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.