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 sur￾rogate 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 membrane￾bound 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 recog￾nizes a not-yet-identified ligand on the stromal-cell mem￾brane, 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 estab￾lishment of an effective pre–B-cell receptor, each pre-B cell undergoes multiple cell divisions, producing 32 to 64 descen￾dants. 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 demon￾strated 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 neces￾sary 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 fac￾tors 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 com￾ponent 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 re￾gions of a number of B-cell–specific genes, including Vpre-B and 5, in a number of Ig switch regions, and in the Ig heavy￾chain enhancer. This indicates that BSAP plays a role beyond the early stages of B-cell development. This factor is also ex￾pressed in the central nervous system, and its absence results in severe defects in mid-brain development. Although the ex￾act 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 stromal￾cell 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 mem￾brane 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 im￾mature B cell no longer expresses the surrogate light chain and in￾stead expresses the or light chain together with the heavy chain.