies is to protect epithelial surfaces from infectious agents, just as IgG antibodies protect the extracellular spaces of the internal tissues. IgA antibodies prevent the attachment of bacteria or toxins to epithelial cells and the absorption of foreign substances, and provide the first line of defense against a wide variety of pathogens. Newborn infants are especially vulnerable to infection, having had no prior exposure to the microbes in the environment they enter at birth. IgA antibodies are secreted in breast milk and are thereby transferred to the gut of the newborn infant, where they provide protection from newly encountered bacteria until the infant can synthesize its own protective antibody.

IgA is not the only protective antibody a mother passes on to her baby. Maternal IgG is transported across the placenta directly into the bloodstream of the fetus during intrauterine life; human babies at birth have as high a level of plasma IgG as their mothers, and with the same range of antigen specificities. The selective transport of IgG from mother to fetus is due to an IgG transport protein in the placenta, FcRn, which is closely related in structure to MHC class I molecules. Despite this similarity, FcRn binds IgG quite differently from the binding of peptide to MHC class I, as its peptide-binding groove is occluded. It binds to the Fc portion of IgG molecules (Fig. 9.21). Two molecules of FcRn bind one molecule of IgG, bearing it across the placenta. In some rodents, FcRn also delivers IgG to the circulation of the neonate from the gut lumen. Maternal IgG is ingested by the newborn animal in its mother's milk and colostrum, the protein-rich fluid secreted by the early postnatal mammary gland. In this case, FcRn transports the IgG from the lumen of the neonate gut into the blood and tissues. Interestingly, FcRn is also found in adults in the gut and liver and on endothelial cells. Its function in adults is to regulate the levels of IgG in serum and other body fluids, which it does by binding circulating antibody, endocytosing it, and then recycling to the cell surface.

By means of these specialized transport systems, mammals are supplied from birth with antibodies against pathogens common in their environments. As they mature and make their own antibodies of all isotypes, these are distributed selectively to different sites in the body (Fig. 9.22). Thus, throughout life, isotype switching and the distribution of isotypes through the body provide effective protection against infection in extracellular spaces.

9-14. High-affinity IgG and IgA antibodies can neutralize bacterial toxins

Many bacteria cause disease by secreting proteins called toxins, which damage or disrupt the function of the host's cells (Fig. 9.23). To have an effect, a toxin must interact specifically with a molecule that serves as a receptor on the surface of the target cell. In many toxins, the receptor-binding domain is on one polypeptide chain whereas the toxic function is carried by a second chain. Antibodies that bind to the receptor-binding site on the toxin molecule can prevent the toxin from binding to the cell and thus protect the cell from attack (Fig. 9.24). Antibodies that act in this way to neutralize toxins are referred to as neutralizing antibodies.

Most toxins are active at nanomolar concentrations: a single molecule of diphtheria toxin can kill a cell. To neutralize toxins, therefore, antibodies must be able to diffuse into the tissues and bind the toxin rapidly and with high affinity. The ability of IgG antibodies to diffuse easily throughout the extracellular fluid and their high affinity make these the principal neutralizing antibodies for toxins found in tissues. IgA antibodies similarly neutralize toxins at the mucosal surfaces of the body.

Diphtheria and tetanus toxins are two bacterial toxins in which the toxic and receptor-binding functions are on separate protein chains. It is therefore possible to immunize individuals, usually as infants, with modified toxin molecules in which the toxic chain has been denatured. These modified toxins, called toxoids, lack toxic activity but retain the receptor-binding site. Thus, immunization with the toxoid induces neutralizing antibodies that protect against the native toxin.

With some insect or animal venoms that are so toxic that a single exposure can cause severe tissue damage or death, the adaptive immune response is too slow to be protective. Exposure to these venoms is a rare event and protective vaccines have not been developed for use in humans. Instead, neutralizing antibodies are generated by immunizing other species, such as horses, with insect and snake venoms to produce anti-venom antibodies (antivenins) for use in protecting humans. Transfer of antibodies in this way is known as passive immunization (see Appendix I, Section A-37).

9-15. High-affinity IgG and IgA antibodies can inhibit the infectivity of viruses

Animal viruses infect cells by binding to a particular cell-surface receptor, often a cell-type-specific protein that determines which cells they can infect. The hemagglutinin of influenza virus, for example, binds to terminal sialic acid residues on the carbohydrates of glycoproteins present on epithelial cells of the respiratory tract. It is known as hemagglutinin because it recognizes and binds to similar sialic acid residues on chicken red blood cells and agglutinates these red blood cells. Antibodies to the hemagglutinin can prevent infection by the influenza virus. Such antibodies are called virus-neutralizing antibodies and, as with the neutralization of toxins, high-affinity IgA and IgG antibodies are particularly important.

Many antibodies that neutralize viruses do so by directly blocking viral binding to surface receptors (Fig. 9.25). However, viruses are sometimes successfully neutralized when only a single molecule of antibody is bound to a virus particle that has many receptor-binding proteins on its surface. In these cases, the antibody must cause some change in the virus that disrupts its structure and either prevents it from interacting with its receptors or interferes with the fusion of the virus membrane with the cell surface after the virus has engaged its surface receptor.

9-16. Antibodies can block the adherence of bacteria to host cells

Many bacteria have cell-surface molecules called adhesins that enable them to bind to the surface of host cells. This adherence is critical to the ability of these bacteria to cause disease, whether they subsequently enter the cell, as do some pathogens such as Salmonella species, or remain attached to the cell surface as extracellular pathogens (Fig. 9.26). Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, has a cellsurface protein known as pilin. Pilin enables the bacterium to adhere to the epithelial cells of the urinary and reproductive tracts and is essential to its infectivity. Antibodies against pilin can inhibit this adhesive reaction and prevent infection.

IgA antibodies secreted onto the mucosal surfaces of the intestinal, respiratory, and reproductive tracts are particularly important in preventing infection by preventing the adhesion of bacteria, viruses, or other pathogens to the epithelial cells lining these surfaces. The adhesion of bacteria to cells within tissues can also contribute to pathogenesis, and IgG antibodies against adhesins can protect from damage much as IgA antibodies protect at mucosal surfaces.

9-17. Antibody:antigen complexes activate the classical pathway of complement by binding to C1q

Another way in which antibodies can protect against infection is by activation of the cascade of complement proteins. We have described these proteins in Chapter 2, as they can also be activated on pathogen surfaces in the absence of antibody, as part of the innate immune response. Complement activation proceeds via a series of proteolytic cleavage reactions, in which inactive components, present in plasma, are cleaved to form proteolytic enzymes that attach covalently to the pathogen surface. All known pathways of complement activation converge to generate the same set of effector actions: the pathogen surface or immune complex is coated with covalently attached fragments (principally C3b) that act as opsonins to promote uptake and removal by phagocytes. At the same time, small peptides with inflammatory and chemotactic activity are released (principally C5a) so that phagocytes are recruited to the site. In addition, the terminal complement components can form a membrane-attack complex that damages some bacteria.

Antibodies initiate complement activation by a pathway known as the classical pathway because it was the first pathway of complement activation to be discovered. The full details of this pathway, and of the other two known pathways of complement activation, are given in Chapter 2, but we will describe here how antibody is able to initiate the classical pathway after binding to pathogen, or after forming immune complexes.

The first component of the classical pathway of complement activation is C1, which is a complex of three proteins called C1q, C1r, and C1s. Two molecules each of C1r and C1s are bound to each molecule of C1q (see Fig. 2.10). Complement activation is initiated when antibodies attached to the surface of a pathogen bind C1q. C1q can be bound by either IgM or IgG antibodies but, because of the structural requirements of binding to C1q, neither of these antibody isotypes can activate complement in solution; the cascade is initiated only when the antibodies are bound to multiple sites on a cell surface, normally that of a pathogen.

The C1q molecule has six globular heads joined to a common stem by long, filamentous domains that resemble collagen molecules; the whole C1q complex has been likened to a bunch of six tulips held together by the stems. Each globular head can bind to one Fc domain, and binding of two or more globular heads activates the C1q molecule. In plasma, the pentameric IgM molecule has a planar conformation that does not bind C1q (Fig. 9.27, left panel); however, binding to the surface of a pathogen deforms the IgM pentamer so that it looks like a staple (see Fig. 9.27, right panel), and this distortion exposes binding sites for the C1q heads. Although C1q binds with low affinity to some subclasses of IgG in solution, the binding energy required for C1q activation is achieved only when a single molecule of C1q can bind two or more IgG molecules that are held within 30–40 nm of each other as a result of binding antigen. This requires many molecules of IgG to be bound to a single pathogen. For this reason, IgM is much more efficient in activating complement than is IgG. The binding of C1q to a single bound IgM molecule, or to two or more bound IgG molecules, leads to the activation of an enzymatic activity in C1r, triggering the complement cascade as shown schematically in Fig. 9.28. This translates antibody binding into the activation of the complement cascade, which, as we learned in Chapter 2, can also be triggered by direct binding of C1q to the pathogen surface.

9-18. Complement receptors are important in the removal of immune complexes from the circulation

Many small soluble antigens form antibody:antigen complexes known as immune complexes that contain too few molecules of IgG to be readily bound to the Fcγ receptors we will discuss in the next part of the chapter. These antigens include toxins bound by neutralizing antibodies and debris from dead microorganisms. Such immune complexes are found after most infections and are removed from the circulation through the action of complement. The soluble immune complexes trigger their own removal by activating complement, again through the binding of C1q, leading to the covalent binding of the activated components C4b and C3b to the complex, which is then cleared from the circulation by the binding of C4b and C3b to CR1 on the surface of erythrocytes. The erythrocytes transport the bound complexes of antigen, antibody, and complement to the liver and spleen. Here, macrophages bearing CR1 and Fc receptors remove the complexes from the erythrocyte surface without destroying the cell, and then degrade them (Fig. 9.29). Even larger aggregates of particulate antigen and antibody can be made soluble by activation of the classical complement pathway, and then removed by binding to complement receptors.

Immune complexes that are not removed tend to deposit in the basement membranes of small blood vessels, most notably those of the renal glomerulus where the blood is filtered to form urine. Immune complexes that pass through the basement membrane of the glomerulus bind to the complement receptor CR1 on the renal podocytes, cells that lie beneath the basement membrane. The functional significance of these receptors in the kidney is unknown; however, they play an important part in the pathology of some autoimmune diseases.

In the autoimmune disease systemic lupus erythematosus, which we will describe in Chapter 13, excessive levels of circulating immune complexes cause huge deposits of antigen, antibody, and complement on the podocytes, damaging the glomerulus; kidney failure is the principal danger in this disease. Immune complexes can also be a cause of pathology in patients with deficiencies in the early components of complement. Such patients do not clear immune complexes effectively and they also suffer tissue damage, especially in the kidneys, in a similar way.

Summary

The T-cell dependent antibody response begins with IgM secretion but quickly progresses to the production of all the different isotypes. Each isotype is specialized both in its localization in the body and in the functions it can perform. IgM antibodies are found mainly in blood; they are pentameric in structure. IgM is specialized to activate complement efficiently upon binding antigen. IgG antibodies are usually of higher affinity and are found in blood and in extracellular fluid, where they can neutralize toxins, viruses, and bacteria, opsonize them for phagocytosis, and activate the complement system. IgA antibodies are synthesized as monomers, which enter blood and extracellular fluids, or as dimeric molecules in the lamina propria of various epithelia. IgA dimers are selectively transported across these epithelia into sites such as the lumen of the gut, where they neutralize toxins and viruses and block the entry of bacteria across the intestinal epithelium. Most IgE antibody is bound to the surface of mast cells that reside mainly just below body surfaces; antigen binding to this IgE triggers local defense reactions. Thus, each of these isotypes occupies a particular site in the body and has a particular role in defending the body against extracellular pathogens and their toxic products. Antibodies can accomplish this by direct interactions with pathogens or their products, for example by binding to active sites of toxins and neutralizing them or by blocking their ability to bind to host cells through specific receptors. When antibodies of the appropriate isotype bind to antigens, they can activate the classical pathway of complement, which leads to the elimination of the pathogen by the various mechanisms described in Chapter 2. Soluble immune complexes of antigen and antibody also fix complement and are cleared from the circulation via complement receptors on red blood cells.

9-19. The Fc receptors of accessory cells are signaling receptors specific for immunoglobulins of different isotypes

The Fc receptors are a family of cell-surface molecules that bind the Fc portion of immunoglobulins. Each member of the family recognizes immunoglobulin of one isotype or a few closely related isotypes through a recognition domain on the α chain of the Fc receptor. Fc receptors are themselves members of the immunoglobulin superfamily. Different accessory cells bear Fc receptors for antibodies of different isotypes, and the isotype of the antibody thus determines which accessory cell will be engaged in a given response. The different Fc receptors, the cells that express them, and their isotype specificity are shown in Fig. 9.30.

Most Fc receptors function as part of a multisubunit complex. Only the α chain is required for specific recognition; the other chains are required for transport to the cell surface and for signal transduction when an Fc region is bound. Signal transduction by many of these Fc receptors is mediated by the γ chain, which is closely related to the ζ chain of the T-cell receptor complex. Some Fcγ receptors, the Fcα receptor, and the high-affinity receptor for IgE use a γ chain for signaling; an exception is human FcγRII-A, a single-chain receptor in which the cytoplasmic domain of the α chain replaces the function of the γ chain. FcγRII-B1 and FcγRII-B2 are also single-chain receptors but function as inhibitory receptors as they contain an ITIM that engages the inositol 5′-phosphatase SHIP (see Section 6-14). Although the most prominent function of Fc receptors is the activation of accessory cells to attack pathogens, they can also contribute in other ways to immune responses. For example, the FcγRII-B receptor negatively regulates B cells, mast cells, macrophages, and neutrophils by adjusting the threshold at which immune complexes will activate these cells. Fc receptors expressed by dendritic cells enable them to ingest antigen:antibody complexes and present antigenic peptides to T cells.

9-20. Fc receptors on phagocytes are activated by antibodies bound to the surface of pathogens and enable the phagocytes to ingest and destroy pathogens

Phagocytes are activated by IgG antibodies, especially IgG1 and IgG3, that bind to specific Fcγ receptors on the phagocyte surface (see Fig. 9.30). As phagocyte activation can initiate an inflammatory response and cause tissue damage, it is essential that the Fc receptors on phagocytes are able to distinguish antibody molecules bound to a pathogen from the much larger number of free antibody molecules that are not bound to anything. This distinction is made possible by the aggregation or multimerization of antibodies that occurs when they bind to multimeric antigens or to multivalent antigenic particles such as viruses and bacteria. Fc receptors on the surface of an accessory cell bind antibody-coated particles with higher avidity than immunoglobulin monomers, and this is probably the principal mechanism by which bound antibodies are distinguished from free immunoglobulin (Fig. 9.31). The result is that Fc receptors enable accessory cells to detect pathogens through bound antibody molecules. Thus, specific antibody together with Fc receptors gives accessory cells that lack intrinsic specificity the ability to identify and remove pathogens and their products from the extracellular spaces.

The most important accessory cells in humoral immune responses are the phagocytic cells of the monocytic and myelocytic lineages, particularly macrophages and neutrophils (see Chapter 2). Many bacteria are directly recognized, ingested, and destroyed by phagocytes, and these bacteria are not pathogenic in normal individuals (see Chapter 2). Bacterial pathogens, however, often have polysaccharide capsules that allow them to resist direct engulfment by phagocytes. These bacteria become susceptible to phagocytosis, however, when they are coated with antibody and complement that engages the Fcγ or Fcα receptors and CR1 on phagocytic cells, triggering bacterial uptake (Fig. 9.32). Phagocytosis by binding to complement receptors is particularly important early in the immune response, before isotypeswitched antibodies have been made. Capsular polysaccharides belong to the TI-2 class of thymus-independent antigens (see Section 9-11) and therefore can stimulate the early production of IgM antibodies. IgM is not an opsonizing antibody in itself, as there are no Fc receptors for IgM, but it is effective at activating the complement system. IgM binding to encapsulated bacteria thus triggers opsonization of these bacteria by complement and their prompt ingestion and destruction by phagocytes bearing complement receptors.

Both the internalization and destruction of microorganisms are greatly enhanced by interactions between the molecules coating an opsonized microorganism and their receptors on the phagocyte surface. When an antibody-coated pathogen binds to Fcγ receptors on the surface of a phagocyte, for example, the cell surface extends around the surface of the particle through successive binding of Fcγ receptors to the antibody Fc regions bound to the pathogen surface. This is an active process triggered by the stimulation of Fcγ receptors. Endocytosis leads to enclosure of the particle in an acidified cytoplasmic vesicle called a phagosome. The phagosome then fuses with one or more lysosomes to generate a phagolysosome, releasing the lysosomal enzymes into the phagosome interior where they destroy the bacterium (see Fig. 9.32). The process of bacterial destruction in the phagolysosome was described in detail in Section 2-3.

Some particles are too large for a phagocyte to ingest; parasitic worms are one example. In this case, the phagocyte attaches to the surface of the antibody-coated parasite via its Fcγ, Fcα, or Fcε receptors, and the lysosomes fuse with the attached surface membrane. This reaction discharges the contents of the lysosome onto the surface of the parasite, damaging it directly in the extracellular space. While the principal phagocytes in the destruction of bacteria are macrophages and neutrophils, large parasites such as helminths are more usually attacked by eosinophils (Fig. 9.33). Thus, Fcγ and Fcα receptors can trigger the internalization of external particles by phagocytosis, or the externalization of internal vesicles by exocytosis. Cross-linking of IgE bound to the high-affinity FcεRI usually results in exocytosis. We will see in the next three sections that natural killer cells and mast cells also release mediators stored in their vesicles when their Fc receptors are aggregated.

9-21. Fc receptors activate natural killer cells to destroy antibody-coated targets

Infected cells are usually destroyed by T cells alerted by foreign peptides bound to cell-surface MHC molecules. However, virus-infected cells can also signal the presence of intracellular infection by expressing on their surfaces viral proteins that can be recognized by antibodies. Cells bound by such antibodies can then be killed by a specialized non-T, non-B lymphoid cell called a natural killer cell (NK cell), which we met earlier in Chapter 2. NK cells are large lymphoid cells with prominent intracellular granules; they make up a small fraction of peripheral blood lymphoid cells. They bear no known antigen-specific receptors but are able to recognize and kill a limited range of abnormal cells. They were first discovered because of their ability to kill some tumor cells but are now known to have an important role in innate immunity.

The destruction of antibody-coated target cells by NK cells is called antibody-dependent cell-mediated cytotoxicity (ADCC) and is triggered when antibody bound to the surface of a cell interacts with Fc receptors on the NK cell (Fig. 9.34). NK cells express the receptor FcγRIII (CD16), which recognizes the IgG1 and IgG3 subclasses and triggers cytotoxic attack by the NK cell on antibodycoated target cells. The mechanism of attack is exactly analogous to that of cytotoxic T cells, involving the release of cytoplasmic granules containing perforin and granzymes (see Section 8-22). The importance of ADCC in defense against infection with bacteria or viruses has not yet been fully established. However, ADCC represents yet another mechanism by which, through engaging an Fc receptor, antibodies can direct an antigen-specific attack by an effector cell that itself lacks specificity for antigen.

9-22. Mast cells, basophils, and activated eosinophils bind IgE antibody via the high-affinity Fcε receptor

When pathogens cross epithelial barriers and establish a local focus of infection, the host must mobilize its defenses and direct them to the site of pathogen growth. One mechanism by which this is achieved is to activate a specialized cell type known as a mast cell. Mast cells are large cells containing distinctive cytoplasmic granules that contain a mixture of chemical mediators, including histamine, that act rapidly to make local blood vessels more permeable. Mast cells have a distinctive appearance after staining with the dye toluidine blue that makes them readily identifiable in tissues (see Fig. 1.4). They are found in particularly high concentrations in vascularized connective tissues just beneath body epithelial surfaces, including the submucosal tissues of the gastrointestinal and respiratory tracts and the dermis that lies just below the surface of the skin.

Mast cells can be activated to release their granules, and to secrete lipid inflammatory mediators and cytokines, via antibody bound to Fc receptors specific for IgE (FcεRI) and IgG (FcγRIII). We have seen earlier that most Fc receptors bind stably to the Fc region of antibodies only when these are bound to antigen. By contrast, FcεRI binds monomeric IgE antibodies with a very high affinity, measured at approximately 10¹⁰ M^-1. Thus, even at the low levels of IgE found circulating in normal individuals, a substantial portion of the total IgE is bound to the FcεRI on mast cells and on circulating basophilic granulocytes or basophils. Eosinophils can also express Fc receptors, but only express FcεRI when activated and recruited to an inflammatory site.

Although mast cells are usually stably associated with bound IgE, they are not activated simply by the binding of monomeric antigens to this IgE. Mast-cell activation only occurs when the bound IgE is cross-linked by multivalent antigen. This signal activates the mast cell to release the contents of its granules, which occurs in seconds (Fig. 9.35), and to synthesize and release lipid mediators such as prostaglandin D₂ and leukotriene C4, and to secrete cytokines such as TNF-α, thereby initiating a local inflammatory response. Degranulation releases the stored histamine, causing a local increase in blood flow and vascular permeability that quickly leads to accumulation of fluid and blood proteins, including antibodies, in the surrounding tissue. Shortly afterwards, there is an influx of blood-borne cells such as polymorphonuclear leukocytes and later macrophages, eosinophils, and effector lymphocytes. This influx can last a few minutes to a few hours and produces an inflammatory response at the site of infection. Thus, mast cells are part of the front-line host defenses against pathogens that enter the body across epithelial barriers.

9-23. IgE-mediated activation of accessory cells has an important role in resistance to parasite infection

Mast cells are thought to serve at least three important functions in host defense. First, their location near body surfaces allows them to recruit both specific and nonspecific effector elements to sites where infectious agents are most likely to enter the internal milieu. Second, they also increase the flow of lymph from sites of antigen deposition to the regional lymph nodes, where naive lymphocytes are first activated. Third, their ability to trigger muscular contraction can contribute to the physical expulsion of pathogens from the lungs or the gut. Mast cells respond rapidly to the binding of antigen to surfacebound IgE antibodies, and their activation leads to the recruitment and activation of basophils and eosinophils, which contribute further to the IgE-mediated response. There is increasing evidence that such IgE-mediated responses are crucial to defense against parasite infestation.

A role for mast cells in the clearance of parasites is suggested by accumulation of mast cells in the intestine, known as mastocytosis, that accompanies helminth infection, and by observations in W/W^V mutant mice, which have a profound mast-cell deficiency caused by mutation of the gene c-kit. These mutant mice show impaired clearance of the intestinal nematodes Trichinella spiralis and Strongyloides species. Clearance of Strongyloides is even more impaired in W/W^V mice that lack IL-3 and therefore, in addition to lacking mast cells, fail to produce basophils. Thus both mast cells and basophils seem to contribute to defense against these helminth parasites. Other evidence also points to the importance of IgE antibodies and eosinophils in defense against parasites. Infections by certain classes of parasite, particularly helminths, are strongly associated with the production of IgE antibodies and the presence of an abnormally large number of eosinophils (eosinophilia) in blood and tissues. Furthermore, experiments in mice show that depletion of eosinophils by using polyclonal anti-eosinophil antisera increases the severity of infection by the parasitic helminth Schistosoma mansoni. Eosinophils seem to be directly responsible for helminth destruction; examination of infected tissues shows degranulated eosinophils adhering to helminths, and experiments in vitro have shown that eosinophils can kill Schistosoma mansoni in the presence of specific IgE (see Fig. 9.33), IgG, or IgA anti-schistosome antibodies.

The role of IgE, mast cells, basophils, and eosinophils can also be seen in resistance to the feeding of blood-sucking ixodid ticks. Normal skin at the site of a tick bite shows degranulated mast cells, and an accumulation of basophils and eosinophils that are degranulated, an indicator of recent activation. Resistance to subsequent feeding by these ticks develops after the first exposure, suggesting a specific immunological mechanism. Mast-cell deficient mice show no such acquired resistance to tick species, and in guinea pigs the depletion of either basophils or eosinophils by specific polyclonal antibodies also reduces resistance to tick feeding. Finally, recent experiments have shown that resistance to ticks in mice is mediated by specific IgE antibody.

Thus, many clinical studies and experiments support a role for this system of IgE binding to the high-affinity FcεRI in host resistance to pathogens that enter across epithelia. We will see later, in Chapter 12, that the same system accounts for many of the symptoms in allergic diseases such as asthma, hayfever, and the life-threatening response known as systemic anaphylaxis.

Summary

Antibody-coated pathogens are recognized by accessory effector cells through Fc receptors that bind to the multiple constant regions (Fc portions) provided by the bound antibodies. Binding activates the accessory cell and triggers destruction of the pathogen. Fc receptors comprise a family of proteins, each of which recognizes immunoglobulins of particular isotypes. Fc receptors on macrophages and neutrophils recognize the constant regions of IgG or IgA antibodies bound to a pathogen and trigger the engulfment and destruction of IgG- or IgA-coated bacteria. Binding to the Fc receptor also induces the production of microbicidal agents in the intracellular vesicles of the phagocyte. Eosinophils are important in the elimination of parasites too large to be engulfed; they bear Fc receptors specific for the constant region of IgG, as well as high-affinity receptors for IgE; aggregation of these receptors triggers the release of toxic substances onto the surface of the parasite. NK cells, tissue mast cells, and blood basophils also release their granule contents when their Fc receptors are engaged. The high-affinity receptor for IgE is expressed constitutively by mast cells and basophils, and is induced in activated eosinophils. It differs from other Fc receptors in that it can bind free monomeric antibody, thus enabling an immediate response to pathogens at their site of first entry into the tissues. When IgE bound to the surface of a mast cell is aggregated by binding to antigen, it triggers the release of histamine and many other mediators that increase the blood flow to sites of infection; it thereby recruits antibodies and effector cells to these sites. Mast cells are found principally below epithelial surfaces of the skin and the digestive and respiratory tracts, and their activation by innocuous substances is responsible for many of the symptoms of acute allergic reactions, as will be described in Chapter 12.