Immunology_Janeway

Immunology_Janeway. Linked recognition relies on the concentration of the appropriate peptide for presentation by MHC class II molecules on the B-cell surface. B cells whose B-cell receptor binds a particular antigen are 10,000 times more efficient at displaying peptide fragments of that antigen on their MHC class II molecules than are B cells that process the antigen through macropinocytosis alone. Linked recognition was originally discovered through studies of the production of antibodies against haptens, which are small chemical groups that cannot elicit antibody responses on their own (see Appendix I, Section A-1). But haptens that are coupled to a carrier protein become immunogenic—known as the hapten carrier effect—for two reasons. The protein can carry multiple hapten groups, allowing it to cross-link B-cell receptors. Also, T cells that are activated against peptides of the carrier protein can become TFH cells and strengthen the antibody response to the hapten. Accidental coupling of a hapten to


Immunology_Janeway

Immunology_Janeway. B-cell receptors. Also, T cells that are activated against peptides of the carrier protein can become TFH cells and strengthen the antibody response to the hapten. Accidental 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 14.


Immunology_Janeway

Immunology_Janeway. Linked recognition works to preserve self-tolerance, since autoreactive antibodies will arise only if self-reactive TFH and self-reactive B cells are present at the same time. This is discussed further in Chapter 15. Vaccine design can take advantage of linked recognition, as in the vaccine used to immunize infants against Haemophilus influenzae type b (see Section 16-26). 10-3 B cells that encounter their antigens migrate toward the lymphoid tissues.


Immunology_Janeway

Immunology_Janeway. 10-3 B cells that encounter their antigens migrate toward the lymphoid tissues. The frequency of naive lymphocytes specific for a particular antigen is extremely low (less than 1 in 10,000). Thus, the chance of a random encounter between a T and a B cell with the same antigen specificity should be less than 1 in 108, making it remarkable that B cells ever interact with TFH cells with similar antigen specificity. For these reasons, linked recognition requires a precise regulation of the migration of activated B and T cells—orchestrated by several sets of ligands and receptors—into specific locations within the lymphoid tissues, which serves to increase the chances of a productive interaction (Fig. 10.5).


Immunology_Janeway

Immunology_Janeway. Naive T cells and B cells express the sphingosine 1-phosphate receptor, S1PR1, which they use to egress from the peripheral lymphoid tissues (see Section 9-7). However, before they exit, they are retained and initially occupy two distinct zones, the T-cell areas and the primary lymphoid follicles (or B-cell areas or B-cell zones), respectively (see Figs 1.18–1.20). These zones are established by different patterns of chemokine receptor expression and chemokine production. Naive T cells express the chemokine receptor CCR7, and localize to zones where its ligands, CCL19 and CCL21, are highly expressed by stromal cells and dendritic cells (see Section 9-3). Circulating naive B cells express CXCR5, and when they migrate into lymphoid tissues, they enter the primary lymphoid follicles, where the chemokine CXCL13 is abundant. Within the follicle, stromal cells and a specialized cell type, the follicular dendritic cell (FDC), secrete CXCL13. The FDC is a nonphagocytic cell of


Immunology_Janeway

Immunology_Janeway. where the chemokine CXCL13 is abundant. Within the follicle, stromal cells and a specialized cell type, the follicular dendritic cell (FDC), secrete CXCL13. The FDC is a nonphagocytic cell of nonhematopoietic origin that bears numerous long processes; it functions by trapping antigen using complement receptors on its cell surface for access by B cells in the follicle.


Immunology_Janeway

Immunology_Janeway. Once in the follicle, naive B cells encounter the soluble TNF-family cytokine BAFF (see Section 8-8), which is secreted by FDCs, stromal cells, and dendritic cells and which acts as a survival factor for B cells. BAFF can act through three


Immunology_Janeway

Immunology_Janeway. Fig. 10.4 T cells and B cells must recognize antigens contained within the same molecular complex in order to interact. In this example, an internal viral protein harbors a peptide epitope (shown as red) that is presented by MHC class II molecules and is recognized by a CD4 T cell. The virus also harbors a native epitope on an external viral coat protein (shown as blue) that is recognized by the surface immunoglobulin on a B cell. If the virus is captured and presented by a dendritic cell, a peptide-specific CD4 T cell (blue) becomes activated (top left panel), whereas nonspecific T cells (green) remain inactive. If the virus is recognized by a specific B cell (top right panel), peptides derived from internal viral proteins are processed and presented by MHC class II molecules. When the activated T cell recognizes its peptide on this B cell (bottom panel), the T cell will deliver various accessory signals to the B cell that promote antibody production against the coat protein. This


Immunology_Janeway

Immunology_Janeway. activated T cell recognizes its peptide on this B cell (bottom panel), the T cell will deliver various accessory signals to the B cell that promote antibody production against the coat protein. This process is known as linked recognition.


Immunology_Janeway

Immunology_Janeway. Fig. 10.5 Antigen-binding B cells meet T cells at the border between the T-cell area and a B-cell follicle in secondary lymphoid tissues. Antigens enter the spleen from the blood and collect in T-cell zones and follicles (first panel). Naive CCR7-positive T cells and CXCR5-positive B cells migrate to distinct regions where the chemokines CCL19 and CCL21, or CXCL13 and 7α, 25-hydroxycholesterol (7α, 25-HC), respectively, are being produced (second panel). If a B cell encounters its antigen, either on a follicular dendritic cell (FDC) or a macrophage, it increases expression of CCR7 and migrates toward the border with the T-cell zone (third panel). T cells activated by antigen-presenting dendritic cells induce expression of CXCR5 and migrate to this same border, where linked recognition induces further B-cell proliferation. After 2 to 3 days, B cells reduce expression of CCR7, but retain EBI2 and migrate in response to 7α, 25-HC to the outer follicle and interfollicular regions (fourth


Immunology_Janeway

Immunology_Janeway. further B-cell proliferation. After 2 to 3 days, B cells reduce expression of CCR7, but retain EBI2 and migrate in response to 7α, 25-HC to the outer follicle and interfollicular regions (fourth panel). After another day or so, some B cells cluster in the interfollicular regions near the red pulp, proliferate, and differentiate into plasmablasts, forming a primary focus with terminal differentiation into antibody-secreting plasma cells. T cells that retain EBI2 expression may remain in the follicle and induce Bcl-6 expression to become TFH cells that participate with B cells there to form a germinal center reaction.


Immunology_Janeway

Immunology_Janeway. Some B cells migrate to form a primary focus and differentiate into plasmablasts, while some T cells induce Bcl-6 and become TFH cells TFH primary focus interfollicular zone CCL21 CXCL13 7˜,25-HC resting T cells resting B cells FDC dendritic cell Interactions with T cells sustain EBI2 expression on B cells, which move to outer follicular and interfollicular regions Activated B cells induce CCR7 and EBI2, and T cells induce CXCR5, and both cells migrate to follicular and interfollicular regions Before activation, resting B cells express CXCR5 and reside in follicles and T cells express CCR7 and reside in T-cell zones activated B cells activated T cells receptors, but its major actions in promoting survival are through BAFF-R (Fig. 10.6). BAFF-R signals through TRAF3 (see Section 3-7) to activate the non-canonical NFκB pathway, as described for CD40 (see Fig. 7.31), and, like CD40 signaling, induces expression of Bcl-2. Two other receptors for BAFF are TACI and BCMA, although BAFF has a


Immunology_Janeway

Immunology_Janeway. the non-canonical NFκB pathway, as described for CD40 (see Fig. 7.31), and, like CD40 signaling, induces expression of Bcl-2. Two other receptors for BAFF are TACI and BCMA, although BAFF has a relatively low affinity for BCMA. TACI and BCMA also bind the related cytokine APRIL, and they signal through TRAF2, 5, and 6 to induce signaling pathways involved in B-cell activation.


Immunology_Janeway

Immunology_Janeway. Antigens derived from microorganisms and viruses are transported into lymph nodes via the afferent lymph, and into the spleen via the blood. Opsonized antigens bearing C3b or C3dg accumulate in the B-cell follicles because they are trapped by complement receptors CR1 and CR2 expressed on the surface of FDCs. Opsonized particulate antigens can also be taken up by specialized macrophages residing in the subcapsular sinus (SCS) of lymph nodes and the marginal sinus of the spleen, regions that are both adjacent to the B-cell


Immunology_Janeway

Immunology_Janeway. BAFF (B-cell activating factor, also called B-lymphocyte stimulator, or BLyS) and APRIL (a proliferation-inducing ligand) are both members of the TNF superfamily of cytokines. They are initially produced as membrane-bound trimers by several cell types. BAFF is produced by FDCs and other cells in the B-cell follicle, where it supports B-cell survival. Its main receptor, BAFF-R, signals in a manner similar to CD40 (see Fig. 7.31) through TRAF3 and NIK to activate both the non-canonical NFκB pathway, leading to the RelB:p52 transcription factor, and the canonical p50:p65 NFκB pathway. BAFF also binds to the receptors TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) and BCMA (B-cell maturation antigen), although its affinity for the latter is relatively weak. These receptors activate the canonical NFκB pathway. The low endocytic and Antigen preservation by


Immunology_Janeway

Immunology_Janeway. Opsonized antigens entering lymph degradative activity of subcapsular macrophages nodes from afferent lymphatics bind subcapsular macrophages also allows antigen to be to the complement receptors on the preserves the antigens transported into the follicle surface of macrophages present in trapped on their surfaces, to become localized on the the subcapsular sinus allowing B cells to surface of follicular follicles (Fig. 10.7). These macrophages seem to retain the antigen on their surface rather than ingesting and degrading it. These antigens can then be sampled and carried by antigen-specific follicular B cells. B cells of any antigen specificity could also acquire antigen from these macrophages via their complement receptors and transport it within the follicle. In the spleen, marginal zone B cells shuttle between that site and the follicle, carrying antigen trapped in the marginal zone for deposition on FDCs. SCS macrophages can also actively function to restrict the dissemination


Immunology_Janeway

Immunology_Janeway. zone B cells shuttle between that site and the follicle, carrying antigen trapped in the marginal zone for deposition on FDCs. SCS macrophages can also actively function to restrict the dissemination of infection. In mice, infection of these macrophages in lymph nodes by vesicular stomatitis virus (VSV), a relative of rabies virus, triggers the cells to produce interferon and to recruit plasmacytoid dendritic cells (pDCs). Type I interferon produced by pDCs restricts further viral spread, which would otherwise eventually pass on to the central nervous system.


Immunology_Janeway

Immunology_Janeway. After a naive follicular B cell first encounters specific antigen displayed by FDCs or macrophages, within a few hours it will become positioned in the outer follicles of lymphoid tissue close to the sites where antigen enters the lymph node or spleen. This positioning is orchestrated by the B cell’s expression of a chemokine receptor, EBI2 (GPR183), whose ligands are oxysterols such as 7α, 25-dihydroxycholesterol. The precise source of these ligands is still unclear, but they are abundant in the outer follicular and interfollicular regions. After sampling antigens there for 6 hours to 1 day, the B cell induces expression of CCR7, which functions together with EBI2 to distribute activated B cells along the interface between the B-cell follicle and the T-cell zone, where CCL21 is expressed.


Immunology_Janeway

Immunology_Janeway. During an immune response, T cells are activated within the T-cell zones by dendritic cells. When naive T cells are activated, some will proliferate, differentiate into effector cells, downregulate expression of S1P1, and exit the lymphoid tissue. However, others will induce expression of CXCR5 and migrate to the border with the B-cell follicle. There, T cells can encounter B cells activated during the same response, increasing the chance that they might recognize linked antigens presented by activated B cells that have recently moved to this location (see Fig. 10.5).


Immunology_Janeway

Immunology_Janeway. Fig. 10.7 Opsonized antigens are captured and preserved by subcapsular sinus macrophages. Macrophages residing in the lymph node subcapsular sinus (SCS) express complement receptors 1 and 2 (CR1 and CR2, respectively), are poorly endocytic, and have reduced levels of lysosomal enzymes compared with macrophages in the medulla. Opsonized antigen arriving from the afferent lymphatics binds to CR1 and CR2 on the surface of SCS macrophages. Instead of being completely degraded by these macrophages, some antigen is retained on the cell surface, where it can be presented and transferred to the surface of follicular B cells. B cells are then able to transport the antigen into the follicle, where it can be trapped on the surfaces of follicular dendritic cells.


Immunology_Janeway

Immunology_Janeway. The SLAM receptor family members SLAM, Ly108, and CD84 are expressed on T cells and B cells and mediate homotypic interactions that lead to adhesion between cells. SLAM can also enhance signaling by the T-cell receptor to augment production of cytokines such as IL-21 that help B cells. The SLAM-associated molecule SAP is a signaling adapter that is required for one SLAM receptor to sustain binding with another. T cells initially express SAP at low levels that are insufficient for sustained adhesion between T and B cells. Fully differentiated TFH cells express high levels of the transcription factor Bcl-6, which induces higher levels of SAP expression. This level is sufficient to sustain cell–cell interactions and allow for the delivery of CD40L and cytokine signals to B cells. 10-4 T cells express surface molecules and cytokines that activate B cells, which in turn promote TFH-cell development.


Immunology_Janeway

Immunology_Janeway. When TFH cells encounter an activating peptide presented by B cells, the TFH cells respond by expressing receptors and cytokines that in turn activate B cells. As mentioned above, the induced expression of CD40L on TFH cells activates CD40 on B cells to increase B-cell survival, and also induces B-cell expression of co-stimulatory molecules, especially those of the B7 family. Activated T cells also express CD30 ligand (CD30L), which binds to CD30 expressed on B cells and promotes B-cell activation. Mice lacking CD30 show reduced proliferation of activated B cells in lymphoid follicles and weaker secondary humoral responses than normal. TFH cells also secrete several cytokines that regulate B-cell proliferation and antibody production. Primary among these is IL-21, which is produced early in immune responses by TFH cells and which activates the transcription factor STAT3 in B cells to support proliferation and differentiation. IL-21 exerts similar autocrine effects on TFH cells. Later


Immunology_Janeway

Immunology_Janeway. in immune responses by TFH cells and which activates the transcription factor STAT3 in B cells to support proliferation and differentiation. IL-21 exerts similar autocrine effects on TFH cells. Later in the antibody response, TFH cells will produce other cytokines, such as IL-4 and IFN-γ, that are characteristic of the other T helper subsets (described in Chapter 9). These will impact B-cell differentiation, particularly class switching, as we discuss later.


Immunology_Janeway

Immunology_Janeway. The ability of TFH cells to successfully deliver these signals to B cells depends on intimate contact between these cells. Specific adhesion molecules, including several Ig superfamily receptors of the SLAM (signaling lymphocyte activation molecule) family, are involved that prolong and stabilize cell–cell contact. TFH cells and B cells both express SLAM (CD150), CD84, and Ly108, which promote cell adhesion through homotypic binding interactions (Fig. 10.8). The cytoplasmic regions of these SLAM family receptors all interact with an adaptor protein, SAP (SLAM-associated protein), which is expressed highly by TFH cells and which is necessary for prolonging cell–cell contact mediated by these receptors. The SAP gene is inactivated in X-linked lymphoproliferative syndrome, which is associated with a T-cell and NK-cell lymphoproliferative disorder and with a defect in antibody production due to failed interactions between TFH cells and B cells in the germinal center, discussed below. The


Immunology_Janeway

Immunology_Janeway. with a T-cell and NK-cell lymphoproliferative disorder and with a defect in antibody production due to failed interactions between TFH cells and B cells in the germinal center, discussed below. The regulated migration of activated B cells and TFH cells to the same location in the peripheral lymphoid organ increases the chance that linked recognition will occur and deliver appropriate help for B-cell differentiation. Antigen-stimulated B cells that fail to interact with T cells that recognize the same antigen die within 24 hours.


Immunology_Janeway

Immunology_Janeway. This first interaction between T and B cells not only provides important help to B cells, but also influences T-cell differentiation by signals provided by the B cell. Activated B cells express ICOSL, a member of the B7 family of co-stimulatory molecules and a ligand for ICOS (inducible co-stimulatory protein), which is expressed by T cells. This Tand B-cell interaction, provided by linked recognition, activates ICOS signaling in T cells and is important for the completion of TFH differentiation (see Section 7-21), leading to induction of the transcription factors Bcl-6 and c-Maf. These transcription factors are required for SAP production and the consequent sustained contact between B and TFH cells. 10-5 Activated B cells differentiate into antibody-secreting plasmablasts and plasma cells.


Immunology_Janeway

Immunology_Janeway. After their initial encounter, B cells that have received T-cell help migrate from the follicle border to continue to proliferate and differentiate. Two to three days after activation, B cells begin to decrease expression of CCR7 and to increase expression of EBI2 (see Fig. 10.5). Decreased expression of CCR7 causes B cells to move away from the boundary with the T-cell zone: EBI2 directs their migration back to the interfollicular regions and the subcapsular sinus in the lymph nodes, or, in the spleen, to the splenic bridging channels, a region between the T-cell area and the red pulp. Here, some B cells will form an emerging aggregate of differentiating B cells called the primary focus, which in lymph nodes is located in the medullary cords, where lymph drains out of the node, and in the spleen can be seen as extrafollicular foci in the splenic red pulp. Primary foci are apparent by about 5 days after an infection or immunization with an antigen not previously encountered.


Immunology_Janeway

Immunology_Janeway. B cells proliferate in the primary focus for several days, and this constitutes the first phase of the primary humoral immune response. Some of these proliferating B cells differentiate into antibody-synthesizing plasmablasts in the primary focus. Not all B cells activated by the initial interaction with TFH cells will move into the primary focus. Some will migrate into the lymphoid follicle, where they may eventually differentiate into plasma cells, as described below. Plasmablasts are cells that have begun to secrete antibody, yet are still dividing and express many of the characteristics of activated B cells that allow their interaction with T cells. After a few more days, the plasmablasts in the primary focus stop dividing and may eventually die. Subsequently, long-lived plasma cells will develop and migrate to the bone marrow, where they will continue antibody production. Since many long-lived plasma cells are generated long after the primary focus has dissipated, it is likely


Immunology_Janeway

Immunology_Janeway. will develop and migrate to the bone marrow, where they will continue antibody production. Since many long-lived plasma cells are generated long after the primary focus has dissipated, it is likely that they do not arise directly from plasmablasts in the primary focus, but rather from B cells that entered the germinal center reaction.


Immunology_Janeway

Immunology_Janeway. The properties of resting B cells, plasmablasts, and plasma cells are compared in Fig. 10.9. The differentiation of a B cell into a plasma cell is accompanied by many morphological changes that reflect a commitment to the production of large amounts of secreted antibody, which can constitute up to 20% of all the protein synthesized by a plasma cell. Plasmablasts and plasma cells have a prominent perinuclear Golgi apparatus and an extensive rough endoplasmic reticulum that is rich in immunoglobulin molecules that are being synthesized and exported into the lumen of the endoplasmic reticulum for secretion. Plasmablasts have relatively large numbers of B-cell receptors on the cell surface, whereas plasma cells have many fewer. This low level of surface immunoglobulin on plasma cells may still be physiologically important, since their survival seems to be determined in part by their ability to continue to bind antigen. Plasmablasts still express B7 co-stimulatory molecules and MHC class


Immunology_Janeway

Immunology_Janeway. be physiologically important, since their survival seems to be determined in part by their ability to continue to bind antigen. Plasmablasts still express B7 co-stimulatory molecules and MHC class II molecules; by contrast, plasma cells turn down the expression of MHC class II molecules. Nevertheless, T cells still provide important signals for plasma-cell differentiation and survival, such as IL-6 and CD40 ligand.


Immunology_Janeway

Immunology_Janeway. Fig. 10.9 Plasma cells secrete antibody at a high rate but can no longer respond to antigen. Resting naive B cells have membrane-bound immunoglobulin (usually IgM and IgD) and MHC class II molecules on their surface. Although their V genes do not carry somatic mutations, B cells can take up antigen and present it to helper T cells. The T cells in return induce the B cells to proliferate and to undergo isotype switching and somatic hypermutation, but B cells do not secrete significant amounts of antibody during this period. Plasmablasts have an intermediate phenotype. They secrete antibody but retain substantial surface immunoglobulin and MHC class II molecules and so can continue to take up and present antigen to T cells. Plasmablasts early in the immune response and those activated by T-independent antigens have usually not undergone somatic hypermutation and class switching, and therefore secrete IgM. Plasma cells are terminally differentiated cells that secrete antibodies. Plasma


Immunology_Janeway

Immunology_Janeway. antigens have usually not undergone somatic hypermutation and class switching, and therefore secrete IgM. Plasma cells are terminally differentiated cells that secrete antibodies. Plasma cells have very low levels of surface immunoglobulin but can express MHC class II molecules and may suppress TFH activity in a negative feedback pathway while differentiating. Early in the immune response they differentiate from unswitched activated B cells and secrete IgM; later in the response they derive from activated B cells that entered the germinal center reaction and underwent class switching and somatic hypermutation. Plasma cells have lost the ability to change the class of their antibody or undergo further somatic hypermutation.


Immunology_Janeway

Immunology_Janeway. Naive B cells travel to the lymph node via the bloodstream and leave via the efferent lymph Plasma cells migrate to the medullary cords or leave via the efferent lymphatics HEVfollicle germinal center primary focus B cells that encounter antigen in the follicle form a primary focus. Some proliferating B cells migrate into the follicle to form a germinal center Plasma cells migrate to the bone marrow bone marrow stromal cell Recent evidence indicates that even the low level of MHC class II molecules expressed on plasma cells functions to present cognate antigen to TFH cells, and acts to suppress IL-21 production and Bcl-6 expression, thus acting as a feedback pathway to regulate ongoing B-cell responses. While some plasma cells survive for only days to a few weeks after their final differentiation, others are very long lived and account for the persistence of antibody responses. 10-6 The second phase of a primary B-cell immune response proliferate to form germinal centers.


Immunology_Janeway

Immunology_Janeway. 10-6 The second phase of a primary B-cell immune response proliferate to form germinal centers. Not all B cells activated by TFH cells will migrate to the outer follicle and eventually establish a primary focus. Instead, some move into a primary lymphoid follicle together with their associated TFH cells (Fig. 10.10), where they continue to proliferate and ultimately form a germinal center; follicles with germinal centers are also called secondary lymphoid follicles. Downregulating EBI2 by B cells appears to favor germinal center formation. In mice lacking EBI2 expression in B cells, antigen-activated B cells remain near the border with the T-cell zone and are able to form germinal centers, but generate fewer plasmablasts.


Immunology_Janeway

Immunology_Janeway. 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 an area of active cell division that forms within a surrounding region of resting B cells in the primary follicle. Proliferating germinal center B cells displace the resting B cells toward the periphery of the follicle, forming a mantle zone of resting cells around the two distinguishable areas of activated B cells, called the light zone and the dark zone (Fig. 10.11, left panel). The germinal center grows in size as the immune response proceeds, and then shrinks and finally disappears when the infection is cleared. Germinal centers are present for about 3–4 weeks after initial antigen exposure.


Immunology_Janeway

Immunology_Janeway. The primary focus and the germinal center reaction differ in the quality of antibody that they produce. Plasmablasts, germinal center B cells, and early memory B cells begin to emerge during the first 4–5 days of an immune response. Plasmablasts in primary foci primarily secrete antibodies of the IgM isotype that offer some immediate protection. In contrast, B cells in the germinal center reaction undergo several processes that produce antibodies that are more effective in eliminating infections. These processes include somatic hypermutation, which alters the V regions of immunoglobulin genes (see below), and which enables a process called affinity maturation, which selects for the survival of mutated B cells that have a high affinity for the antigen.


Immunology_Janeway

Immunology_Janeway. Fig. 10.10 Activated B cells form germinal centers in lymphoid follicles. Activation of B cells in a lymph node is shown here. Top panel: naive circulating B cells enter lymph nodes from the blood via high endothelial venules (HEV) and are attracted by chemokines into the primary lymphoid follicle; if these B cells do not encounter antigen in the follicle, they leave via the efferent lymphatic vessel. Second panel: B cells that have bound antigen move to the border with the T-cell area, where they may encounter activated helper T cells specific for the same antigen; these T cells interact with the B cells and activate them to start proliferation and differentiation into plasmablasts. Some B cells activated at the T-cell–B-cell border migrate to form a primary focus of antibody-secreting plasmablasts in the interfollicular regions (spleen) or medullary cords (lymph nodes), whereas others move back into the follicle, where they continue to proliferate and form a germinal center.


Immunology_Janeway

Immunology_Janeway. plasmablasts in the interfollicular regions (spleen) or medullary cords (lymph nodes), whereas others move back into the follicle, where they continue to proliferate and form a germinal center. Germinal centers are sites of sustained B-cell proliferation and differentiation. Follicles in which germinal centers have formed are known as secondary follicles. Within the germinal center, B cells begin their differentiation into either antibody-secreting plasma cells or memory B cells. Third and fourth panels: plasma cells leave the germinal center and migrate to the medullary cords, or leave the lymph node altogether via the efferent lymphatics and migrate to the bone marrow.


Immunology_Janeway

Immunology_Janeway. Schematic representation of a follicle Cyclic reentry of cells into the dark zone with a germinal center is dependent on reexpression of CXCR4 on centrocytes Fig. 10.11The structure of a germinal center. The germinal center is a specialized microenvironment in which B-cell proliferation, somatic hypermutation, and selection for strength of antigen binding all occur. Closely packed centroblasts, which express CXCR4 and CXCR5, form the ‘dark zone’ of the germinal center; the less densely packed ‘light zone’ contains centrocytes, which express only CXCR5. Stromal cells in the dark zone produce CXCL12, which attracts the CXCR4-expressing centroblasts. Cyclic reentry describes the process by which B cells can lose and gain expression of CXCR4 and thus move from the light zone to the dark zone and back again.


Immunology_Janeway

Immunology_Janeway. In addition, class switching allows the selected B cells to produce antibodies with a variety of effector functions. These B cells will differentiate either into plasma cells that secrete higher-affinity and class-switched antibody in the latter part of the primary immune response, or into memory B cells as described in Chapter 11.


Immunology_Janeway

Immunology_Janeway. B cells in the germinal center divide rapidly, every 6–8 hours. Initially, these rapidly proliferating B cells, called centroblasts, express the chemokine receptors CXCR4 and CXCR5 but markedly reduce their expression of surface immunoglobulin, particularly of IgD. Centroblasts proliferate in the dark zone of the germinal center, named for its densely packed appearance (Fig. 10.12). Stromal cells in the dark zone produce CXCL12 (SDF-1), a ligand for CXCR4 that acts to retain centroblasts in this region. As time goes on, some centroblasts reduce their rate of cell division, enter the growth phase, pausing at the G2/M phase of the cell cycle, reduce CXCR4 expression, and begin to produce higher levels of surface immunoglobulin. These B cells are termed centrocytes. The loss of CXCR4 allows centrocytes to move into the light zone, a less densely packed area containing abundant FDCs that produce the chemokine CXCL13 (BLC), a ligand for CXCR5 (see Fig. 10.11, right panel). The B cells


Immunology_Janeway

Immunology_Janeway. centrocytes to move into the light zone, a less densely packed area containing abundant FDCs that produce the chemokine CXCL13 (BLC), a ligand for CXCR5 (see Fig. 10.11, right panel). The B cells proliferate in the light zone, but to a lesser extent than in the dark zone.


Immunology_Janeway

Immunology_Janeway. Fig. 10.12Germinal centers are sites of intense cell proliferation and cell death. The photomicrograph (first panel) shows a section through a human tonsillar germinal center. Closely packed centroblasts, seen in the lower part of this photomicrograph, form the so-called dark zone of the germinal center. Above this region is the less densely packed light zone. The second panel shows immunofluorescent staining of a germinal center. B cells are found in the dark zone, light zone, and mantle zone. Proliferating cells are stained green for Ki67, an antigen expressed in nuclei of dividing cells, revealing the rapidly proliferating centroblasts in the dark zone. The dense network of follicular dendritic cells, stained red, mainly occupies the light zone. Centrocytes in the light zone proliferate to a lesser degree than centroblasts. Small recirculating B cells occupy the mantle zone at the edge of the B-cell follicle. Large masses of CD4 T cells, stained blue, can be seen in the T-cell


Immunology_Janeway

Immunology_Janeway. to a lesser degree than centroblasts. Small recirculating B cells occupy the mantle zone at the edge of the B-cell follicle. Large masses of CD4 T cells, stained blue, can be seen in the T-cell zones, which separate the follicles. There are also significant numbers of T cells in the light zone of the germinal center; CD4 staining in the dark zone is associated mainly with CD4-positive phagocytes, that digest B cells that die there. Photographs courtesy of I. MacLennan.


Immunology_Janeway

Immunology_Janeway. Light micrograph of germinal center (high power) Germinal center stained to show T cells, follicular dendritic cells, and proliferating B cells MOVIE 10.1


Immunology_Janeway

Immunology_Janeway. MOVIE 10.1 Fig. 10.13 The primary antibody repertoire is diversified by three processes that modify the rearranged immunoglobulin gene. First panel: the primary antibody repertoire is initially composed of IgM-containing variable regions (red) produced by V(D)J recombination and constant regions (blue) from the μ gene segment. The range of reactivity of this primary repertoire can be further modified by somatic hypermutation, by class switch recombination at the immunoglobulin loci, and in some species by gene conversion (not shown). Second panel: somatic hypermutation results in mutations (shown as black lines) being introduced into the heavy-chain and light-chain V regions (red), altering the affinity of the antibody for its antigen. Third panel: in class switch recombination, the initial μ heavy-chain C regions (blue) are replaced by heavy-chain regions of another isotype (shown as yellow), modifying the effector activity of the antibody but not its antigen specificity.


Immunology_Janeway

Immunology_Janeway. hypermutation, and cells with mutations that improve affinity for antigen are selected. Somatic hypermutation introduces mutations that change anywhere from one to a few amino acids in the immunoglobulin, producing closely related B-cell clones that differ subtly in specificity and antigen affinity (Fig. 10.13). These mutations in the V genes are initiated by an enzyme called activation-induced cytidine deaminase, or AID, which is expressed only by germinal center B cells. Before describing the enzymatic mechanisms initiated by AID, we first present a general overview of this process in which random mutations can improve antibody affinity.


Immunology_Janeway

Immunology_Janeway. The immunoglobulin V-region genes accumulate mutations at a rate of about one base pair change per 103 base pairs per cell division, while the rate of mutations in the rest of the cell’s DNA is much lower: around one base pair change per 1010 base pairs per cell division. Somatic hypermutation also affects some DNA flanking the rearranged V gene, but does not generally extend into the C-region exons. Since each V region is encoded by about 360 base pairs and about three out of every four base changes will alter the amino acid encoded, there is about a 50% chance during each B-cell division that a mutation will occur to the receptor.


Immunology_Janeway

Immunology_Janeway. The point mutations accumulate in a stepwise manner as the descendants of each B cell proliferate in the germinal center to form B-cell clones (Fig. 10.14). An altered receptor 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. Most mutations have a negative impact on the ability of the B-cell receptor to bind the original antigen, by preventing the correct folding of the immunoglobulin molecule or by blocking the complementarity-determining regions from binding antigen. Detrimental mutations may alter conserved framework regions (see Fig. 4.7) and disrupt basic immunoglobulin structure. Cells that harbor such detrimental mutations are eliminated by apoptosis in a process of negative selection, either because they can no longer make a functional B-cell receptor or because they cannot take up antigen as well as sibling B cells (Fig. 10.15). Germinal centers are filled with apoptotic B cells that are quickly engulfed by


Immunology_Janeway

Immunology_Janeway. make a functional B-cell receptor or because they cannot take up antigen as well as sibling B cells (Fig. 10.15). Germinal centers are filled with apoptotic B cells that are quickly engulfed by macrophages, giving rise to the characteristic tingible body macrophages. These contain dark-staining nuclear debris in their cytoplasm. Negative selection is implied 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. This process prevents rapidly dividing B cells from expanding to numbers that would overwhelm the lymphoid tissues. Less frequently, mutations may improve the affinity of a B-cell receptor for antigen, and these mutations will be selectively expanded (see Fig. 10.15) because the cells expressing receptors with such mutations will have an increased survival rate compared with low-affinity cells. Positive selection is evident in


Immunology_Janeway

Immunology_Janeway. expanded (see Fig. 10.15) because the cells expressing receptors with such mutations will have an increased survival rate compared with low-affinity cells. Positive selection is evident in the accumulation of numerous amino acid replacements in the complementarity-determining regions, which determine antibody specificity


Immunology_Janeway

Immunology_Janeway. Fig. 10.14 Somatic hypermutation introduces mutations into the rearranged immunoglobulin variable (V) regions that improve antigen binding. The process of somatic hypermutation can be tracked by sequencing immunoglobulin V regions from hybridomas (clones of antibody-producing cells; see Appendix I, Section A-7) established at different time points after the experimental immunization of mice. The result of one experiment is depicted here. Each V region sequenced is represented by a horizontal line. The complementarity-determining regions CDR1, CDR2, and CDR3 are shown by pink shading. Mutations that change the amino acid sequence are represented by red bars. Within a few days of immunization, the V regions within a particular clone of responding B cells begin to acquire mutations, and over the course of the next week more mutations accumulate (top panels). B cells whose V regions have accumulated deleterious mutations and can no longer bind antigen die. B cells whose V regions have


Immunology_Janeway

Immunology_Janeway. the course of the next week more mutations accumulate (top panels). B cells whose V regions have accumulated deleterious mutations and can no longer bind antigen die. B cells whose V regions have acquired mutations that improve the affinity of the B-cell receptor for antigen are able to compete more effectively for antigen, and receive signals that drive their proliferation and expansion. The antibodies they produce also have this improved affinity. This process of mutation and selection can continue in the lymph node germinal center through multiple cycles in response to secondary and tertiary immune responses elicited by further immunization with the same antigen (center and bottom panels). In this way, the antigen-binding efficiency of the antibody response is improved over time.


Immunology_Janeway

Immunology_Janeway. and affinity (see Fig. 10.14), a process we discuss in the next section. The result of selection for enhanced binding to antigen is that the nucleotide changes that alter amino acid sequences, and thus protein structure, tend to be clustered in the CDRs of the immunoglobulin V-region genes, whereas silent, or neutral, mutations that preserve amino acid sequence and do not alter protein structure are scattered throughout the V region.


Immunology_Janeway

Immunology_Janeway. Fig. 10.15 Selection for high-affinity mutants in the germinal center relies on help provided by TFH cells. After activated B cells interact with TFH cells at the follicle border, they migrate to germinal centers (GCs), where the following events depicted here occur. In the dark zone of the GC, somatic hypermutation alters the immunoglobulin V regions (first panel). In some B cells (yellow), the mutated B-cell receptor (BCR) will have low or no affinity for the antigen, while in other B cells (orange) the mutated BCR affinity may be higher. After exiting the dark zone, the B cells with higher-affinity BCRs will capture antigen (red) trapped on follicular dendritic cells (FDCs) and then process and present it on MHC class II molecules (second panel). B cells with low-affinity BCRs will fail to capture and present antigen. B cells that present linked antigen epitopes to TFH cells will receive help through CD40L and IL-21, which promote survival and proliferation. B cells that lack


Immunology_Janeway

Immunology_Janeway. fail to capture and present antigen. B cells that present linked antigen epitopes to TFH cells will receive help through CD40L and IL-21, which promote survival and proliferation. B cells that lack antigen on MHC class II molecules receive no help and will eventually die (third panel). Some of the proliferating B cells undergo repeated cycles of entry to the dark zone, mutation, and selection (fourth panel), and other progeny B cells undergo differentiation to either memory B cells or plasma cells (not shown).


Immunology_Janeway

Immunology_Janeway. Fig. 10.16 Antigens are trapped in immune complexes that bind to the surface of follicular dendritic cells. Radiolabeled antigen localizes to, and persists in, lymphoid follicles of draining lymph nodes (see the light micrograph and the schematic representation (middle panel), showing a germinal center in a lymph node). The intense dark staining shows the localization in the germinal center of radiolabeled antigen that had been injected 3 days previously. The antigen is in the form of antigen:antibody:complement complexes bound to Fc receptors and to complement receptors CR1 or CR2 on the surface of the follicular dendritic cell (FDC), as depicted in the right-hand panel and inset. These complexes are not internalized, as such antigen can persist in this form for long periods. Photograph courtesy of J. Tew. 10-8 Positive selection of germinal center B cells involves contact with TFH cells and CD40 signaling.


Immunology_Janeway

Immunology_Janeway. Selection of B cells with improved affinity for antigen occurs in increments. It was originally discovered in vitro that resting 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 by TFH cells, respectively. The details of selection in the germinal center have become more clear recently from in vivo two-photon microscopic studies (see Appendix I, A-10) that show that positive selection of a B cell depends on the B cell’s ability to take up antigen, and to receive signals delivered by TFH cells. It is thought that somatic hypermutation occurs in the centroblasts in the dark zone; when a centroblast reduces its rate of proliferation and becomes a centrocyte, it increases the number of B-cell receptors on its surface and moves to the light zone, where there are abundant FDCs. Antigen can be trapped and stored for long periods in the form of immune complexes on FDCs


Immunology_Janeway

Immunology_Janeway. the number of B-cell receptors on its surface and moves to the light zone, where there are abundant FDCs. Antigen can be trapped and stored for long periods in the form of immune complexes on FDCs (Fig. 10.16 and Fig. 10.17). The centrocyte’s ability to bind antigen determines its relative ability to acquire


Immunology_Janeway

Immunology_Janeway. Fig. 10.17 Immune complexes bound to FDCs form iccosomes, which are released and can be taken up by B cells in the germinal center. FDCs have a prominent cell body and many dendritic processes. Immune complexes, bound to complement receptors and Fc receptors on the FDC surface, become clustered, forming prominent ‘beads’ along the dendrites (a). An intermediate form of FDC is shown, which has both straight filiform dendrites and others that are becoming beaded. These beads are shed from the cell as iccosomes (immune complex-coated bodies), which can bind to a B cell in the germinal center (b) and be taken up by it (c). In panels b and c, the iccosome has been formed with immune complexes containing horseradish peroxidase, which is electron-dense and therefore appears dark in the transmission electron micrographs. Photographs courtesy of A.K. Szakal.


Immunology_Janeway

Immunology_Janeway. antigen, in competition with the other clonally related centrocytes harboring different mutations. Centrocytes whose receptors bind antigen better will capture and present more peptides on their surface MHC class II molecules. TFH cells in the germinal center recognize these peptides and, as before, are activated to deliver signals to the B cell that promote survival. Centrocytes whose mutations reduce antigen-binding affinity will take up less antigen, and so will receive weaker survival signals from TFH cells. Successful B cells will reexpress CXCR4 and return to the dark zone, where they will undergo additional rounds of division, in effect becoming centroblasts again. Germinal center B cells that fail to acquire sufficient antigen from FDCs to engage TFH cells will become apoptotic and be lost. This process of B-cell migration within the germinal center is known as the cyclic reentry model (see Fig. 10.11, right panel). In this way, the affinity and specificity of B cells are


Immunology_Janeway

Immunology_Janeway. and be lost. This process of B-cell migration within the germinal center is known as the cyclic reentry model (see Fig. 10.11, right panel). In this way, the affinity and specificity of B cells are continually refined during the germinal center response, through affinity maturation (see Section 10-6). The selection process can be quite stringent: although 50–100 B cells may seed the germinal center, most of them 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.


Immunology_Janeway

Immunology_Janeway. In the germinal center, TFH cells and B cells interact to deliver signals that are important for both cells (see Section 10-4). Mice that lack ICOS are deficient in the germinal center reaction and have severely reduced class-switched antibody responses due to defective TFH-cell function. CD40 signaling in B cells is activated by CD40L on TFH cells and increases expression of the survival molecule Bcl-XL, a relative of Bcl-2. These interactions also include signaling by SLAM family receptors through the adapter protein SAP, as discussed above. Two-photon intravital microscopy has revealed that mice lacking the SLAM receptor CD84 have reduced numbers of conjugates between antigen-specific T cells and B cells in germinal centers, and these mice also have a reduced humoral response to antigen. 10-9 Activation-induced cytidine deaminase (AID) introduces mutations into genes transcribed in B cells.


Immunology_Janeway

Immunology_Janeway. 10-9 Activation-induced cytidine deaminase (AID) introduces mutations into genes transcribed in B cells. Now that we have discussed the cellular processes involved in somatic hypermutation and affinity maturation, we will delve into the details of the mutation process itself. The enzyme AID is important for both somatic hyper-mutation and class switch recombination, as mice lacking AID have defects in both processes. People with mutations in the AID gene that inactivate the enzyme—that is, have activation-induced cytidine deaminase deficiency, or AID deficiency—also lack both somatic hypermutation and class switching. This condition leads to the production of predominantly IgM antibodies and the absence of affinity maturation, a syndrome known as hyper IgM type 2 immunodeficiency (discussed in Chapter 13).


Immunology_Janeway

Immunology_Janeway. AID is related to enzymes that deaminate cytosine to uracil in making nucleotide precursors for RNA and DNA synthesis. Its closest homolog, APOBEC1 (apolipoprotein B mRNA editing catalytic polypeptide 1), is an RNA-editing enzyme that deaminates cytosine in the context of RNA. However, AID fulfills its activity in antibody gene diversification by acting on cytosine in the DNA of the immunoglobulin locus. When AID deaminates cytidine residues in the immunoglobulin V regions, somatic hypermutation is initiated; when cytidine residues in switch regions are deaminated, class switch recombination is initiated.


Immunology_Janeway

Immunology_Janeway. AID can deaminate cytidine residues in single-stranded DNA but not double-stranded DNA (Fig. 10.18). For AID to act, AID target genes are typically being transcribed, so that the DNA double helix is temporarily unwound. Since AID is expressed only in germinal center B cells, targeting of the immunoglobulin genes takes place only in these cells and in the actively transcribed rearranged The activity of AID, which is expressed only in B cells, requires access to the cytidine side chain of a single-stranded DNA molecule (first panel), which is normally prevented by the hydrogen bonding in double-stranded DNA. AID initiates a nucleophilic attack on the exposed cytosine ring (second panel), which is resolved by the deamination of the cytidine to form uridine (third panel). Fig. 10.19 AID initiates DNA lesions whose repair leads to somatic hypermutation, class switch recombination, or gene conversion.


Immunology_Janeway

Immunology_Janeway. When AID converts a cytidine (C) to uridine (U) in the DNA of an immunoglobulin gene, the final mutation produced depends on which repair pathways are used. Somatic hypermutation can result from either the mismatch repair (MSH2/6) pathway combined with error-prone polymerase activity of Polη, or the base-excision repair (UNG) pathway. Acting together, these can generate point mutations at and around the site of the original C:G pair. REV1 is a DNA repair enzyme that can synthesize DNA, or recruit other enzymes that can synthesize DNA, over the abasic sites in damaged DNA. REV1 itself will insert only C opposite the abasic site, but it can help recruit other polymerases that can also insert A, G, and T. The end result is insertion of a random nucleotide at the C:G residues where AID initially acted. Both class switch recombination and gene conversion require the formation of a single-strand break in the DNA. A single-strand break is formed when apurinic/apyrimidinic endonuclease 1


Immunology_Janeway

Immunology_Janeway. acted. Both class switch recombination and gene conversion require the formation of a single-strand break in the DNA. A single-strand break is formed when apurinic/apyrimidinic endonuclease 1 (APE1) removes a damaged residue from the DNA as part of the repair process (see Fig. 10.20, bottom two panels). In class switch recombination, single-strand breaks made in two of the so-called switch regions upstream of the C-region genes are converted to double-strand breaks. The cell’s machinery for repairing double-strand breaks, which is very similar to the later stages of V(D)J recombination, then rejoins the DNA ends in a way that leads to a recombination event in which a different C-region gene is brought adjacent to the rearranged V region. Gene conversion results from the broken DNA strand using homologous sequences flanking the immunoglobulin gene as a template for repair DNA synthesis, thus replacing part of the gene with new sequences.


Immunology_Janeway

Immunology_Janeway. V regions where RNA polymerase generates transient single-stranded regions. Somatic hypermutation does not occur in loci that are not being actively transcribed. Rearranged VH and VL genes are mutated even if they are ‘nonproductive’ rearrangements and are not being expressed as protein, as long as they are being transcribed. Some actively transcribed genes in B cells besides those for immunoglobulins can also be affected by the somatic mutation process, but at a much lower rate. 10-10 Mismatch and base-excision repair pathways contribute to somatic hypermutation following initiation by AID.


Immunology_Janeway

Immunology_Janeway. The uridine produced by AID represents a dual lesion in DNA; not only is uridine foreign to normal DNA, but it is now a mismatch with the guanosine nucleoside on the opposite DNA strand. The presence of uridine in DNA can trigger several types of DNA repair—including the mismatch repair and the base-excision repair pathways—which further alter the DNA sequence. The various repair processes lead to different mutational outcomes (Fig. 10.19). In the mismatch repair pathway, the presence of uridine is detected by the mismatch repair proteins MSH2 and MSH6 (MSH2/6). They recruit nucleases that remove the complete uridine nucleotide along with several adjacent nucleotides from the damaged DNA strand. This is followed by a fill-in ‘patch repair’ by a DNA polymerase; unlike the process in all other cells, in B cells this DNA synthesis is error-prone and tends to introduce mutations at nearby A:T base pairs.


Immunology_Janeway

Immunology_Janeway. The initial steps in the base-excision repair pathway are shown in Fig. 10.20. In this pathway, the enzyme uracil-DNA glycosylase (UNG) removes the uracil base from the uridine to create an abasic site in the DNA. If no further modification is made, this will result at the next round of DNA replication in the random insertion of a nucleotide opposite the abasic site by DNA polymerase, leading to mutation. The action of UNG may, however, be followed by the action of another enzyme, apurinic/apyrimidinic endonuclease 1 (APE1), which excises the abasic residue to create a single-strand discontinuity (known as a single-strand nick) in the DNA at the site of the original cytidine. Repair of the single-strand nick proceeding through double-strand breaks may result in gene conversion. Gene conversion is not used in the diversification of immunoglobulin genes in humans and mice, but is of importance in some other mammals and in birds.


Immunology_Janeway

Immunology_Janeway. Fig. 10.20 The base-excision repair pathway produces single-strand nicks in DNA by the sequential actions of AID, uracil-DNA glycosylase (UNG), and apurinic/ apyrimidinic endonuclease 1 (APE1). Double-stranded DNA (first panel) can be made accessible to AID by transcription that unwinds the DNA helix locally (second panel). AID, which is specifically expressed in activated B cells, converts cytidine residues to uridines (third panel). The ubiquitous base-excision repair enzyme UNG can then remove the uracil ring from uridine, creating an abasic site (fourth panel). The repair endonuclease APE1 then cuts the sugar–phosphate DNA backbone next to the abasic residue (fifth panel), thereby forming a single-strand nick in the DNA (sixth panel). APE1 does not excise ribose to form a single-strand nick in DNA, but rather cuts the DNA backbone to yield a 5ʹ-deoxyribosephosphate terminus that is then removed by, for example, DNA polymerase b.


Immunology_Janeway

Immunology_Janeway. Somatic hypermutation involves both mutation at the original cytidines targeted by AID and mutation at nearby non-cytidine nucleotides. If the original U:G mismatch is recognized by UNG, then an abasic site will be generated in the DNA (see Fig. 10.19). If no further modification is made to this site, it can be replicated without instructive base pairing from the template strand by a class of error-prone ‘translesion’ DNA polymerases that normally repair gross damage to DNA, such as that caused by ultraviolet (UV) radiation. These polymerases can incorporate any nucleotide into the new DNA strand opposite the abasic site, and after a further round of DNA replication this can result in a stable mutation at the site of the original C:G base pair.


Immunology_Janeway

Immunology_Janeway. In the mismatch repair pathway in B cells, but not in other cell types, the DNA lesion is repaired by error-prone DNA polymerases rather than by more accurate polymerases that faithfully copy the undamaged template strand. Individuals with a defect in the translesion polymerase Polη have relatively fewer mutations than usual at A:T, but not at C:G, in their hypermutated immunoglobulin V regions. This fact suggests that Polη is the repair polymerase involved in this pathway of somatic hypermutation. These individuals also have a form of xeroderma pigmentosum, a condition resulting from the inability of their cells to repair DNA damage caused by UV radiation. 10-11 AID initiates class switching to allow the same assembled VH exon to be associated with different CH genes in the course of an immune response.


Immunology_Janeway

Immunology_Janeway. VH exon to be associated with different CH genes in the course of an immune response. All the progeny of a particular B cell activated in an immune response will express the same VH gene that was generated during its development in the bone marrow, although the gene may be modified by somatic hypermutation. In contrast, that B cell’s progeny may express several different C-region isotypes as the cells mature and proliferate during the immune response. The first antigen receptors expressed by B cells are IgM and IgD, and the first antibody produced in an immune response is always IgM. Later in the immune response, the same assembled V region may be expressed in IgG, IgA, or IgE antibodies. This change is known as class switching (or isotype switching), and, unlike the expression of IgD, it involves irreversible DNA recombination. It is stimulated in the course of an immune response by external signals such as cytokines released by TFH cells.


Immunology_Janeway

Immunology_Janeway. Switching from IgM to the other immunoglobulin classes occurs only after MOVIE 5.2 B cells have been stimulated by antigen. It is achieved through class switch recombination, which is a type of nonhomologous DNA recombination that is guided by stretches of repetitive DNA known as switch regions. Switch regions lie in the intron between the JH gene segments and the Cμ gene, and at equivalent sites upstream of the genes for each of the other heavy-chain isotypes, with the exception of the δ gene, which does not require DNA rearrangement for its expression (Fig. 10.21, first panel). When a B cell switches from the coexpression of IgM and IgD to the expression of another subtype, DNA recombination occurs between Sμ and the S region immediately upstream of


Immunology_Janeway

Immunology_Janeway. Fig. 10.21 Class switching involves recombination between specific switch signals. The top panel shows the organization of a rearranged immunoglobulin heavy-chain locus before class switching. Second panel: this figure illustrates switching between the μ and ε isotypes in the mouse heavy-chain locus. Switch regions (S) are repetitive DNA sequences that guide class switching and are found upstream of each of the immunoglobulin C-region genes, with the exception of the δ gene. Switching is guided by the initiation of transcription by RNA polymerase (shaded circle) through these regions from promoters (shown as arrows) located upstream of each S. Due to the repetitive sequences, RNA polymerase can stall within the S regions, allowing these regions to serve as substrates for AID, and subsequently for UNG and APE1. Third panel: these enzymes introduce a high density of single-strand nicks into the non-template DNA strand and the template strand. Staggered nicks are converted to


Immunology_Janeway

Immunology_Janeway. subsequently for UNG and APE1. Third panel: these enzymes introduce a high density of single-strand nicks into the non-template DNA strand and the template strand. Staggered nicks are converted to double-strand breaks by a mechanism that is not yet understood. Fourth panel: these breaks are then recognized by the cell’s double-strand break repair machinery, which involves DNA-PKcs, Ku proteins, and other repair proteins. Bottom two panels: the two switch regions, in this case Sμ and Sε, are brought together by the repair proteins, and class switching is completed by excision of the intervening region of DNA (including Cμ and Cδ) and ligation of the Sμ and Sε regions.


Immunology_Janeway

Immunology_Janeway. the new constant-region gene. In such a recombination event, the Cμ coding regions and the entire intervening DNA between Cμ and the S region undergoing rearrangement are deleted. Figure 10.21 illustrates switching from Cμ to Cε in the mouse. All switch recombination events produce genes that can encode a functional protein, because the switch sequences lie in introns and therefore cannot cause frameshift mutations. The enzyme AID initiates class switch recombination, and acts only on regions of DNA being transcribed. Certain properties of the switch region sequences


Immunology_Janeway

Immunology_Janeway. Fig. 10.22 RNA processed from switch region introns interacts with AID and guides its activity. Top panel: promoters upstream of each switch region initiate transcription by RNA polymerase upstream of a rearranged VH gene, as in the case of Cμ, shown here, or a noncoding exon for all other constant regions. In all cases, the switch region itself lies within an intron upstream of the exons encoding the constant regions. This intronic switch region RNA is removed from the primary RNA transcript by splicing at specific splice acceptor and donor sites. Middle panel: after splicing, the switch region RNA is further processed and its repetitive elements allow the formation of putative G-quadruplex structures. Evidence indicates that these RNAs are able to bind AID, as implied in the cartoon. Bottom panel: the RNA acts as a guide to bring AID to the switch region by the ability of the G-quadruplex to hybridize with the original DNA template strand from which it was transcribed.


Immunology_Janeway

Immunology_Janeway. promote the accessibility to AID when they are being transcribed. Each switch region consists of many repeats of a G-rich sequence element on the non-template strand. For example, Sμ consists of about 150 repeats of the sequence (GAGCT)n(GGGGGT), where n is usually 3 but can be as many as 7. The sequences of the other switch regions (Sγ, Sα, and Sε) differ in exact sequence but all contain repeats of the GAGCT and GGGGGT sequences. It appears that movement of RNA polymerase through this highly repetitive region is occasionally halted—called polymerase stalling. This may be caused by bubble-like structures, called R-loops, that form when the transcribed RNA displaces the non-template strand of the DNA double helix (see Fig. 10.21, third panel) due to having many G residues in tandem on one strand.


Immunology_Janeway

Immunology_Janeway. Polymerase stalling seems closely connected with the recruitment of AID to specific switch regions being transcribed. A multisubunit RNA processing/ degradation complex, the RNA exosome, associates with AID and accumulates on transcribed switch regions, and the protein Spt5 associates with the stalled polymerase; both are necessary for AID to generate double-stranded breaks. Recent evidence indicates that AID is selectively guided to the transcribed switch by an additional mechanism. After an RNA polymerase has completed transcription of one RNA template, the intron RNA harboring the switch region is spliced out. This RNA is processed to generate an RNA structure, called a G-quadruplex, that is based on the G-rich repetitive element of the switch region (Fig. 10.22). This G-quadruplex serves a dual purpose, both binding to AID and also associating with the switch region from which it was transcribed, based on its sequence complementarity. Thus the G-quadruplex guides AID to the


Immunology_Janeway

Immunology_Janeway. serves a dual purpose, both binding to AID and also associating with the switch region from which it was transcribed, based on its sequence complementarity. Thus the G-quadruplex guides AID to the appropriate switch region, where particular palindromic sequences, such as AGCT, act as good substrates to allow its cytidine deaminase activity to act on both strands concurrently. In this way, the G-quadruplex functions in a manner similar to the synthetic guide RNAs that deliver the Cas9 endonuclease to specific genomic regions, as described in Appendix I, Section A-35).


Immunology_Janeway

Immunology_Janeway. Following the generation of double-stranded breaks in switch regions, general cellular mechanisms for repairing these breaks lead to the nonhomologous recombination between switch regions that results in class switching (see Fig. 10.21, fourth and fifth panels). The ends to be joined are brought together by the alignment of repetitive sequences common to the different switch regions, and rejoining of the DNA ends then leads to excision of all DNA between the two switch regions and the formation of a chimeric region at the junction. Loss of AID completely blocks class switching, but deficiency of UNG in both mice and humans severely impairs class switching, suggesting sequential actions of AID and UNG in generating DNA breaks. Joining of DNA ends is probably mediated by classic nonhomologous end joining (as in V(D) J recombination) as well as by a poorly understood alternative end-joining pathway. Class switching is sometimes impaired in the disease ataxia telangiectasia, which is


Immunology_Janeway

Immunology_Janeway. end joining (as in V(D) J recombination) as well as by a poorly understood alternative end-joining pathway. Class switching is sometimes impaired in the disease ataxia telangiectasia, which is caused by mutations in the DNA-PKcs-family kinase ATM, a known DNA repair protein. The role of ATM in class switching is not yet entirely clear, however.


Immunology_Janeway

Immunology_Janeway. 10-12 Cytokines made by TFH cells direct the choice of isotype for class switching in T-dependent antibody responses. Now that we understand the general mechanisms that control DNA rearrangements of class switching, we are ready to explain how a particular heavy-chain is selected during an immune response. It is the choice of antibody isotype that ultimately determines the effector function of antibodies, and we will see that this choice is largely controlled by the cytokines that are produced by TFH cells in the germinal center reaction.


Immunology_Janeway

Immunology_Janeway. As discussed above, interactions between germinal center B cells and TFH cells are essential for class switching to occur. The required interactions occur through the interplay of CD40 on B cells with CD40 ligand on activated helper T cells. Genetic deficiency of CD40 ligand greatly reduces class switching and causes abnormally high levels of plasma IgM, a condition known as hyper IgM syndrome. People with this defect lack antibodies of classes other than IgM and exhibit severe humoral immunodeficiency, manifested as repeated infections with common bacterial pathogens. Much of the IgM in hyper IgM syndromes may be induced by thymus-independent antigens on the pathogens that chronically infect these patients. Nevertheless, people with CD40 ligand deficiency can make IgM antibodies in response to thymus-dependent antigens, which indicates that in the B-cell response, CD40L–CD40 interac tions are most important in enabling a sustained response that includes class switching and affinity


Immunology_Janeway

Immunology_Janeway. to thymus-dependent antigens, which indicates that in the B-cell response, CD40L–CD40 interac tions are most important in enabling a sustained response that includes class switching and affinity maturation, rather than in the initial activation of B cells.


Immunology_Janeway

Immunology_Janeway. The selection of the particular C region for class switch recombination is not random but is regulated by the cytokines produced by TFH cells and other cells during the immune response. Different cytokines preferentially induce switching to different isotypes (Fig. 10.23). Cytokines induce class switching in part by inducing the production of RNA transcripts through the switch regions that lie 5ʹ to each heavy-chain C gene segment. When activated B cells are exposed to IL-4, for example, transcription from promoters that lie upstream of the switch regions of Cγ1 and Cε can be detected a day or two before switching occurs. This will make it possible for switch to occur to either of these two heavy-chain C genes, but in any particular germinal center B cell, recombination will occur in only one. In the example of class switching shown in Fig. 10.21, transcription through the Sε regions caused the rearrangement between the Sμ and Sε regions, making the IgE isotype antibody. This results


Immunology_Janeway

Immunology_Janeway. one. In the example of class switching shown in Fig. 10.21, transcription through the Sε regions caused the rearrangement between the Sμ and Sε regions, making the IgE isotype antibody. This results because IL-4 signaling activates the transcription factor STAT6, which initiates transcription of the Iε promoter upstream of the Sε region. Other cytokines activate other promoters upstream of other switch regions to produce other antibody classes. TFH cells also produce IL-21, which promotes switching to IgG1 and IgG3. Transforming growth factor (TGF)-β induces switching to IgG2b (Cγ2b) and IgA (Cα). IL-5 promotes switching to IgA, and interferon (IFN)-γ induces switching to IgG2a and IgG3.


Immunology_Janeway

Immunology_Janeway. The individual cytokines induce (violet) or inhibit (red) the production of certain antibody classes. Much of the inhibitory effect is probably the result of directed switching to a different class. The actions of IL-21 on class switching are regulated by IL-4. These data are drawn from experiments with mouse cells. Fig. 10.23 Different cytokines induce switching to different antibody classes. InducesInhibitsInhibits Induces Induces InducesInduces Induces Induces Induces Induces Inhibits Inhibits Inhibits Inhibits Inhibits Inhibits IgEIgG2aIgG3IgM IgG1 IgG2b IgACytokines IL-4 IL-5 IFN-˜TGF-°Role of cytokines in regulating expression of antibody classes Augments production IL-21 10-13 B cells that survive the germinal center reaction eventually differentiate into either plasma cells or memory cells.


Immunology_Janeway

Immunology_Janeway. When B cells have undergone affinity maturation and class switching, some eventually exit from the light zone and start to differentiate into plasma cells that produce large amounts of antibody. In B cells, the transcription factors Pax5 and Bcl-6 inhibit the expression of transcription factors required for plasma-cell differentiation, and both Pax5 and Bcl-6 are downregulated when the B cell starts differentiating. The transcription factor IRF4 then induces the expression of BLIMP-1, a transcriptional repressor that switches off genes required for B-cell proliferation, class switching, and affinity maturation. B cells in which BLIMP-1 is induced become plasma cells; they cease proliferating, increase the synthesis and secretion of immunoglobulins, and change their cell-surface properties. Plasma cells downregulate CXCR5 and upregulate CXCR4 and α4:β1 integrins so that they can leave the germinal centers and home to peripheral tissues.


Immunology_Janeway

Immunology_Janeway. Some plasma cells deriving from germinal centers in lymph nodes or spleen migrate to the bone marrow, where a subset live for a long period, whereas others migrate to the medullary cords in lymph nodes or splenic red pulp. B cells that have been activated in germinal centers in mucosal tissues, and which are predominantly switched to IgA production, stay within the mucosal system. A splice variant of XBP1 (X-box binding protein 1) is expressed in plasma cells and helps to regulate their secretory capacity. Plasma cells in bone marrow receive signals from stromal cells that are essential for their survival, and they can be very long lived, whereas plasma cells in the medullary cords or red pulp are not long lived. XBP1 is also required for plasma cells to colonize bone marrow successfully. Plasma cells in the bone marrow are the source of long-lasting high-affinity class-switched antibody.


Immunology_Janeway

Immunology_Janeway. Other germinal center B cells differentiate into memory B cells. Memory B cells are long-lived descendants of cells that were once stimulated by antigen and had proliferated in the germinal center. They divide very slowly if at all; they express surface immunoglobulin but secrete no antibody, or do so only at a low rate. Because the precursors of some memory B cells arise from the germinal center reaction, memory B cells can inherit the genetic changes that occur there, including somatic hypermutation and the gene rearrangements that result in a class switch. The signals that control which path of differentiation a B cell takes are still being investigated. We will briefly return to memory B cells in Chapter 11. 10-14 Some antigens do not require T-cell help to induce B-cell responses.


Immunology_Janeway

Immunology_Janeway. 10-14 Some antigens do not require T-cell help to induce B-cell responses. Humans and mice with T-cell deficiencies are able to produce antibodies against thymus-independent (TI) antigens, which we introduced in Section 10-1. These antigens include certain bacterial polysaccharides, polymeric proteins, and lipopolysaccharides, which are able to stimulate naive B cells in the absence of T-cell help. These nonprotein bacterial products cannot elicit classical T-cell responses, yet they induce antibody responses in normal individuals. In addition, there are TI antigens that are not derived from bacteria; these include plant-derived mitogens and lectins, viral antigens, and superantigens, and some parasite-derived antigens.


Immunology_Janeway

Immunology_Janeway. Thymus-independent antigens fall into two classes, TI-1 and TI-2, which activate B cells by two different mechanisms. TI-1 antigens rely on activity that can directly induce B-cell division without T-cell help. We now understand that TI-1 antigens contain molecules that cause the proliferation and differentiation of most B cells regardless of their antigen specificity; this is known as polyclonal activation (Fig. 10.24, top panels). TI-1 antigens are therefore


Immunology_Janeway

Immunology_Janeway. Fig. 10.24 TI-1 antigens induce polyclonal B-cell responses at high concentrations, and antigen-specific antibody responses at low concentrations. At high antigen concentration, the signal delivered by the B-cell-activating moiety of TI-1 antigens is sufficient to induce B-cell proliferation and antibody secretion in the absence of specific antigen binding to surface immunoglobulin. Thus, all B cells respond (top panels). At low concentration, only B cells specific for the TI-1 antigen bind enough of it to focus its B-cell activating properties onto the B cell; this gives a specific antibody response to epitopes on the TI-1 antigen (lower panels).


Immunology_Janeway

Immunology_Janeway. often called B-cell mitogens, a mitogen being a substance that induces cells to undergo mitosis. For example, LPS and bacterial DNA are both TI-1 antigens because they activate TLRs expressed by B cells (see Section 3-5) and can act as a mitogen. Naive murine B cells express most TLRs constitutively, but naive human B cells do not express high levels of most TLRs until they receive stimulation through the B-cell receptor. So by the time a B cell has been stimulated by antigen through its B-cell receptor, it is likely to express several TLRs and be responsive to stimulation by TLR ligands that accompany the antigens. 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 specifically bind the TI-1 antigen become activated. At these low concentrations, amounts of TI-1 antigen sufficient for B-cell activation can only be concentrated on the B-cell surface with the


Immunology_Janeway

Immunology_Janeway. specifically bind the TI-1 antigen become activated. At these low concentrations, amounts of TI-1 antigen sufficient for B-cell activation can only be concentrated on the B-cell surface with the aid of this specific binding (see Fig. 10.24, bottom panels). B-cell responses to TI-1 antigens in the early stages of an infection may be important in defense against several extracellular pathogens, but they do not lead to affinity maturation or memory B cells, both of which require antigen-specific T-cell help.


Immunology_Janeway

Immunology_Janeway. The second class of thymus-independent antigens—TI-2 antigens—consists of molecules that have highly repetitive structures, such as bacterial capsular polysaccharides. These 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 Section 8-6, are inactivated by encounter with repetitive epitopes. Infants and young children up to about 5 years of age do not make fully effective antibody responses against polysaccharide antigens, and this might be because most of their B cells are immature.


Immunology_Janeway

Immunology_Janeway. Responses to several TI-2 antigens are prominently made by marginal zone B cells, a subset of nonrecirculating B cells that line the border of the splenic white pulp, and by B-1 cells (see Section 8-9). Marginal zone B cells are rare at birth and accumulate with age; they might therefore be responsible for most physiological TI-2 responses, which increase in efficiency with age. TI-2 antigens probably act by simultaneously cross-linking a critical number of B-cell receptors on the surface of antigen-specific mature B cells (Fig. 10.25, left


Immunology_Janeway

Immunology_Janeway. Fig. 10.25 B-cell activation by TI-2 antigens requires, or is greatly enhanced by, cytokines. Multiple cross-linking of the B-cell receptor by TI-2 antigens can lead to IgM antibody production (left panels), but there is evidence that in addition cytokines greatly augment these responses, and also lead to isotype switching (right panels). It is not clear where such cytokines originate, but one possibility is that dendritic cells, which may be able to bind the antigen through innate immune-system receptors on their surface and so present it to the B cells, secrete a soluble TNF-family cytokine called BAFF, which can activate class switching by the B cell.


Immunology_Janeway

Immunology_Janeway. lgGlgMActivated dendritic cells release a cytokine, BAFF, that augments production of antibody against TI-2 antigens and induces class switching TI-2 antigens alone can signal B cells to produce IgM antibody BBBAFFIgMIgM12 panels). Dendritic cells and macrophages can provide co-stimulatory signals for activation of B cells by TI-2 antigens. One of these co-stimulatory signals is BAFF, which can be secreted by dendritic cells and interacts with the receptor TACI on the B cell (see Fig. 10.25, right panels). The density of TI-2 antigen epitopes is critical; excessive cross-linking of B-cell receptors renders mature B cells unresponsive or anergic, as in immature B cells, while too low a density may be insufficient for activation.


Immunology_Janeway

Immunology_Janeway. An important class of TI-2 antigens arises during infection by capsulated bacteria. 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 against bacterial peptides presented by macrophages. IgM antibodies rapidly produced against the capsular polysaccharide independent of peptide-specific T-cell help will coat the bacteria, promoting their ingestion and destruction by phagocytes early in the infection.


Immunology_Janeway

Immunology_Janeway. Not all antibodies against bacterial polysaccharides are produced strictly through this TI-2 mechanism. We mentioned earlier the importance of antibodies against the capsular polysaccharide of Haemophilus influenzae type b in protective immunity to this bacterium. The immunodeficiency disease Wiskott–Aldrich syndrome is caused by defects in T cells that impair their interaction with B cells (see Chapter 13). Patients with Wiskott–Aldrich syndrome respond poorly to protein antigens, but, unexpectedly, also fail to make IgM and IgG antibody against polysaccharide antigens and are highly susceptible to infection with encapsulated bacteria such as H. influenzae. The failure to make IgM seems to be due in part to greatly reduced development of the marginal zone of the spleen, which contains B cells responsible for making much of the ‘natural’ IgM antibody against ubiquitous carbohydrate antigens. Thus, IgM and IgG antibodies induced by TI-2 antigens are likely to be an important part of


Immunology_Janeway

Immunology_Janeway. B cells responsible for making much of the ‘natural’ IgM antibody against ubiquitous carbohydrate antigens. Thus, IgM and IgG antibodies induced by TI-2 antigens are likely to be an important part of the humoral immune response in many bacterial infections, and in humans at least, the production of class-switched antibodies to TI-2 antigens might normally rely on some degree of T-cell help.


Immunology_Janeway

Immunology_Janeway. As well as producing IgM, TI responses can include switching to certain other antibody classes, such as IgG3 in the mouse. This is probably the result of help from dendritic cells (see Fig. 10.25, right panels), which provide secreted cytokines such as BAFF and membrane-bound signals to proliferating plasmablasts as they respond to TI antigens. The distinguishing features of thymus-dependent, TI-1, and TI-2 antibody responses are summarized in Fig. 10.26. Summary.


Immunology_Janeway

Immunology_Janeway. Summary. B-cell activation by many antigens 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 cells and displayed by the B cells as peptide:MHC class II complexes. Follicular helper T cells stimulate B cells by conjugation in germinal centers, with binding of CD40 ligand on the T cell to CD40 on the B cell, and by their release of cytokines, such as IL-21. Activated B cells also express molecules, such as ICOSL, that can stimulate T cells. The initial interaction between B and T cells occurs at the border of the T-cell and B-cell areas of secondary lymphoid tissue, to which antigen-activated helper T cells and B cells migrate in response to chemokines. Further interactions between T cells and B cells continue after migration into the follicle and the formation of a germinal center.


Immunology_Janeway

Immunology_Janeway. Fig. 10.26 Properties of different classes of antigen that elicit antibody responses. Some data indicate a minor role for T cells in antibody responses to TI-2 antigens; robust responses to TI-2 antigens can be observed in T-cell-deficient mice.


Immunology_Janeway

Immunology_Janeway. T cells induce a phase of vigorous B-cell proliferation in the germinal center reaction and direct the differentiation of clonally expanded B cells into either antibody-secreting plasma cells or memory B cells. Immunoglobulin genes expressed in B cells are diversified in the germinal center reaction by somatic hypermutation and class switching, initiated by activation-induced cytidine deaminase (AID). Unlike V(D)J recombination, these processes occur only in B cells. Somatic hypermutation diversifies the V region through the introduction of point mutations that are selected for providing greater affinity for the antigen as the immune response proceeds. Class switching does not affect the V region but increases the functional diversity of immunoglobulins by replacing the Cμ region in the immunoglobulin gene, which is first expressed with another heavy-chain C region to produce IgG, IgA, or IgE antibodies. Class switching provides antibodies with the same antigen specificity but


Immunology_Janeway

Immunology_Janeway. the immunoglobulin gene, which is first expressed with another heavy-chain C region to produce IgG, IgA, or IgE antibodies. Class switching provides antibodies with the same antigen specificity but distinct effector capacities. The switching to different antibody isotypes is regulated by cytokines released from helper T cells. Some nonprotein antigens stimulate B cells in the absence of linked recognition by peptide-specific helper T cells. Responses to these thymus-independent antigens are accompanied by only limited class switching and do not induce memory B cells. However, such responses have a crucial role in host defense against pathogens whose surface antigens cannot elicit peptide-specific T-cell responses.


Immunology_Janeway

Immunology_Janeway. The distributions and functions of immunoglobulin classes.


Immunology_Janeway

Immunology_Janeway. Extracellular pathogens can invade most sites within the body, and so antibodies must be equally widely distributed to combat them. Most classes of antibodies are distributed by diffusion from their site of synthesis, but specialized transport mechanisms are required to deliver antibodies across the epithelial surfaces lining the mucosa of organs such as the lungs and intestine. The particular heavy-chain isotype of the antibody can either limit antibody diffusion or engage specific transporters that deliver the antibody across epithelia. This part of the chapter describes these mechanisms and the antibody classes that use them to enter compartments of the body where their particular effector functions are appropriate. Here we restrict our discussion to the protective functions of antibodies that result solely from their binding to pathogens, and in the next part of the chapter, we discuss the effector cells and molecules that are specifically engaged by different antibody classes.


Immunology_Janeway

Immunology_Janeway. 10-15 Antibodies of different classes 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. Less often, insects, wounds, or hypodermic needles introduce microorganisms directly into the blood. Antibodies protect all the body’s mucosal surfaces, tissues, and blood from such infections; these antibodies serve to neutralize the pathogen or promote its elimination before it can establish a significant infection.


Immunology_Janeway

Immunology_Janeway. The different classes of antibodies (see Fig. 5.19) are adapted to function in different compartments of the body. Their functional activities and distributions are listed in Fig. 10.27. Because a given V region can become associated with any C region through class switching, the progeny of a single B cell can produce antibodies that share the same specificity yet provide all of the protective functions appropriate for each body compartment. All naive B cells express cell-surface IgM and IgD. IgM is the first antibody secreted by activated B cells but is less than 10% of the immunoglobulin found in plasma. Little IgD antibody is produced at any time, while IgE contributes a small but biologically important part of the immune response. IgG and IgA are the predominant antibody classes. IgE contributes a small but biologically important part of the response. The overall predominance of IgG is also due in part to its longer lifetime in the plasma (see Fig. 5.20).


Immunology_Janeway

Immunology_Janeway. The major effector functions of each class (+++) are shaded in dark red, whereas lesser functions (++) are shown in dark pink, and very minor functions (+) in pale pink. The distributions are marked similarly, with actual average levels in serum being shown in the bottom row. IgA has two subclasses, IgA1 and IgA2. The IgA column refers to both. *IgG2 can act as an opsonin in the presence of an Fc receptor of the appropriate allotype, found in about 50% of people of Caucasian descent.


Immunology_Janeway

Immunology_Janeway. IgM antibodies are produced first in a humoral immune response and tend to be of low affinity. However, IgM molecules form pentamers that are stabilized by a single J-chain molecule (see Fig. 5.23) and have 10 antigen-binding sites, conferring higher overall avidity when binding to multivalent antigens such as bacterial capsular polysaccharides. This higher avidity of the pentamer compensates for the low affinity of the individual antigen-binding site within the IgM monomers. Because of the large size of the pentamers, IgM is found mainly in the bloodstream and, to a lesser extent, in the lymph, rather than in intercellular spaces within tissues. 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. IgM hexamers can also form, and these fix complement much more efficiently than pentamers, possibly because C1q is also a hexamer. However, the in vivo role of IgM hexamers in protecting against


Immunology_Janeway

Immunology_Janeway. IgM hexamers can also form, and these fix complement much more efficiently than pentamers, possibly because C1q is also a hexamer. However, the in vivo role of IgM hexamers in protecting against infections has not been fully established.


Immunology_Janeway

Immunology_Janeway. 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 produced by conventional B cells that have not undergone class switching, but most is produced by B-1 cells residing in the peritoneal cavity and pleural spaces and by marginal zone B cells of the spleen. These cells secrete antibodies against commonly encountered carbohydrate antigens, including those of bacteria, and do not require T-cell help; they therefore provide a preformed repertoire of IgM antibodies in blood and body cavities that can recognize invading pathogens (see Section 8-9).


Immunology_Janeway

Immunology_Janeway. Antibodies of the other classes—IgG, IgA, and IgE—are smaller, and diffuse easily out of the blood into the tissues. IgA can form dimers (see Fig. 5.23), but 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 classes have been selected in the germinal centers for their increased affinity for antigen after somatic hypermutation. IgG4 is the least abundant of the IgG subclasses, but has the unusual ability to form hybrid antibodies. One IgG4 heavy chain and attached light chain can split from the original heavy-chain dimer and reassociate with a different IgG4 heavy chain–light chain pair, forming a bivalent IgG4 antibody with two distinct antigen specificities.


Immunology_Janeway

Immunology_Janeway. IgG is the principal class of antibody in blood and extracellular fluid, whereas IgA is the principal class in secretions, the most important being those from the epithelia lining the intestinal and respiratory tracts. IgG efficiently opsonizes pathogens for engulfment by phagocytes and activates the complement system, but IgA is a less potent opsonin and a weak activator of complement. IgG operates mainly in the tissues, where accessory cells and molecules are available, whereas dimeric IgA operates mainly on epithelial surfaces, where complement and phagocytes are not normally present; therefore IgA functions chiefly as a neutralizing antibody. Monomeric IgA can be produced by plasma cells that differentiate from class-switched B cells in lymph nodes and spleen, and it acts as a neutralizing antibody in extracellular spaces and in the blood. This monomeric IgA is predominantly of the subclass IgA1; the ratio of IgA1 to IgA2 in the blood is 10:1. The IgA antibodies produced by plasma


Immunology_Janeway

Immunology_Janeway. antibody in extracellular spaces and in the blood. This monomeric IgA is predominantly of the subclass IgA1; the ratio of IgA1 to IgA2 in the blood is 10:1. The IgA antibodies produced by plasma cells in the gut are dimeric and predominantly of subclass IgA2; the ratio of IgA2 to IgA1 in the gut is 3:2.


Immunology_Janeway

Immunology_Janeway. 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 cell-associated IgE triggers mast cells to release powerful chemical mediators that induce reactions such as coughing, sneezing, and vomiting, which in turn can expel infectious agents, as discussed later in this chapter. 10-16 Polymeric immunoglobulin receptor binds to the Fc regions of IgA and IgM and transports them across epithelial barriers.


Immunology_Janeway

Immunology_Janeway. In the mucosal immune system, IgA-secreting plasma cells are found predominantly in 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 of the bronchi (Fig. 10.28). IgA antibody synthesized in the lamina propria is secreted as a dimeric IgA molecule associated with a single J chain. This polymeric form of IgA binds specifically to a receptor called the polymeric immunoglobulin receptor (pIgR), which is present on the basolateral surfaces of the overlying epithelial cells. When the pIgR has bound a molecule of dimeric IgA, the complex is internalized and carried in a transport vesicle through the cytoplasm of the epithelial cell to its luminal surface. This process is called transcytosis. IgM also binds to the pIgR and can be secreted into the gut by the same mechanism. Upon reaching the luminal surface of


Immunology_Janeway

Immunology_Janeway. epithelial cell to its luminal surface. This process is called transcytosis. IgM also binds to the pIgR and can be secreted into the gut by the same mechanism. Upon reaching the luminal surface of the enterocyte, the antibody is released into the mucous layer covering the gut lining by proteolytic cleavage of the extracellular domain of the pIgR. The cleaved extracellular domain of the pIgR is known as secretory component (frequently abbreviated to SC) and remains associated with the antibody. Secretory component is bound to the part of the Fc region of IgA that contains the binding site for the Fcα receptor I, which is why secretory IgA does not bind to this receptor. Secretory component serves several physiological roles. It binds to mucins in mucus, acting as ‘glue’ to bind secreted IgA to the mucous layer on the luminal surface of the gut epithelium, where the antibody binds and neutralizes gut pathogens and their toxins (see Fig. 10.28). Secretory component also protects the


Immunology_Janeway

Immunology_Janeway. IgA to the mucous layer on the luminal surface of the gut epithelium, where the antibody binds and neutralizes gut pathogens and their toxins (see Fig. 10.28). Secretory component also protects the antibodies against cleavage by gut enzymes.


Immunology_Janeway

Immunology_Janeway. 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 inside tissues. By binding bacteria, virus particles, and toxins, IgA antibodies prevent the attachment of bacteria and viruses to epithelial cells and the uptake of toxins, and provide the first line of defense against a wide variety of pathogens. IgA is also thought to have an additional role in the gut, that of regulating the gut microbiota (see Chapter 12). The alveolar spaces in the lower respiratory tract lack the thicker mucosal layer characteristic of the upper respiratory tract, because efficient gas diffusion would be impeded by a mucous layer covering the alveolar epithelium. IgG can rapidly transudate into these


Immunology_Janeway

Immunology_Janeway. mucosal layer characteristic of the upper respiratory tract, because efficient gas diffusion would be impeded by a mucous layer covering the alveolar epithelium. IgG can rapidly transudate into these spaces and is the major isotype responsible for protection there.