I chapter 11 B-Cell generation Activation and Differentiation HE DEVELOPMENTAL PROCESS THAT RESULTS IN production of plasma cells and memory B cells can be divided into three broad stages: generation of mature, immunocompetent B cells(maturation), activa- Initial Contact between b and T cells tion of mature B cells when they interact with antigen, and differentiation of activated B cells into plasma cells and memory B cells. In many vertebrates, ng humans B-Cell maturation and mice, the bone marrow generates B cells. This process is an orderly sequence of Ig-gene rearrangements, which a B-Cell Activation and Proliferation progresses in the absence of antigen. This is the antigen The Humoral Response independent phase of B-cell development. A mature b cell leaves the bone marrow expres In Vivo Sites for Induction of Humoral Responses brane-bound immunoglobulin(mIgM and mlgD) with a m Germinal Centers and Antigen-Induced B-Cell ingle antigenic specificity. These naive B cells, which have Differentiation not encountered antigen, circulate in the blood and lymph and are carried to the secondary lymphoid organs, most no- m Regulation of B-Cell Development tably the spleen and lymph nodes(see Chapter 2). If a B cell Regulation of the Immune Effector Response is activated by the antigen specific to its membrane-bound antibody, the cell proliferates(clonal expansion)and differen- tiates to generate a population of antibody-secreting plasma cells and memory B cells. In this activation stage, affinity maturation is the progressive increase in the average affinity Some aspects of B-cell developmental processes have of the antibodies produced and class switching is the change been described in previous chapters. The overall pathway, in the isotype of the antibody produced by the b cell from u beginning with the earliest distinctive B-lineage cell, is de to y, a, or E. Since B cell activation and differentiation in the scribed in sequence in this chapter Figure 11-1 presents an periphery require antigen, this stage comprises the antigen- overview of the major events in humans and mice. Most of dependent phase of B-cell development. his chapter applies to humans and mice, but important Many B cells are produced in the bone marrow through- departures from these developmental pathways have been out life but very few of these cells mature. In mice, the size of shown to occur in some other vertebrates. Finally nis cha the recirculating pool of B cells is about 2 X 10 cells. Most ter will consider the regulation of B-cell development at var- of these cells circulate as naive b cells, which have short life ious stages. spans(half lives of less than 3 days to about 8 weeks) if they fail to encounter antigen or lose in the competition with other B cells for residence in a supportive lymphoid environ- B-Cell Maturate ment. Given that the immune system is able to generate a to- tal antibody diversity that exceeds 10, clearly only a small The generation of mature B cells first occurs in the embryo fraction of this potential repertoire is displayed at any time and continues throughout life. Before birth, the yolk sac, by membrane immunoglobulin on recirculating B cells In- fetal liver, and fetal bone marrow are the major sites of B-cell deed, throughout the life span of an animal, only a small maturation; after birth, generation of mature B cells occurs fraction of the possible antibody diversity is ever generated. in the bone marrow
■ B-Cell Maturation ■ B-Cell Activation and Proliferation ■ The Humoral Response ■ In Vivo Sites for Induction of Humoral Responses ■ Germinal Centers and Antigen-Induced B-Cell Differentiation ■ Regulation of B-Cell Development ■ Regulation of the Immune Effector Response Initial Contact Between B and T Cells B-Cell Generation, Activation, and Differentiation T production of plasma cells and memory B cells can be divided into three broad stages: generation of mature, immunocompetent B cells (maturation), activation of mature B cells when they interact with antigen, and differentiation of activated B cells into plasma cells and memory B cells. In many vertebrates, including humans and mice, the bone marrow generates B cells. This process is an orderly sequence of Ig-gene rearrangements, which progresses in the absence of antigen. This is the antigenindependent phase of B-cell development. A mature B cell leaves the bone marrow expressing membrane-bound immunoglobulin (mIgM and mIgD) with a single antigenic specificity. These naive B cells, which have not encountered antigen, circulate in the blood and lymph and are carried to the secondary lymphoid organs, most notably the spleen and lymph nodes (see Chapter 2). If a B cell is activated by the antigen specific to its membrane-bound antibody, the cell proliferates (clonal expansion) and differentiates to generate a population of antibody-secreting plasma cells and memory B cells. In this activation stage, affinity maturation is the progressive increase in the average affinity of the antibodies produced and class switching is the change in the isotype of the antibody produced by the B cell from to , , or . Since B cell activation and differentiation in the periphery require antigen, this stage comprises the antigendependent phase of B-cell development. Many B cells are produced in the bone marrow throughout life, but very few of these cells mature. In mice, the size of the recirculating pool of B cells is about 2 108 cells. Most of these cells circulate as naive B cells, which have short life spans (half lives of less than 3 days to about 8 weeks) if they fail to encounter antigen or lose in the competition with other B cells for residence in a supportive lymphoid environment. Given that the immune system is able to generate a total antibody diversity that exceeds 109 , clearly only a small fraction of this potential repertoire is displayed at any time by membrane immunoglobulin on recirculating B cells. Indeed, throughout the life span of an animal, only a small fraction of the possible antibody diversity is ever generated. Some aspects of B-cell developmental processes have been described in previous chapters. The overall pathway, beginning with the earliest distinctive B-lineage cell, is described in sequence in this chapter. Figure 11-1 presents an overview of the major events in humans and mice. Most of this chapter applies to humans and mice, but important departures from these developmental pathways have been shown to occur in some other vertebrates. Finally, this chapter will consider the regulation of B-cell development at various stages. B-Cell Maturation The generation of mature B cells first occurs in the embryo and continues throughout life. Before birth, the yolk sac, fetal liver, and fetal bone marrow are the major sites of B-cell maturation; after birth, generation of mature B cells occurs in the bone marrow. chapter 11
248 PART I Generation of B-Cell and T-Cell Responses VISUALIZING CONCEPTS ANTIGEN-INDEPENDENT PHASE CD45R Selection> Bone marrow marker ANTIGEN-DEPENDENT PHASE Tu cll +ag (-10% Cell death Activated (-90% Affinity Plasma FIGURE 11-1 Overview of B-cell development. During the anti- tivated TH cells. Once activated, B cells proliferate within sec- gen-independent maturation phase, immunocompetent B cells ondary lymphoid organs. Those bearing high-affinity mlg differ expressing membrane igM and igD are generated in the bone entiate into plasma cells and memory B cells, which may express marrow. Only about 10% of the potential B cells reach maturity different isotypes because of class switching. The numbers cited and exit the bone marrow. Naive B cells in the periphery die within refer to B-cell development in the mouse, but the overall princi a few days unless they encounter soluble protein antigen and ac- ples apply to humans as well Progenitor B Cells Proliferate shaft of a bone. Proliferation and differentiation of pro-B in Bone marrow cells into precursor B cells(pre-B cells)requires the micro environment provided by the bone-marrow stromal cells. If B-cell development begins as lymphoid stem cells differenti- pro-B cells are removed from the bone marrow and cultured ate into the earliest distinctive B-lineage cell-the progeni-. in vitro, they will not progress to more mature B-cell stage tor B cell (pro-B cell)which expresses a transmembrane unless stromal cells are present. The stromal cells play two tyrosine phosphatase called CD45R(sometimes called B220 important roles: they interact directly with pro-B and pre-B in mice). Pro-B cells proliferate within the bone marrow, fill- cells, and they secrete various cytokines, notably IL-7, that ing the extravascular spaces between large sinusoids in the support the developmental process
Progenitor B Cells Proliferate in Bone Marrow B-cell development begins as lymphoid stem cells differentiate into the earliest distinctive B-lineage cell—the progenitor B cell (pro-B cell)—which expresses a transmembrane tyrosine phosphatase called CD45R (sometimes called B220 in mice). Pro-B cells proliferate within the bone marrow, filling the extravascular spaces between large sinusoids in the shaft of a bone. Proliferation and differentiation of pro-B cells into precursor B cells (pre-B cells) requires the microenvironment provided by the bone-marrow stromal cells. If pro-B cells are removed from the bone marrow and cultured in vitro, they will not progress to more mature B-cell stages unless stromal cells are present. The stromal cells play two important roles: they interact directly with pro-B and pre-B cells, and they secrete various cytokines, notably IL-7, that support the developmental process. 248 PART II Generation of B-Cell and T-Cell Responses VISUALIZING CONCEPTS ~5 × 106 per day Bone marrow CD45R (B220) surface marker Peripheral lymphoid organ Plasma cell Ig-gene rearrangement Selection Progenitor B cell Mature B cell Secreted Ab Cell death (~90%) Memory B cell Activated B cell Affinity maturation Class switching TH cell –Ag +Ag (~10%) Naive B cell ANTIGEN-INDEPENDENT PHASE (maturation) ANTIGEN-DEPENDENT PHASE (activation and differentiation) FIGURE 11-1 Overview of B-cell development. During the antigen-independent maturation phase, immunocompetent B cells expressing membrane IgM and IgD are generated in the bone marrow. Only about 10% of the potential B cells reach maturity and exit the bone marrow. Naive B cells in the periphery die within a few days unless they encounter soluble protein antigen and activated TH cells. Once activated, B cells proliferate within secondary lymphoid organs. Those bearing high-affinity mIg differentiate into plasma cells and memory B cells, which may express different isotypes because of class switching. The numbers cited refer to B-cell development in the mouse, but the overall principles apply to humans as well
B-Cell Generation. Activation and Differentiation CHAPTEr 11 249 Immature B cells Pre-B cells Pro-B cells IL-7 VLA-4 SCF mlgM VCAM-1 Bone-marrow FIGURE 11-2 Bone-marrow stromal cells are required for matura- al cell, which triggers a signal, mediated by the tyrosine kinase ion of progenitor B cells into precursor B cells. Pro-B cells bind to of c-Kit, that stimulates the pro-B cell to express receptors for stromal cells by means of an interaction between VCAM-1 on the IL-7 IL-7 released from the stromal cell then binds to the IL-7recep stromal cell and VLA-4 on the pro B cell. This interaction promotes tors, inducing the pro-B cell to mature into a pre-B cell Proliferation the binding of c-Kit on the pro-B cell to stem cell factor(SCF)on the and differentiation evenutally produces immature B cells At the earliest developmental stage, pro-B cells require di- ment continues on the other chromosome. Upon completion rect contact with stromal cells in the bone marrow. This in- of heavy-chain rearrangement, the cell is classified as a pre-B teraction is mediated by several cell-adhesion molecules, cell. Continued development of a pre-B cell into an imma including VLA-4 on the pro-B cell and its ligand, VCAM-1, ture B cell requires a productive light-chain gene rearrange on the stromal cell(Figure 11-2). After initial contact is ment. Because of allelic exclusion, only one light-chain isotype made, a receptor on the pro-B cell called c-Kit interacts with is expressed on the membrane of a B cell. Completion of a a stromal-cell surface molecule known as stem-cell factor productive light-chain rearrangement commits the now im- (SCF). This interaction activates c-Kit, which is a tyrosine mature B cell to a particular antigenic specificity determined kinase, and the pro-B cell begins to divide and differentiate by the cells heavy-chain VDj sequence and light-chain VJ into a pre-B cell and begins expressing a receptor for IL-7. sequence. Immature B cells express mlgM(membrane IgM) The IL-7 secreted by the stromal cells drives the maturation on the cell surface process, eventually inducing down-regulation of the adhe- As would be expected, the recombinase enzymes RAG-1 sion molecules on the pre-B cells, so that the proliferating and RAG-2, which are required for both heavy-chain and cells can detach from the stromal cells. At this stage, pre-B light-chain gene rearrangements, are expressed during the cells no longer require direct contact with stromal cells but pro-B and pre-B cell stages(see Figure 11-3). The enzyme continue to require IL-7 for growth and maturation. terminal deoxyribonucleotidyl transferase(tdt), which cat alyzes insertion of N-nucleotides at the dh-JH and VH-DH" Gene Rearrangment Produces JH coding joints, is active during the pro-B cell stage and Immature b cells ceases to be active early in the pre-B-cell stage. Because TdT expression is turned off during the part of the pre-B-cell B-cell maturation depends on rearrangement of the immuno- stage when light-chain rearrangement occurs, N-nucleotides globulin DNA in the lymphoid stem cells. The mechanisms are not usually found in the Vl-Ji coding joints of Ig-gene rearrangement were described in Chapter 5. First The bone-marrow phase of B-cell development culmi to occur in the pro-B cell stage is a heavy-chain DH-to-JH nates in the production of an Ig M-bearing immature B cell.At gene rearrangement; this is followed by a VH-to-DHJH this stage of development the b cell is not fully functional, and rearrangement(Figure 11-3). If the first heavy-chain re- antigen induces death or unresponsiveness(anergy) rather arrangement is not productive, then VH-DH-JH rearrange- than division and differentiation. Full maturation is signaled
At the earliest developmental stage, pro-B cells require direct contact with stromal cells in the bone marrow. This interaction is mediated by several cell-adhesion molecules, including VLA-4 on the pro-B cell and its ligand, VCAM-1, on the stromal cell (Figure 11-2). After initial contact is made, a receptor on the pro-B cell called c-Kit interacts with a stromal-cell surface molecule known as stem-cell factor (SCF). This interaction activates c-Kit, which is a tyrosine kinase, and the pro-B cell begins to divide and differentiate into a pre-B cell and begins expressing a receptor for IL-7. The IL-7 secreted by the stromal cells drives the maturation process, eventually inducing down-regulation of the adhesion molecules on the pre-B cells, so that the proliferating cells can detach from the stromal cells. At this stage, pre-B cells no longer require direct contact with stromal cells but continue to require IL-7 for growth and maturation. Ig-Gene Rearrangment Produces Immature B Cells B-cell maturation depends on rearrangement of the immunoglobulin DNA in the lymphoid stem cells. The mechanisms of Ig-gene rearrangement were described in Chapter 5. First to occur in the pro-B cell stage is a heavy-chain DH-to-JH gene rearrangement; this is followed by a VH-to-DHJH rearrangement (Figure 11-3). If the first heavy-chain rearrangement is not productive, then VH-DH-JH rearrangement continues on the other chromosome. Upon completion of heavy-chain rearrangement, the cell is classified as a pre-B cell. Continued development of a pre-B cell into an immature B cell requires a productive light-chain gene rearrangement. Because of allelic exclusion, only one light-chain isotype is expressed on the membrane of a B cell. Completion of a productive light-chain rearrangement commits the now immature B cell to a particular antigenic specificity determined by the cell’s heavy-chain VDJ sequence and light-chain VJ sequence. Immature B cells express mIgM (membrane IgM) on the cell surface. As would be expected, the recombinase enzymes RAG-1 and RAG-2, which are required for both heavy-chain and light-chain gene rearrangements, are expressed during the pro-B and pre-B cell stages (see Figure 11-3). The enzyme terminal deoxyribonucleotidyl transferase (TdT), which catalyzes insertion of N-nucleotides at the DH-JH and VH-DHJH coding joints, is active during the pro-B cell stage and ceases to be active early in the pre–B-cell stage. Because TdT expression is turned off during the part of the pre–B-cell stage when light-chain rearrangement occurs, N-nucleotides are not usually found in the VL-JL coding joints. The bone-marrow phase of B-cell development culminates in the production of an IgM-bearing immature B cell.At this stage of development the B cell is not fully functional, and antigen induces death or unresponsiveness (anergy) rather than division and differentiation. Full maturation is signaled B-Cell Generation, Activation, and Differentiation CHAPTER 11 249 Pro-B cells Pre-B cells c-Kit VLA-4 SCF VCAM-1 IL-7 receptor IL-7 mIgM Immature B cells Bone-marrow stromal cell FIGURE 11-2 Bone-marrow stromal cells are required for maturation of progenitor B cells into precursor B cells. Pro-B cells bind to stromal cells by means of an interaction between VCAM-1 on the stromal cell and VLA-4 on the pro-B cell. This interaction promotes the binding of c-Kit on the pro-B cell to stem cell factor (SCF) on the stromal cell, which triggers a signal, mediated by the tyrosine kinase activity of c-Kit, that stimulates the pro-B cell to express receptors for IL-7. IL-7 released from the stromal cell then binds to the IL-7 receptors, inducing the pro-B cell to mature into a pre-B cell. Proliferation and differentiation evenutally produces immature B cells
PART II Generation of B-Cell and T-Cell Responses chain of pre-Bc Duj PRO-B CELL PRE-B CELL MMATURE B CELL NAIVE B CELL MATURE B CELL STEM CELL H-chain ge Germ line l VHDHJH L-chain genes VLJL Vpre-B andλ5 Vpre- B andλ5 Germ-line Germ-line RAG-1/2 Membrane ig avy cha u+8 ht chain light chain Pu. 1. Ikaros BSAP(Pax-5) factors others E2A Kit arkers CDI9 CD43 CD25 FIGURE11-3 Sequence of events and characteristics of the stages thesis of both membrane- bound igM and lgD by mature B cells in B-cell maturation in the bone marrow. The pre-B cell expresses a RAG-1/2=two enzymes encoded by recombination-activating genes membrane immunoglobulin consisting of a heavy(H)chain and sur- TdT terminal deoxyribonucleotidyl transferase. A number of B-cell- rogate light chains, Vpre-B and A5. Changes in the RNA processing associated transcription factors are important at various stages of of heavy-chain transcripts following the pre-B cell stage lead to syn- B-cell development:: some are indicated by the co-expression of IgD and IgM on the membrane. This The Pre-B-Cell Receptor Is Essential progression involves a change in RNA processing of the for B-Cell Development heavy-chain primary transcript to permit production of two mRNAS, one encoding the membrane form of the u chain As we saw in Chapter 10, during one stage in T-cell develop- and the other encoding the membrane form of the 8 chain ment, the p chain of the T-cell receptor associates with pre- (see Figure 5-19). Although IgD is a characteristic cell-surface Ta to form the pre-T-cell receptor(see Figure 10-1).A marker of mature naive B cells, its function is not clear How- parallel situation occurs during B-cell development. In the ever, since immunoglobulin 8 knockout mice have essentially pre-B cell, the membrane u chain is associated with the sur- normal numbers of fully functional B cells, IgD is not essen- rogate light chain, a complex consisting of two proteins: a tial to either B-cell development or antigen responsiveness. V-like sequence called Vpre- B and a C-like sequence called
by the co-expression of IgD and IgM on the membrane. This progression involves a change in RNA processing of the heavy-chain primary transcript to permit production of two mRNAs, one encoding the membrane form of the chain and the other encoding the membrane form of the chain (see Figure 5-19). Although IgD is a characteristic cell-surface marker of mature naive B cells, its function is not clear. However, since immunoglobulin knockout mice have essentially normal numbers of fully functional B cells, IgD is not essential to either B-cell development or antigen responsiveness. The Pre–B-Cell Receptor Is Essential for B-Cell Development As we saw in Chapter 10, during one stage in T-cell development, the chain of the T-cell receptor associates with preT to form the pre–T-cell receptor (see Figure 10-1). A parallel situation occurs during B-cell development. In the pre-B cell, the membrane chain is associated with the surrogate light chain, a complex consisting of two proteins: a V-like sequence called Vpre-B and a C-like sequence called 250 PART II Generation of B-Cell and T-Cell Responses FIGURE 11-3 Sequence of events and characteristics of the stages in B-cell maturation in the bone marrow. The pre-B cell expresses a membrane immunoglobulin consisting of a heavy (H) chain and surrogate light chains, Vpre-B and 5. Changes in the RNA processing of heavy-chain transcripts following the pre-B cell stage lead to synthesis of both membrane-bound IgM and IgD by mature B cells. RAG-1/2 = two enzymes encoded by recombination-activating genes; TdT = terminal deoxyribonucleotidyl transferase. A number of B-cell– associated transcription factors are important at various stages of B-cell development; some are indicated here. IgM IgD IgM VL J VHDH L JH PRO-B CELL PRE-B CELL MATURE B CELL IMMATURE B CELL H-chain genes L-chain genes DH JH VHDH JH DH JH RAG-1/2 TdT VL JL Surrogate Vpre-B and λ5 Germ-line κ and λ + + − − + − − − Heavy chain − µ µ + δ Membrane Ig Transcription factors Surface markers BSAP(Pax-5) Sox-4 EBF E2A Oct-2 c-Kit CD45R, CD19, HSA(CD24), Ig-α/Ig-β IL-7R CD43 Light chain Surrogate light chain Surrogate light chain LYMPHOID STEM CELL Germ line Germ line − − − Pu.1, Ikaros, others − κ or λ CD25 mIgM mIgD IgM Periphery antigen-dependent Bone marrow antigen-independent NAIVE B CELL − − Surrogate Vpre-B and λ5 Germ-line κ and λ Surrogate light chain of pre-BCR Ig-α/Ig-β
B-Cell Generation Activation and Differentiation CHAPTER 11 251 Pro-B cell Pre-B cell Immature b cell VH DuJuc VHDHJHC lgw/lgβ Crosslinking by stromal cell ligand Stops VH?DHJH (allelic exclusion)? FIGURE Schematic diagram of sequential expression of mem- and a A5 polypeptide, which are noncovalently associated. The im- brane immunoglobulin and surrogate light chain at different stages mature B cell no longer expresses the surrogate light chain and in- of B-cell differentiation in the bone marrow. The pre-B-cell receptor stead expresses the K or A light chain together with the u heav contains a surrogate light chain consisting of a Vpre-B polypeptide chain A5, which associate noncovalently to form a light-chain-like factors are knocked out by gene disruption have shown that struc four such factors, E2A, early B-cell factor(EBF), B-cell- The membrane-bound complex of u heavy chain and sur- specific activator protein(BSAP), and Sox-4 are particularly rogate light chain appears on the pre- B cell associated with the important for B-cell development (see Figure 11-3). Mice Ig-o/Ig-B heterodimer to form the pre-B-cell receptor(Figure that lack E2a do not express RAG-l, are unable to make 11-4). Only pre-B cells that are able to express membrane- DHH rearrangements, and fail to express x5, a critical com bound u heavy chains in association with surrogate ponent of the surrogate light chain. A similar pattern is seen chains are able to proceed along the maturation pathway in EBF-deficient mice. These findings point to important There is speculation that the pre-B-cell receptor recog- roles for both of these transcription factors early in B-cell nizes a not-yet-identified ligand on the stromal-cell mem- development, and they may play essential roles in the early brane, thereby transmitting a signal to the pre-B cell that stages of commitment to the B-cell lineage. Knocking out the prevents VH to DHH rearrangement of the other heavy-chain Pax-5 gene, whose product is the transcription factor BSAP, allele, thus leading to allelic exclusion. Following the estab- also results in the arrest of B-cell development at an early lishment of an effective pre-B-cell receptor, each pre-B cell stage. Binding sites for BSAP are found in the promoter re- undergoes multiple cell divisions, producing 32 to 64 descen- gions of a number of B-cell-specific genes, including Vpre-B dants. Each of these progeny pre-B cells may then rearrange and A5, in a number of lg switch regions, and in the Ig heavy- different light-chain gene segments, thereby increasing the chain enhancer. This indicates that BSAP plays a role beyond overall diversity of the antibody repertoire. the early stages of B-cell development. This factor is also The critical role of the pre-B-cell receptor was demon- pressed in the central nervous system, and its absence results strated with knockout mice in which the gene encoding the A5 in severe defects in mid-brain development. Although the ex- protein of the receptor was disrupted. B-cell development in act site of action of Sox- 4 is not known, it affects early stages these mice was shown to be blocked at the pre-B stage, which of B-cell activation. While Figure 11-3 shows that all of these suggests that a signal generated through the receptor is neces- transcription factors affect development at an early stage sary for pre-B cells to proceed to the immature B-cell stage. some of them are active at later stages also Knockout Experiments Identified Essential Cell-Surface Markers Identify Transcription Factors Development stages As described in Chapter 2, many different transcription fac- The developmental progression from progenitor to mature tors act in the development of hematopoietic cells. Nearly a B cell is typified dozen of them have so far been shown to play roles in B-cell Figure 11-3). At the pro-B stage, the cells do not display the development. Experiments in which particular transcription heavy or light chains of antibody but they do express CD45R
5, which associate noncovalently to form a light-chain–like structure. The membrane-bound complex of heavy chain and surrogate light chain appears on the pre-B cell associated with the Ig-/Ig- heterodimer to form the pre–B-cell receptor (Figure 11-4). Only pre-B cells that are able to express membranebound heavy chains in association with surrogate light chains are able to proceed along the maturation pathway. There is speculation that the pre–B-cell receptor recognizes a not-yet-identified ligand on the stromal-cell membrane, thereby transmitting a signal to the pre-B cell that prevents VH to DHJH rearrangement of the other heavy-chain allele, thus leading to allelic exclusion. Following the establishment of an effective pre–B-cell receptor, each pre-B cell undergoes multiple cell divisions, producing 32 to 64 descendants. Each of these progeny pre-B cells may then rearrange different light-chain gene segments, thereby increasing the overall diversity of the antibody repertoire. The critical role of the pre–B-cell receptor was demonstrated with knockout mice in which the gene encoding the 5 protein of the receptor was disrupted. B-cell development in these mice was shown to be blocked at the pre-B stage, which suggests that a signal generated through the receptor is necessary for pre-B cells to proceed to the immature B-cell stage. Knockout Experiments Identified Essential Transcription Factors As described in Chapter 2, many different transcription factors act in the development of hematopoietic cells. Nearly a dozen of them have so far been shown to play roles in B-cell development. Experiments in which particular transcription factors are knocked out by gene disruption have shown that four such factors, E2A, early B-cell factor (EBF), B-cell– specific activator protein (BSAP), and Sox-4 are particularly important for B-cell development (see Figure 11-3). Mice that lack E2A do not express RAG-1, are unable to make DHJH rearrangements, and fail to express 5, a critical component of the surrogate light chain. A similar pattern is seen in EBF-deficient mice. These findings point to important roles for both of these transcription factors early in B-cell development, and they may play essential roles in the early stages of commitment to the B-cell lineage. Knocking out the Pax-5 gene, whose product is the transcription factor BSAP, also results in the arrest of B-cell development at an early stage. Binding sites for BSAP are found in the promoter regions of a number of B-cell–specific genes, including Vpre-B and 5, in a number of Ig switch regions, and in the Ig heavychain enhancer. This indicates that BSAP plays a role beyond the early stages of B-cell development. This factor is also expressed in the central nervous system, and its absence results in severe defects in mid-brain development. Although the exact site of action of Sox-4 is not known, it affects early stages of B-cell activation. While Figure 11-3 shows that all of these transcription factors affect development at an early stage, some of them are active at later stages also. Cell-Surface Markers Identify Development Stages The developmental progression from progenitor to mature B cell is typified by a changing pattern of surface markers (see Figure 11-3). At the pro-B stage, the cells do not display the heavy or light chains of antibody but they do express CD45R, B-Cell Generation, Activation, and Differentiation CHAPTER 11 251 Immature B cell κ or λ Crosslinking by antigen Activation Death Pre-B cell Crosslinking by stromalcell ligand Pro-B cell λ5 Stops VH DH JH (allelic exclusion) ? Induces Vκ Jκ ? VHDH JHCµ Ig-α/Ig-β Vpre-B VHDH JHCµ Surrogate light chain FIGURE 11-4 Schematic diagram of sequential expression of membrane immunoglobulin and surrogate light chain at different stages of B-cell differentiation in the bone marrow. The pre–B-cell receptor contains a surrogate light chain consisting of a Vpre-B polypeptide and a 5 polypeptide, which are noncovalently associated. The immature B cell no longer expresses the surrogate light chain and instead expresses the or light chain together with the heavy chain.
PART II Generation of B-Cell and T-Cell Responses which is a form of the protein tyrosine phophatase found on ation of some B-1 cells in sites outside the bone marrow to leukocytes, and the signal-transducing molecules Ig-a/Ig-B, form additional naive B-1 cells. The B-1 population responds which are found in association with the membrane forms of poorly to protein antigens but much better to carbohydrate antibody in later stages of B-cell development. Pro-B cells ones. Most of its members are IgM-bearing cells, and this also express CD19 (part of the B-cell coreceptor), CD43 population undergoes much less somatic hypermutation and Ceukosialin), and CD24 a molecule also known as heat heat- class switching than the B-2 set of B cells does. Consequenth stable antigen(HSA)on the surface. At this stage, c-Kit, a re- the antibodies produced by a high proportion of B-1 cells are ceptor for a growth-promoting ligand present on stromal cells, of rather low affinity is also found on the surface of pro-B cells. As cells progres from the pro-B to the pre-B stage, they express many of the Self-Reactive B Cells Are Selected Against same markers that were present during the pro-B stage; how- in Bone Marrow press CD25, the a chain of the IL-2 receptor. The display of It is estimated that in the mouse the bone marrow produces the pre-B-cell receptor(pre-BCR)is a salient feature of the about 5 X 10B cells/day but only 5 X 10(or about 10%) pre-B cell stage. After rearrangement of the light chain, sur- are actually recruited into the recirculating B-cell pool. This face immunoglobulin containing both heavy and light chains means that 90% of the B cells produced each day die without appears, and the cells, now classified as immature B cells, lose ever leaving the bone marrow. Some of this loss is attribut- the pre-BCR and no longer express CD25 Monoclonal anti- able to negative selection and subsequent elimination( clonal bodies are available that can recognize all of these antigenic deletion)of immature b cells that express auto-antibodi arkers, making it possible to recognize and isolate the vari- against self-antigens in the bone marrow. ous stages of B-cell development by the techniques of im- It has long been established that the crosslinkage of mIgM munohistology and flow cytometry described in Chapter 6. on immature B cells, demonstrated experimentally by treat- ing immature B cells with antibody against the u constant re- B-1 B Cells Are a Self-Renewing B-Cell Subset gion, can cause the cells to die by apoptosis within the bone There is a subset of B cells, called B-1 B cells, that arise before immature B cells that express self-reactive mIgM bind to self- B-2 B cells, the major group of B cells in humans and mice. In antigens in the bone marrow. For example, D. A. Nemazee humans and mice, B-1 B cells compose about 5% of the total and K Burki introduced a transgene encoding the heavy and B-cell population. They appear during fetal life express sur- light chains of an IgM antibody specific for K an H-2 class face IgM but little or no IgD, and are marked by the display of I MHC molecule, into H-2and H-2mice(Figure 11-5a, b) CD5. However, CD5 is not an indispensable component of the Since class I MHC molecules are expressed on the membrane B-1lineage, it does not appear on the B-1 cells of rats, and mice of all nucleated cells, the endogenous H-2 and H-2 class I that lack a functional CD5 gene still produce B-1 cells In ani- MHC molecules would be present on bone-marrow stromal mals whose B-2 B cells are the major B-cell population, B-1 cells in the transgenic mice. In the H-2dmice, which do not cells are minor populations in such secondary tissues as express kk, 25%-50% of the mature, peripheral B cells ex- nodes and spleen. Despite their scarcity in many lymphoid pressed the transgene-encoded anti-k both as a membrane sites, they are the major B-cell type found in the peritoneum. antibody and as secreted antibody. In contrast, in the H-2 Although there is not a great deal of definitive informa- mice, which express K no mature, peripheral B cells ex- tion on the function of B-1 cells, several features set them pressed the transgene-encoded antibody to H-2(Table 11-1) apart from the B-2 B cells of humans and mice During fetal These results suggest that there is negative selection against life, B-1 cells arise from stem cells in bone marrow. However, any immature B cells expressing auto-antibodies on their in postnatal life this population renews itself by the prolifer- membranes because these antibodies react with self-antigen TABLE Expression of transgene encoding IgM antibody to H-2 class I MHC molecules EXPRESSION OF TRANSGENE Experimental animal Number of animals tested As membrane al As secreted Ab (ug/ml) Nontransgenics H-2 transgenics (+) 930 SOURCE: Adapted from D A Nemazee and K Burki, 1989, Nature 337: 562
which is a form of the protein tyrosine phophatase found on leukocytes, and the signal-transducing molecules Ig-/Ig-, which are found in association with the membrane forms of antibody in later stages of B-cell development. Pro-B cells also express CD19 (part of the B-cell coreceptor), CD43 (leukosialin), and CD24, a molecule also known as heatstable antigen (HSA) on the surface. At this stage, c-Kit, a receptor for a growth-promoting ligand present on stromal cells, is also found on the surface of pro-B cells. As cells progress from the pro-B to the pre-B stage, they express many of the same markers that were present during the pro-B stage; however, they cease to express c-Kit and CD43 and begin to express CD25, the chain of the IL-2 receptor. The display of the pre–B-cell receptor (pre-BCR) is a salient feature of the pre-B cell stage. After rearrangement of the light chain, surface immunoglobulin containing both heavy and light chains appears, and the cells, now classified as immature B cells, lose the pre-BCR and no longer express CD25. Monoclonal antibodies are available that can recognize all of these antigenic markers, making it possible to recognize and isolate the various stages of B-cell development by the techniques of immunohistology and flow cytometry described in Chapter 6. B-1 B Cells Are a Self-Renewing B-Cell Subset There is a subset of B cells, called B-1 B cells, that arise before B-2 B cells, the major group of B cells in humans and mice. In humans and mice, B-1 B cells compose about 5% of the total B-cell population. They appear during fetal life, express surface IgM but little or no IgD, and are marked by the display of CD5. However, CD5 is not an indispensable component of the B-1 lineage, it does not appear on the B-1 cells of rats, and mice that lack a functional CD5 gene still produce B-1 cells. In animals whose B-2 B cells are the major B-cell population, B-1 cells are minor populations in such secondary tissues as lymph nodes and spleen. Despite their scarcity in many lymphoid sites, they are the major B-cell type found in the peritoneum. Although there is not a great deal of definitive information on the function of B-1 cells, several features set them apart from the B-2 B cells of humans and mice. During fetal life, B-1 cells arise from stem cells in bone marrow. However, in postnatal life this population renews itself by the proliferation of some B-1 cells in sites outside the bone marrow to form additional naive B-1 cells. The B-1 population responds poorly to protein antigens but much better to carbohydrate ones. Most of its members are IgM-bearing cells, and this population undergoes much less somatic hypermutation and class switching than the B-2 set of B cells does. Consequently, the antibodies produced by a high proportion of B-1 cells are of rather low affinity. Self-Reactive B Cells Are Selected Against in Bone Marrow It is estimated that in the mouse the bone marrow produces about 5 107 B cells/day but only 5 106 (or about 10%) are actually recruited into the recirculating B-cell pool. This means that 90% of the B cells produced each day die without ever leaving the bone marrow. Some of this loss is attributable to negative selection and subsequent elimination (clonal deletion) of immature B cells that express auto-antibodies against self-antigens in the bone marrow. It has long been established that the crosslinkage of mIgM on immature B cells, demonstrated experimentally by treating immature B cells with antibody against the constant region, can cause the cells to die by apoptosis within the bone marrow. A similar process is thought to occur in vivo when immature B cells that express self-reactive mIgM bind to selfantigens in the bone marrow. For example, D. A. Nemazee and K. Burki introduced a transgene encoding the heavy and light chains of an IgM antibody specific for Kk , an H-2k class I MHC molecule, into H-2d and H-2d/k mice (Figure 11-5a,b). Since class I MHC molecules are expressed on the membrane of all nucleated cells, the endogenous H-2k and H-2d class I MHC molecules would be present on bone-marrow stromal cells in the transgenic mice. In the H-2d mice, which do not express Kk , 25%–50% of the mature, peripheral B cells expressed the transgene-encoded anti-Kk both as a membrane antibody and as secreted antibody. In contrast, in the H-2d/k mice, which express Kk , no mature, peripheral B cells expressed the transgene-encoded antibody to H-2k (Table 11-1). These results suggest that there is negative selection against any immature B cells expressing auto-antibodies on their membranes because these antibodies react with self-antigen 252 PART II Generation of B-Cell and T-Cell Responses TABLE 11-1 Expression of transgene encoding IgM antibody to H-2k class I MHC molecules EXPRESSION OF TRANSGENE Experimental animal Number of animals tested As membrane Ab As secreted Ab (g/ml) Nontransgenics 13 (–) <0.3 H-2d transgenics 7 (+) 93.0 H-2d/k transgenics 6 (–) <0.3 SOURCE: Adapted from D. A. Nemazee and K. Burki, 1989, Nature 337:562.
B-Cell Generation Activation, and Differentiation CHAPTER 11 253 k 属 -)25-50% of mature B cells expres (c)H-2 dk transgenics Light-chain editing mature b cells with new light chains no longer bind Kk GURE11-5 Experimental evidence for negative selection(clonal mature so that 25%-50% of the splenic B cells expressed the trans. deletion) of self-reactive B cells during maturation in the bone mar- gene-encoded anti-K as membrane lg. More detailed analysis of the row. The presence or absence of mature peripheral B cells expressing H-2dktransgenics revealed a few peripheral B cells that expressed the a transgene- encoded igM against the H-2 class I molecule K was transgene-encoded u chain but a different light chain( c). Apparently determined in H-2 mice(a)and H-2 mice(b). In the H-2a trans- a few immature b cells underwent light-chain editing, so they no genic, the immature B cells recognized the self-antigen K and were longer bound the K molecule and consequently escaped negative se. deleted by negative selection. In the H-2transgenics, the immature lection. Adapted from D A Nemazee and K Burki, 1989, Nature 337. B cells did not bind to a self-antigen and consequently went on to 562: S.L. Tiegs et al., 1993, JEM 177: 1009. (e.g, the K molecule in H-2transgenics) present on stro- does not always result in their immediate deletion(Figure mal cells, leading to crosslinking of the antibodies and subse- 11-5c). Instead, maturation of the self-reactive cell is arrested quent death of the immature B cells while the B cell"edits"the light-chain gene of its receptor In this case, the H-2 transgenics produced a few mature Self-Reactive B Cells May Be Rescued B cells that expressed mlgm containing the u chain encoded by Editing of Light-Chain Genes in the transgene, but different, endogenous light chains. One explanation for these results is that when some immature Later work using the transgenic system described by Nemazee B cells bind a self-antigen, maturation is arrested; the cells nd Burki showed that negative selection of immature B cells up-regulate RAG-1 and RAG-2 expression and begin further
(e.g., the Kk molecule in H-2d/k transgenics) present on stromal cells, leading to crosslinking of the antibodies and subsequent death of the immature B cells. Self-Reactive B Cells May Be Rescued by Editing of Light-Chain Genes Later work using the transgenic system described by Nemazee and Burki showed that negative selection of immature B cells does not always result in their immediate deletion (Figure 11-5c). Instead, maturation of the self-reactive cell is arrested while the B cell “edits” the light-chain gene of its receptor. In this case, the H-2d/k transgenics produced a few mature B cells that expressed mIgM containing the chain encoded in the transgene, but different, endogenous light chains. One explanation for these results is that when some immature B cells bind a self-antigen, maturation is arrested; the cells up-regulate RAG-1 and RAG-2 expression and begin further B-Cell Generation, Activation, and Differentiation CHAPTER 11 253 Kd (b) H-2d transgenics 25–50% of mature B cells express anti -Kk (c) H-2 d/k transgenics A few mature B cells with new light chains no longer bind Kk Light-chain editing Immature B cells Bone-marrow stromal cell Anti-Kk Kk (a) H-2d/k transgenics No mature B cells express anti-Kk Kd FIGURE 11-5 Experimental evidence for negative selection (clonal deletion) of self-reactive B cells during maturation in the bone marrow. The presence or absence of mature peripheral B cells expressing a transgene-encoded IgM against the H-2 class I molecule Kk was determined in H-2d/k mice (a) and H-2d mice (b). In the H-2d/k transgenics, the immature B cells recognized the self-antigen Kk and were deleted by negative selection. In the H-2d transgenics, the immature B cells did not bind to a self-antigen and consequently went on to mature, so that 25%–50% of the splenic B cells expressed the transgene-encoded anti-Kk as membrane Ig. More detailed analysis of the H-2d/k transgenics revealed a few peripheral B cells that expressed the transgene-encoded chain but a different light chain (c). Apparently, a few immature B cells underwent light-chain editing, so they no longer bound the Kk molecule and consequently escaped negative selection. [Adapted from D. A. Nemazee and K. Burki, 1989, Nature 337: 562; S. L. Tiegs et al., 1993, JEM 177:1009.]
254 PART II Generation of B-Cell and T-Cell Responses rearrangement of their endogenous light-chain DNA. Some TI-2 antigens activate B cells by extensively crosslinking of these cells succeed in replacing the k light chain of the self- the mlg receptor. However, TI-2 antigens differ from TI-1 antigen reactive antibody with a x chain encoded by endoge- antigens in three important respects. First, they are not B-cell nous A-chain gene segments. As a result, these cells will begin mitogens and so do not act as polyclonal activators. Second to express an"edited"mlg M with a different light chain and a TI-l antigens will activate both mature and immature B cells, specificity that is not self-reactive. These cells escape negative but TI-2 antigens activate mature B cells and inactivate im selection and leave the bone marrow mature B cells. Third, although the B-cell response to TI-2 antigens does not require direct involvement of TH cells cytokines derived from TH cells are required for efficient B-Cell Activation and Proliferation B-cell proliferation and for class switching to isotypes other than IgM. This can be shown by comparing the effect of TI-2 After export of B cells from the bone marrow, activation, pro- antigens in mice made T-cell-deficient in various ways. In liferation, and differentiation occur in the periphery and re- nude mice, which lack thymus-derived Tcells but do contain quire antigen Antigen-driven activation and clonal selection a few t cells that arise from other sites that probably lie in the of naive B cells leads to generation of plasma cells and mem- intestine, TI-2 antigens do elicit B-cell responses. TI-2 anti- ory B cells. In the absence of antigen-induced activation, gens do not induce antibody production in mice that cannot naive B cells in the periphery have a short life span, dying express either aB or y8 TCRs because the genes encoding the within a few weeks by apoptosis(see Figure 11-1) TCR B and 8 chains have been knocked out. Administration of a few t cells to these tcr-knockout mice restores their Thymus-Dependent and Thymus Independent Antigen Have Different ability to elicit B山 esponses to ncin dependent antigens is Requirements for Response different from the response to thymus-dependent antigens Table 11-2). The response to Ti antigens is generally weaker, Depending on the nature of the antigen, B-cell activation pro- no memory cells are formed, and igM is the predominant ceeds by two different routes, one dependent upon TH cells, the antibody secreted, reflecting a low level of class switching other not. The B-cell response to thymus-dependent (TD)an- These differences highlight the important role played by th tigens requires direct contact with TH cells, not simply expo- cells in generating memory B cells, affinity maturation, and sure to TH-derived cytokines. Antigens that can activate B cells class switching to other isotypes in the absence of this kind of direct participation by TH cells are known as thymus-independent (TD) antigens. TI antigens are Two Types of Signals Drive B Cells into divided into types 1 and 2, and they activate B cells by different and Through the Cell Cycle mechanisms. Some bacterial cell-wall components, including lipopolysaccharide(LpS), function as type 1 thymus-independent Naive, or resting, B cells are nondividing cells in the go stage of (TI-1)antigens. Type 2 thymus-independent (T1-2) antigens are the cell cycle. Activation drives the resting cell into the cell cy highly repetitious molecules such as polymeric proteins(e.g, cle, progressing through Gn into the S phase, in which dNA is bacterial flagellin or bacterial cell-wall polysaccharides with replicated. The transition from g1 to S is a critical restriction repeating polysaccharide units. point in the cell cycle. Once a cell has reached S, it completes Most TI-1 antigens are polyclonal B-cell activators(mito- the cell cycle, moving through Ga and into mitosis(M) gens); that is, they are able to activate b cells regardless of Analysis of the progression of lymphocytes from go to the their antigenic specificity. At high concentrations, some TI-1 S phase revealed similarities with the parallel sequence in fi- antigens will stimulate proliferation and antibody secretion broblast cells. These events could be grouped into two cate- which TI-1 antigens activate B cells is not well understood. tence signals drive the b cell from Go into early GI, rendering When B cells are exposed to lower concentrations of TI-l the cell competent to receive the next level of signals. Pro- antigens, only those B cells specific for epitopes of the antigen gression signals then drive the cell from gi into S and ulti will be activated. These antigens can stimulate antibody pro- mately to cell division and differentiation. Competence is duction in nude mice(which lack a thymus and thus are achieved by not one but two distinct signaling events, which greatly deficient in T cells), and the response is not greatly are designated signal 1 and signal 2. These signaling events are augmented by transferring T cells into these athymic mice, generated by different pathways with thymus-independent indicating that Ti-1 antigens are truly T-cell independent. and thymus-dependent antigens, but both pathways include The prototypic TI-1 antigen is lipopolysaccharide(LPS), a signals generated when multivalent antigen binds and cross- major component of the cell walls of gram-negative bacteria. links mlg(Figure 11-6). Once the B cell has acquired an ef- At low concentrations, LPS stimulates the production of fective competence signal in early activation, the interaction antibodies specific for LPS. At high concentrations, it is a of cytokines and possibly other ligands with the B-cell mem- lyclonal B-cell activator. brane receptors provides progression signals
rearrangement of their endogenous light-chain DNA. Some of these cells succeed in replacing the light chain of the selfantigen reactive antibody with a chain encoded by endogenous -chain gene segments. As a result, these cells will begin to express an “edited” mIgM with a different light chain and a specificity that is not self-reactive. These cells escape negative selection and leave the bone marrow. B-Cell Activation and Proliferation After export of B cells from the bone marrow, activation, proliferation, and differentiation occur in the periphery and require antigen. Antigen-driven activation and clonal selection of naive B cells leads to generation of plasma cells and memory B cells. In the absence of antigen-induced activation, naive B cells in the periphery have a short life span, dying within a few weeks by apoptosis (see Figure 11-1). Thymus-Dependent and ThymusIndependent Antigen Have Different Requirements for Response Depending on the nature of the antigen, B-cell activation proceeds by two different routes, one dependent upon TH cells, the other not. The B-cell response to thymus-dependent (TD) antigens requires direct contact with TH cells, not simply exposure to TH-derived cytokines. Antigens that can activate B cells in the absence of this kind of direct participation by TH cells are known as thymus-independent (TI) antigens. TI antigens are divided into types 1 and 2, and they activate B cells by different mechanisms. Some bacterial cell-wall components, including lipopolysaccharide (LPS), function as type 1 thymus-independent (TI-1) antigens. Type 2 thymus-independent (TI-2) antigens are highly repetitious molecules such as polymeric proteins (e.g., bacterial flagellin) or bacterial cell-wall polysaccharides with repeating polysaccharide units. Most TI-1 antigens are polyclonal B-cell activators (mitogens); that is, they are able to activate B cells regardless of their antigenic specificity. At high concentrations, some TI-1 antigens will stimulate proliferation and antibody secretion by as many as one third of all B cells. The mechanism by which TI-1 antigens activate B cells is not well understood. When B cells are exposed to lower concentrations of TI-1 antigens, only those B cells specific for epitopes of the antigen will be activated. These antigens can stimulate antibody production in nude mice (which lack a thymus and thus are greatly deficient in T cells), and the response is not greatly augmented by transferring T cells into these athymic mice, indicating that TI-1 antigens are truly T-cell independent. The prototypic TI-1 antigen is lipopolysaccharide (LPS), a major component of the cell walls of gram-negative bacteria. At low concentrations, LPS stimulates the production of antibodies specific for LPS. At high concentrations, it is a polyclonal B-cell activator. TI-2 antigens activate B cells by extensively crosslinking the mIg receptor. However, TI-2 antigens differ from TI-1 antigens in three important respects. First, they are not B-cell mitogens and so do not act as polyclonal activators. Second, TI-1 antigens will activate both mature and immature B cells, but TI-2 antigens activate mature B cells and inactivate immature B cells. Third, although the B-cell response to TI-2 antigens does not require direct involvement of TH cells, cytokines derived from TH cells are required for efficient B-cell proliferation and for class switching to isotypes other than IgM. This can be shown by comparing the effect of TI-2 antigens in mice made T-cell–deficient in various ways. In nude mice, which lack thymus-derived T cells but do contain a few T cells that arise from other sites that probably lie in the intestine, TI-2 antigens do elicit B-cell responses. TI-2 antigens do not induce antibody production in mice that cannot express either or TCRs because the genes encoding the TCR and chains have been knocked out. Administration of a few T cells to these TCR-knockout mice restores their ability to elicit B-cell responses to TI-2 antigens. The humoral response to thymus-independent antigens is different from the response to thymus-dependent antigens (Table 11-2). The response to TI antigens is generally weaker, no memory cells are formed, and IgM is the predominant antibody secreted, reflecting a low level of class switching. These differences highlight the important role played by TH cells in generating memory B cells, affinity maturation, and class switching to other isotypes. Two Types of Signals Drive B Cells into and Through the Cell Cycle Naive, or resting, B cells are nondividing cells in the G0 stage of the cell cycle. Activation drives the resting cell into the cell cycle, progressing through G1 into the S phase, in which DNA is replicated. The transition from G1 to S is a critical restriction point in the cell cycle. Once a cell has reached S, it completes the cell cycle, moving through G2 and into mitosis (M). Analysis of the progression of lymphocytes from G0 to the S phase revealed similarities with the parallel sequence in fibroblast cells. These events could be grouped into two categories, competence signals and progression signals. Competence signals drive the B cell from G0 into early G1, rendering the cell competent to receive the next level of signals. Progression signals then drive the cell from G1 into S and ultimately to cell division and differentiation. Competence is achieved by not one but two distinct signaling events, which are designated signal 1 and signal 2. These signaling events are generated by different pathways with thymus-independent and thymus-dependent antigens, but both pathways include signals generated when multivalent antigen binds and crosslinks mIg (Figure 11-6). Once the B cell has acquired an effective competence signal in early activation, the interaction of cytokines and possibly other ligands with the B-cell membrane receptors provides progression signals. 254 PART II Generation of B-Cell and T-Cell Responses
B-Cell Generation. Activation and Differentiation CHAPTER 11 255 TABLE 11-2 Properties of thymus-dependent and thymus-independent antigens TI ANTIGENS Property TD antigen Type 2 Chemical nature Soluble protein Polymeric protein antigen capsular polysaccharides Humoral re Isotype switching Affinity maturation monologic memory Polyclonal activation No Yes(high doses) No Transduction of Activating signals Involves erodimer associates with a single mlg molecule to form the Ig-a/Ig-B Heterodimers receptor complex(Figure 11-7). Thus the BCR is function ally divided into the ligand-binding immunoglobulin mole For many years, immunologists questioned how engagement cule and the signal-transducing Ig-a/Ig-B heterodimer. A of the Ig receptor by antigen could activate intracellular sig- similar functional division marks the pre-BCR, which trans naling pathways. All isotypes of mlg have very short cyto- duces signals via a complex consisting of an Ig-a/Ig-B het- plasmic tails. Both mlgM and mlg D on B cells extend into the rodimer and u heavy chains combined with the surrogate cytoplasm by only three amino acids; the mIgA tail consists light chain(see Figure 11-4). The Ig-a chain has a long cyto- of 14 amino acids, and the mlgG and mIgE tails contains 28 plasmic tail containing 61 amino acids; the tail of the amino acids. In each case, the cytoplasmic tail is too short to chain contains 48 amino acids. The cytoplasmic tails of bot be able to generate a signal by associating with intracellular Ig-a and Ig-B contain the 18-residue motif termed the signaling molecules, such as tyrosine kinases and G proteins. immunoreceptor tyrosine-based activation motif (ITAM: The discovery that membrane Ig is associated with the disul- see Figure 11-7) which has already been described in several fide-linked heterodimer Ig-a/Ig-B, forming the B-cell recep- molecules of the T-cell-receptor complex(see Figure 10-11) tor(BCR), solved this longstanding puzzle. Though it was Interactions with the cytoplasmic tails of Ig-a/Ig-B trans originally thought that two Ig-a/Ig-B heterodimers associ- duce the stimulus produced by crosslinking of mlg molecules ated with one mlg to form the B-cell receptor, careful bio- into an effective intracellular signal chemical analysis has shown that only one Ig-a/Ig-B het- In the bCr and the tCR, as well as in a number of recep tors for the Fc regions of particular lg classes(fcERI for ige; FcyRIIA, FcyRIIC, FcyRIIIA for IgG), ligand binding and signal transduction are mediated by a multimeric complex of proteins that is functionally compartmentalized The ligand- (a)TI-l antigen (b) TD antigen binding portions of these complexes (mlg in the case of the BCR) is on the surface of the cell, and the signal-transducing TH cell portion is mostly or wholly within the cell. As is true of the TCR, signaling from the BCR is mediated by protein ty kinases(PTKs). Furthermore, like the TCR, the bCr itself has no PtK activity; this activity is acquired by recruitment of a number of different kinases, from nearby locations within CD4O/CD4OL the cell, to the cytoplasmic tails of the signal Phosphorylation B cell of tyrosines within the ITAMs of the BCr by receptor associ- ated PtKs is among the earliest events in B-cell activation GURE 11-6 An effective signal for B-cell activation involves two and plays a key role in bringing other critical PTKs to the distinct signals induced by membrane events. Binding of a type 1 BCR and in their activation. The antigen-mediated crosslink thymus-independent(TI-1)antigen to a B cell provides both signals. ing of BCRs initiates a number of signaling cascades that ulti A thymus-dependent(TD)antigen provides signal 1 by crosslinking mately result in the cells mlg, but a separate interaction between CD40 on the B cell and surface immunoglobulin by antigen. The crosslinking of BCRs CD40L on an activated TH cell is required to generate signal 2 results in the induction of many signal-transduction pathways
Transduction of Activating Signals Involves Ig-/Ig- Heterodimers For many years, immunologists questioned how engagement of the Ig receptor by antigen could activate intracellular signaling pathways. All isotypes of mIg have very short cytoplasmic tails. Both mIgM and mIgD on B cells extend into the cytoplasm by only three amino acids; the mIgA tail consists of 14 amino acids; and the mIgG and mIgE tails contains 28 amino acids. In each case, the cytoplasmic tail is too short to be able to generate a signal by associating with intracellular signaling molecules, such as tyrosine kinases and G proteins. The discovery that membrane Ig is associated with the disulfide-linked heterodimer Ig-/Ig-, forming the B-cell receptor(BCR), solved this longstanding puzzle. Though it was originally thought that two Ig-/Ig- heterodimers associated with one mIg to form the B-cell receptor, careful biochemical analysis has shown that only one Ig-/Ig- heterodimer associates with a single mIg molecule to form the receptor complex. (Figure 11-7). Thus the BCR is functionally divided into the ligand-binding immunoglobulin molecule and the signal-transducing Ig-/Ig- heterodimer. A similar functional division marks the pre-BCR, which transduces signals via a complex consisting of an Ig-/Ig- hetrodimer and heavy chains combined with the surrogate light chain (see Figure 11-4). The Ig- chain has a long cytoplasmic tail containing 61 amino acids; the tail of the Ig- chain contains 48 amino acids. The cytoplasmic tails of both Ig- and Ig- contain the 18-residue motif termed the immunoreceptor tyrosine-based activation motif (ITAM; see Figure 11-7) which has already been described in several molecules of the T-cell–receptor complex (see Figure 10-11). Interactions with the cytoplasmic tails of Ig-/Ig- transduce the stimulus produced by crosslinking of mIg molecules into an effective intracellular signal. In the BCR and the TCR, as well as in a number of receptors for the Fc regions of particular Ig classes (FcRI for IgE; FcRIIA, FcRIIC, FcRIIIA for IgG), ligand binding and signal transduction are mediated by a multimeric complex of proteins that is functionally compartmentalized. The ligandbinding portions of these complexes (mIg in the case of the BCR) is on the surface of the cell, and the signal-transducing portion is mostly or wholly within the cell. As is true of the TCR, signaling from the BCR is mediated by protein tyrosine kinases (PTKs). Furthermore, like the TCR, the BCR itself has no PTK activity; this activity is acquired by recruitment of a number of different kinases, from nearby locations within the cell, to the cytoplasmic tails of the signal. Phosphorylation of tyrosines within the ITAMs of the BCR by receptor associated PTKs is among the earliest events in B-cell activation and plays a key role in bringing other critical PTKs to the BCR and in their activation. The antigen-mediated crosslinking of BCRs initiates a number of signaling cascades that ultimately result in the cell’s responses to the crosslinking of its surface immunoglobulin by antigen. The crosslinking of BCRs results in the induction of many signal-transduction pathways B-Cell Generation, Activation, and Differentiation CHAPTER 11 255 TABLE 11-2 Properties of thymus-dependent and thymus-independent antigens TI ANTIGENS Property TD antigens Type 1 Type 2 Chemical nature Soluble protein Bacterial cell-wall Polymeric protein antigens; components (e.g., LPS) capsular polysaccharides Humoral response Isotype switching Yes No Limited Affinity maturation Yes No No Immunologic memory Yes No No Polyclonal activation No Yes (high doses) No 1 2 2 (a) TI-1 antigen (b) TD antigen B cell B cell CD40/CD40L TH cell 1 FIGURE 11-6 An effective signal for B-cell activation involves two distinct signals induced by membrane events. Binding of a type 1 thymus-independent (TI-1) antigen to a B cell provides both signals. A thymus-dependent (TD) antigen provides signal 1 by crosslinking mIg, but a separate interaction between CD40 on the B cell and CD40L on an activated TH cell is required to generate signal 2.
PART II Generation of B-Cell and T-Cell Responses Antige Resting B cell Crosslinked B-cell nembrane lg/g阝 马圆 ITAM D/EX, D/EX2Y X2LX,Y X2L ITAM sequence FIGURE. The initial stages of signal transduction by an activated enzymes phosphorylate tyrosine residues on the cytoplasmic tails of B-cell receptor(BCR). The BCR comprises an antigen-binding mlg the lg-a/lg-s heterodimer, creating docking sites for Syk kinase, which and one signal-transducing Ig-a/g-p heterodimer. Following antigen is then also activated. The highly conserved sequence motif of ITAMs is crosslinkage of the BCR, the immunoreceptor tyrosine-based activation shown with the tyrosines Y)in blue. D/E indicates that an aspartate or motifs(ITAMs) interact with several members of the Src family of tyro. a glutamate can appear at the indicated position, and X indicates that sine kinases(Fyn, Blk, and Lck), activating the kinases. The activated the position can be occupied by any amino acid and the activation of the B cell Figure 11-8 shows many paral of the second messengers IP3 and DAG. IP3 causes the lels between B-cell and T-cell activation These include release of ca+ from intracellular stores and dag activates PKC. a third important set of signaling Compartmentalization of function within receptor pathways are those governed by the small G proteins Ra subunits: Both the B-cell and T-cell pathways begin wit and Rac that are also activated by signals received antigen receptors that are composed of an antigen through the tCR or BCr binding and a signaling unit. The antigen-binding confers specificity, but has cytoplasmic tails too short to Changes in gene expression: One of the important transduce signals to the cytoplasm of the cell. The outcomes of signal-transduction processes set in motion signaling unit has long cytoplasmic tails that are the with engagement of the BCR or the TCR is the signal transducers of the receptor complex generation or translocation to the nucleus of active transcription factors that stimulate or inhibit the Activation by membrane-associated Src protein tyrosine transcription of specific genes kinases: The receptor-associated PTKs (Lck in T cells and Lyn, Blk, and Fyn in B cells) catalyze phosphorylations Failures in signal transduction can have severe conse- during the early stages of signal transduction that are quences for the immune system. The Clinical Focus on essential to the formation of a functional receptor X-linked agammaglobulinemia describes the effect of defec tive signal transduction on the development of B cells n Assembly of a large signaling complex with protein The B-Cell-Coreceptor Complex Can tyrosine-kinase activity: The phosphorylated tyrosines in Enhance B-Cell Responses he ITAMs of the BCR and TCR provide docking sites for he molecules that endow these receptors with PTK Stimulation through antigen receptors can be modified sig- activity, ZAP-70 in T cells and Syk in B cells nificantly by signals through coreceptors. Recall that co- stimulation through CD28 is an essential feature of effective Recruitment of other signal-transduction pathways: positive stimulation of T lymphocytes, while signaling Signals from the BCR and TCR result in the production through CTLA-4 inhibits the response of the T cell In b ce
and the activation of the B cell. Figure 11-8 shows many parallels between B-cell and T-cell activation. These include: ■ Compartmentalization of function within receptor subunits: Both the B-cell and T-cell pathways begin with antigen receptors that are composed of an antigenbinding and a signaling unit. The antigen-binding unit confers specificity, but has cytoplasmic tails too short to transduce signals to the cytoplasm of the cell. The signaling unit has long cytoplasmic tails that are the signal transducers of the receptor complex. ■ Activation by membrane-associated Src protein tyrosine kinases: The receptor-associated PTKs (Lck in T cells and Lyn, Blk, and Fyn in B cells) catalyze phosphorylations during the early stages of signal transduction that are essential to the formation of a functional receptor signaling complex. ■ Assembly of a large signaling complex with proteintyrosine-kinase activity: The phosphorylated tyrosines in the ITAMs of the BCR and TCR provide docking sites for the molecules that endow these receptors with PTK activity; ZAP-70 in T cells and Syk in B cells. ■ Recruitment of other signal-transduction pathways: Signals from the BCR and TCR result in the production of the second messengers IP3 and DAG. IP3 causes the release of Ca2+ from intracellular stores, and DAG activates PKC. A third important set of signaling pathways are those governed by the small G proteins Ras and Rac that are also activated by signals received through the TCR or BCR. ■ Changes in gene expression: One of the important outcomes of signal-transduction processes set in motion with engagement of the BCR or the TCR is the generation or translocation to the nucleus of active transcription factors that stimulate or inhibit the transcription of specific genes. Failures in signal transduction can have severe consequences for the immune system. The Clinical Focus on X-linked agammaglobulinemia describes the effect of defective signal transduction on the development of B cells. The B-Cell–Coreceptor Complex Can Enhance B-Cell Responses Stimulation through antigen receptors can be modified significantly by signals through coreceptors. Recall that costimulation through CD28 is an essential feature of effective positive stimulation of T lymphocytes, while signaling through CTLA-4 inhibits the response of the T cell. In B cells 256 PART II Generation of B-Cell and T-Cell Responses P P P Src Fyn Blk Lck Syk Kinases D/E X7 D/E X2Y X2L X7Y X2L ITAM sequence P P P ITAM Cytoplasm B-cell membrane Resting B cell Antigen Crosslinked B cell Ig-α/Ig-β P P FIGURE 11-7 The initial stages of signal transduction by an activated B-cell receptor (BCR). The BCR comprises an antigen-binding mIg and one signal-transducing Ig-/Ig- heterodimer. Following antigen crosslinkage of the BCR, the immunoreceptor tyrosine-based activation motifs (ITAMs) interact with several members of the Src family of tyrosine kinases (Fyn, Blk, and Lck), activating the kinases. The activated enzymes phosphorylate tyrosine residues on the cytoplasmic tails of the Ig-/Ig- heterodimer, creating docking sites for Syk kinase, which is then also activated. The highly conserved sequence motif of ITAMs is shown with the tyrosines (Y) in blue. D/E indicates that an aspartate or a glutamate can appear at the indicated position, and X indicates that the position can be occupied by any amino acid
