Chapter 8: The Development of B and T Lymphocytes PDF

The Development of B and T Lymphocytes The production of new lymphocytes, or lymphopoiesis, takes place in specialized lymphoid tissues—the central (or primary) lymphoid tissues— which are the bone marrow for most B cells and the thymus for most T cells. Precursors for both populations originate in...

The Development of B and T Lymphocytes The production of new lymphocytes, or lymphopoiesis, takes place in specialized lymphoid tissues—the central (or primary) lymphoid tissues— which are the bone marrow for most B cells and the thymus for most T cells. Precursors for both populations originate in the bone marrow, but whereas B cells complete most of their development there, the precursors of most T cells migrate to the thymus, where they develop into mature T cells. A major goal of lymphopoiesis is to generate a diverse repertoire of B-cell receptors and T-cell receptors on circulating B and T cells, respectively, thereby enabling an individual to make adaptive immune responses against the wide range of pathogens encountered during a lifetime. In the fetus and the juvenile, the central lymphoid tissues are the sources of large numbers of new lymphocytes, which migrate to populate the peripheral lymphoid tissues (also called secondary lymphoid tissues) such as lymph nodes, spleen, and mucosal lymphoid tissue. In mature individuals, the development of new T cells in the thymus slows down, and peripheral T-cell numbers are maintained by the division of mature T cells outside the central lymphoid organs. New B cells, in contrast, are continually produced from the bone marrow, even in adults. This chapter will focus on the development of T cells and B cells from their uncommitted progenitors, with an emphasis on the major populations of CD4+ and CD8+ T cells and B cells. The development of additional subsets of T cells and B cells, such as invariant NKT (iNKT) cells, Treg cells, γ:δ TCR+ T cells, B-1 B cells, and marginal zone B cells will be briefly discussed. The structure of the antigen-receptor genes expressed by B cells and T cells, and the mechanisms by which a complete antigen receptor is assembled, were described in Chapters 4 and 5. Once an antigen receptor has been formed, rigorous testing is required to select lymphocytes that carry useful antigen receptors—that is, antigen receptors that can recognize a wide spectrum of pathogens and yet will not react against an individual’s own cells. Given the incredible diversity of receptors that the rearrangement process can generate, it is important that those lymphocytes that mature are likely to be useful in recognizing and responding to foreign antigens, especially as an individual can express only a small fraction of the total possible receptor repertoire in his or her lifetime. We describe how the specificity and affinity of the receptor for self ligands are tested to determine whether the immature lymphocyte will either survive and join the mature repertoire, or die. In general, it seems that developing lymphocytes whose receptors interact weakly with self antigens, or that bind self antigens in a particular way, receive a signal that enables them to survive. This process, known as positive selection, is particularly critical in the development of α:β T cells, which recognize composite antigens consisting of peptides bound to MHC molecules, because it ensures that an individual’s T cells will be able to respond to peptides bound to one’s own MHC molecules. In contrast, lymphocytes with strongly self-reactive receptors must be eliminated to prevent autoimmune reactions; this process of negative selection is one of the ways in which the immune system is made self-tolerant. The default fate of developing lymphocytes, in the absence of any signal being received from the receptor, is death by apoptosis, and as we will see, the vast ERRNVPHGLFRVRUJ 8 IN THIS CHAPTER Development of B lymphocytes. Development of T lymphocytes. Positive and negative selection of T cells. 295 296 Chapter 8: The Development of B and T lymphocytes majority of developing lymphocytes die before emerging from the central lymphoid organs or before completing maturation in the peripheral lymphoid organs. In this chapter we describe the different stages of the development of B cells and T cells in mice and humans from the uncommitted stem cell in the bone marrow up to the mature, functionally specialized lymphocyte with its unique antigen receptor ready to respond to a foreign antigen. The final stages in the life history of a mature lymphocyte, in which an encounter with its antigen activates it to become an effector or memory lymphocyte, are discussed in Chapters 9–11. We now know that the B- and T-cell development that predominates during late fetal life and after birth is distinct from waves of lymphocyte development that take place earlier in fetal ontogeny. These earlier waves originate from stem cells found in the fetal liver and in even more primitive hematopoietic tissues in the developing embryo. Unlike the lymphocytes that develop from bone marrow stem cells, B and T cells that develop from these early fetal progenitors generally populate mucosal and epithelial tissues and function in innate immune responses. In the adult, these subsets of lymphocytes are minority populations in secondary lymphoid tissues. This chapter will focus on B and T cells that develop from bone marrow stem cells and that comprise the cells of the adaptive immune response (see Figs 1.7 and 1.20). The chapter is divided into three parts. The first two describe B-cell and T-cell development, respectively. In the third section, we discuss the positive and negative selection of T cells in the thymus. Fig. 8.1 B cells develop in the bone marrow and migrate to peripheral lymphoid organs, where they can be activated by antigens. In the first phase of development, progenitor B cells in the bone marrow rearrange their immunoglobulin genes. This phase is independent of antigen but is dependent on interactions with bone marrow stromal cells (first panels). It ends in an immature B cell that carries an antigen receptor in the form of cell-surface IgM (second panels), and in the second phase it can now interact with antigens in its environment. Immature B cells that are strongly stimulated by antigen at this stage either die or are inactivated in a process of negative selection, thus removing many self-reactive B cells from the repertoire. In the third phase of development, the surviving immature B cells emerge into the periphery and mature to express IgD as well as IgM. They can now be activated by encounter with their specific foreign antigen in a peripheral lymphoid organ (third panels). Activated B cells proliferate, and differentiate into antibody-secreting plasma cells and long-lived memory cells (fourth panels). B-cell precursor rearranges its immunoglobulin genes Development of B lymphocytes. The main phases of a B lymphocyte’s life history are shown in Fig. 8.1. The stages in both B-cell and T-cell development are defined mainly by the successive steps in the assembly and expression of functional antigen-receptor genes. Immature B cell bound to self cell-surface antigen is removed from the repertoire Mature B cell bound to foreign antigen is activated bone marrow cell Activated B cells give rise to plasma cells and memory cells plasma cell B-cell precursor multivalent foreign antigen self antigen IgM cytokines IgD memory cell bone marrow stromal cell gastrointestinal tract bone marrow heart Generation of B-cell receptors in the bone marrow Negative selection in the bone marrow Migration of B cells through the circulatory system to lymphoid organs and B-cell activation Immunobiology | chapter 8 | 08_001 Murphy et al | Ninth edition © Garland Science design by blink studio limited ERRNVPHGLFRVRUJ Antibody secretion and memory cells in bone marrow and lymphoid tissue Development of B lymphocytes. At each step of lymphocyte development, the progress of gene rearrangement is monitored; the major recurring theme is that successful gene rearrangement leads to the production of a protein chain that serves as a signal for the cell to progress to the next stage. We will see that a developing B cell is presented with opportunities for multiple rearrangements that increase the likelihood of expressing a functional antigen receptor, but that there are also checkpoints that reinforce the requirement that each B cell express just one receptor specificity. We will start by looking at how the earliest recognizable cells of the B-cell lineage develop from the multipotent hematopoietic stem cells in the bone marrow, and at what point the B-cell and T-cell lineages diverge. 8-1 Lymphocytes derive from hematopoietic stem cells in the bone marrow. The cells of the lymphoid lineage—B cells, T cells, and innate lymphoid cells (ILCs)—are all derived from common lymphoid progenitor cells, which themselves derive from the multipotent hematopoietic stem cells (HSCs) that give rise to all blood cells (see Fig. 1.3). Development from the precursor stem cell into cells that are committed to becoming B cells or T cells follows the basic principles of cell differentiation. Properties that are essential for the function of the mature cell are gradually acquired, along with the loss of properties that are more characteristic of the immature cell. In the case of lymphocyte development, cells become committed first to the lymphoid lineage, as opposed to the myeloid, and then to either the B-cell or the T-cell lineage (Fig. 8.2). The specialized microenvironment of the bone marrow provides signals both for the development of lymphocyte progenitors from hematopoietic stem cells and for the subsequent differentiation of B cells. Such signals act on the developing lymphocytes to switch on key genes that direct the developmental program and are produced by the network of specialized nonlymphoid connective tissue stromal cells that are in intimate contact with the developing lymphocytes (Fig. 8.3). The contribution of the stromal cells is twofold. First, they form specific adhesive contacts with the developing lymphocytes by interactions between cell-adhesion molecules and their ligands. Second, they provide soluble and membrane-bound cytokines and chemokines that control lymphocyte differentiation and proliferation. The hematopoietic stem cells first differentiate into multipotent progenitor cells (MPPs), which can produce both lymphoid and myeloid cells but are no longer self-renewing stem cells. Multipotent progenitors express a cell-surface receptor tyrosine kinase known as FLT3 that binds the membrane-bound FLT3 ligand on stromal cells. Additionally, MPPs express transcription factors and Fig. 8.2 A multipotent hematopoietic stem cell generates all the cells of the immune system. In the bone marrow or other hematopoietic sites, the multipotent stem cell gives rise to cells with progressively more limited potential. A simplified progression is shown here. The multipotent progenitor (MPP), for example, has lost its stem-cell properties. The first branch leads to cells with myeloid and erythroid potential, on the one hand (CMPs and MEPs), and, on the other, to the common lymphoid progenitors (CLPs), with lymphoid potential. The former give rise to all nonlymphoid cellular blood elements, including circulating monocytes and granulocytes, as well as the macrophages and dendritic cells that reside in tissues and peripheral lymphoid organs (not shown). The CLP population is heterogeneous and single cells can give rise to NK cells, T cells, or B cells through successive stages of differentiation in either the bone marrow or thymus. There may be considerable plasticity in these pathways, in that in certain circumstances progenitor cells may switch their commitment. For example, a progenitor cell may give rise to either B cells or macrophages; however, for simplicity these alternative pathways are not shown. Some dendritic cells are also thought to be derived from the lymphoid progenitor. Hematopoietic stem cell HSC Multipotent progenitor MPP Common granulocyte/ megakaryocyte/ erythrocyte progenitor CMP/MEP Further development to mature blood cells Common lymphoid progenitor CLP Pre-NK cell Pre-B cell Thymocyte NK cell B cell T cell Immunobiology | chapter 8 | 08_002 Murphy et al | Ninth edition ERRNVPHGLFRVRUJ © Garland Science design by blink studio limited 297 298 Chapter 8: The Development of B and T lymphocytes Multipotent progenitor cell Common lymphoid progenitor Early pro-B cell Late pro-B cell Pre-B cell Immature B cell FLT3 IL-7 receptor FLT3 ligand CAMs CXCL12 CAMs IL-7 VLA-4 VCAM-1 lgM Kit SCF bone marrow stromal cell Ikaros PU.1 E2A, EBF, Pax5/BSAP Immunobiology | chapter 8 | 08_003 Murphy8.3 et alThe | Ninth editionstages of B-cell development are dependent Fig. early Science design by blink studio limited © onGarland bone marrow stromal cells. Interaction of B-cell progenitors with bone marrow stromal cells is required for development to the immature B-cell stage. The designations pro-B cell and pre-B cell refer to defined phases of B-cell development, as described in Fig. 8.4. Multipotent progenitor cells express the receptor tyrosine kinase FLT3, which binds to its ligand on stromal cells. Signaling through FLT3 is required for differentiation to the next stage, the common lymphoid progenitor. The chemokine CXCL12 (SDF-1) acts to retain stem cells and lymphoid progenitors at appropriate stromal cells in the bone marrow. The receptor for interleukin-7 (IL-7) is present at this stage, and IL-7 produced by stromal cells is required for the development of B-lineage cells. Progenitor cells bind to the adhesion molecule VCAM-1 on stromal cells through the integrin VLA-4 and also interact through other cell-adhesion molecules (CAMs). The adhesive interactions promote the binding of the receptor tyrosine kinase Kit (CD117) on the surface of the pro-B cell to stem-cell factor (SCF) on the stromal cell, which activates the kinase and induces the proliferation of B-cell progenitors. The actions of the listed transcription factors in B-cell development are discussed in the text. The pink horizontal bands denote the expression of particular proteins at the indicated stages of development. receptors that are required for the development of multiple hematopoietic lineages, such as the transcription factor PU.1 and the receptor c-kit. In the next stage, MPPs produce two subsets of progenitor cells that give rise to all the lymphocyte lineages. One progenitor cell, as yet unnamed, produces the ILC subsets, ILC1, ILC2, and ILC3 cells. A second progenitor cell arising from the MPP is known as the common lymphoid progenitor (CLP). Differentiation of MPPs into CLPs requires signaling through the FLT3 receptor expressed on MPPs. Progenitor cell transfer and lineage repopulation experiments have shown that the CLP population is actually heterogeneous and represents a continuum of cells with decreasing multipotent potential. A subset of CLP cells with the broadest potential is able to generate B cells, T cells, and NK cells. A second subset of CLPs is able to generate only B cells and T cells, and a third subset of CLPs is committed exclusively to the B-cell lineage. B-cell-committed CLPs give rise to pro-B cells (see Fig. 8.3). The production of lymphocyte progenitors from the multipotent progenitor cell is accompanied by expression of the receptor for interleukin-7 (IL-7), which is induced by FLT3 signaling together with the activity of PU.1. The cytokine IL-7, secreted by bone marrow stromal cells, is essential for the growth and survival of developing B cells in mice (but possibly not in humans). The IL-7 receptor is composed of two polypeptides, the IL-7 receptor α chain and the common cytokine receptor γ chain (γ-c), so called because it is also a subunit of five additional cytokine receptors. This family of cytokine receptors includes the receptors for IL-2, IL-4, IL-9, IL-15, and IL-21, in addition to IL-7. These receptors also share the tyrosine kinase Jak3, a signaling protein that binds exclusively to γ-c and is required for productive signaling by each of the receptors. ERRNVPHGLFRVRUJ Development of B lymphocytes. Due to the importance of IL-7 for murine B-cell development, mice with a genetic deficiency in IL-7, IL-7 receptor α, γ-c, or Jak3 all exhibit a severe block in B-cell development. Another essential factor for B-cell development is stem-cell factor (SCF), a membrane-bound cytokine present on bone marrow stromal cells that stimulates the growth of hematopoietic stem cells and the earliest B-lineage progenitors. SCF interacts with the receptor tyrosine kinase Kit on the precursor cells (see Fig. 8.3). The chemokine CXCL12 (stromal cell-derived factor 1, SDF‑1) is also essential for the early stages of B-cell development. It is produced constitutively by bone marrow stromal cells, and one of its roles may be to retain developing B-cell precursors in the marrow microenvironment. Thymic stroma-derived lymphopoietin (TSLP) resembles IL-7 and binds a receptor that includes the IL-7 receptor α chain, but not γ-c. Despite its name, TSLP may promote B-cell development in the embryonic liver and, in the peri­ natal period at least, in the mouse bone marrow. A definitive B-cell stage, the pro-B cell, is specified by induction of the B-lineage-specific transcription factor E2A. It is not clear what initiates the expression of E2A in some progenitors, but it is known that the transcription factors PU.1 and Ikaros are required for E2A expression. E2A then induces the expression of the early B-cell factor (EBF). IL-7 signaling promotes the survival of these committed progenitors, while E2A and EBF act together to drive the expression of proteins that determine the pro-B-cell state. As B-lineage cells mature, they migrate within the marrow, remaining in contact with the stromal cells. The earliest stem cells lie in a region called the endosteum, which lines the inner cavity of the long bones such as the femur and tibia. Developing B-lineage cells make contact with reticular stromal cells in the trabecular spaces, and as they mature they move toward the central sinus of the marrow cavity. The final stages of development of immature B cells into mature B cells occur in peripheral lymphoid organs such as the spleen, which we describe in Sections 8-7 and 8-8 of this chapter. 8-2 B-cell development begins by rearrangement of the heavychain locus. The stages of B-cell development are, in the order they occur, early pro-B cell, late pro-B cell, large pre-B cell, small pre-B cell, immature B cell, and mature B cell (Fig. 8.4). Rearrangement of the heavy-chain locus is initiated in the pro-B cell when E2A and EBF induce the expression of several key proteins that enable gene rearrangement to occur, including the RAG-1 and RAG-2 components of the V(D)J recombinase (see Chapter 5). Only one gene locus is rearranged at a time, in a fixed sequence. The first rearrangement to take place is the joining of a D gene segment to a J segment at the immunoglobulin heavy-chain (IgH) locus. D to JH rearrangement takes place mostly in the early pro-B-cell stage, but can be seen as early as the common lymphoid progenitor. In the absence of E2A or EBF this initial rearrangement event fails to occur. Another key protein induced by E2A and EBF is the transcription factor Pax5, one isoform of which is known as the B-cell activator protein (BSAP) (see Fig. 8.3). Among the targets of Pax5 are the gene for the B-cell co-receptor component CD19 and the gene for Igα, a signaling component of both the pre-B-cell receptor and the B-cell receptor (see Section 7-7). In the absence of Pax5, pro-B cells fail to develop further down the B-cell pathway but can be induced to give rise to T cells and myeloid cell types, indicating that Pax5 is required for commitment of the pro-B cell to the B-cell lineage. Pax5 also induces the expression of the B-cell linker protein (BLNK), an SH2-containing scaffold protein that is required for further development of the pro-B cell and for signaling from the mature B-cell antigen receptor ERRNVPHGLFRVRUJ X-linked Severe Combined Immunodeficiency 299 300 Chapter 8: The Development of B and T lymphocytes Stem cell Early pro-B cell Late pro-B cell Large pre-B cell Small pre-B cell pre-B receptor lgD lgM Germline D–J rearranging V–DJ rearranging VDJ rearranged VDJ rearranged VDJ rearranged VDJ rearranged L-chain genes Germline Germline Germline Germline V –J rearranging VJ rearranged VJ rearranged Absent μ chain transiently at surface as part of pre-B-cell receptor. Mainly intracellular Intracellular μ chain IgM expressed on cell surface IgD and IgM made from alternatively spliced H-chain transcripts Protein Function RAG-1 RAG-2 Lymphoidspecific recombinase TdT N-nucleotide addition VpreB lgM Mature B cell H-chain genes Surface Ig λ5 Immature B cell Absent Absent Surrogate light-chain components Igα Igβ CD45R Signal transduction Btk CD19 Kit IL-7R CD43 Growth factor receptor Unknown CD24 BP-1 Aminopeptidase Fig. 8.4 The| development Immunobiology chapter 8 | 08_004 of a B-lineage cell proceeds through Murphy et alstages | Ninth edition several marked by the rearrangement and expression design by blink studiogenes. limited © ofGarland the Science immunoglobulin The stem cell has not yet begun to rearrange its immunoglobulin (Ig) gene segments; they are in the germline configuration found in all nonlymphoid cells. The heavychain (H-chain) locus rearranges first. Rearrangement of a D gene segment to a JH gene segment starts in the common lymphoid progenitor and occurs mostly in early pro-B cells, generating late pro-B cells in which VH to DJH rearrangement occurs. A successful VDJH rearrangement leads to the expression of a complete immunoglobulin heavy chain as part of the pre-B-cell receptor, which signals via Igα, Igβ, and Btk (see Fig 7.27). Once this occurs, the cell is stimulated to become a large pre-B cell, which proliferates to become small resting pre-B cells; at this point the cells cease expression of the surrogate light chains (λ5 and VpreB) and express the μ heavy chain alone in the cytoplasm. Small pre-B cells reexpress the RAG proteins and start to rearrange the light-chain (L-chain) genes. Upon successfully assembling a light-chain gene, a cell becomes an immature B cell that expresses a complete IgM molecule at the cell surface, which also signals via Igα and Igβ. Mature B cells produce a δ heavy chain as well as a μ heavy chain, by a mechanism of alternative mRNA splicing (see Fig. 5.17), and are marked by the additional appearance of IgD on the cell surface. All stages through the development of immature B cells takes place in the bone marrow; the final maturation to IgM+IgD+ mature B cells occurs in the spleen. The earliest B-lineage surface markers are CD19 and CD45R (B220 in the mouse), which are expressed throughout B-cell development. A pro-B cell is also distinguished by the expression of CD43 (a marker of unknown function), Kit (CD117), and the IL-7 receptor. A late pro-B cell starts to express CD24 (a marker of unknown function). A pre-B cell is phenotypically distinguished by the expression of the enzyme BP-1, whereas Kit is no longer expressed. ERRNVPHGLFRVRUJ Development of B lymphocytes. (see Section 7-20). The temporal expressions of some of the transcription factors, surface proteins, and receptors required for B-cell development are listed in Fig. 8.3 and Fig. 8.4. Although the V(D)J recombinase system operates in both B- and T-lineage cells and uses the same core enzymes, rearrangements of T-cell receptor genes do not occur in B-lineage cells, nor do complete rearrangements of immunoglobulin genes occur in T cells. The ordered rearrangement events that do occur are associated with lineage-specific low-level transcription of the gene segments about to be joined. The initial D to JH rearrangements in the immunoglobulin heavy-chain locus (Fig. 8.5) typically occur on both alleles, at which point the cell becomes a late pro-B cell. Most D to JH joins in humans are potentially useful, because most human D gene segments can be translated in all three reading frames without encountering a stop codon. Thus, there is no need for a special mechanism to distinguish successful D to JH joints, and at this early stage there is also no need to ensure that only one allele undergoes rearrangement. Indeed, given the likely rate of failure at later stages, starting off with two successfully rearranged D–JH sequences may be an advantage. Genes Proteins Cells Early pro-B cell VH DJH Cμ VL JL CL No functional μ protein expressed VH to DJH rearrangements occur in late pro-B cells Large pre-B cell Cμ VDJH VL JL CL pre-B receptor Cμ VH Igβ Igα VpreB λ5 surrogate light chain Stop heavy-chain gene rearrangement; progression to light-chain gene rearrangement in small pre-B cells Immature B cell VDJH Cμ VJL CL VL IgM Cμ VH Igβ Igα CL Stop light-chain gene rearrangement Mature B cell Immunobiology | chapter 8 | 08_006 Murphy et al | Ninth edition © Garland Science design by blink studio limited ERRNVPHGLFRVRUJ Fig. 8.5 A productively rearranged immunoglobulin gene is immediately expressed as a protein by the developing B cell. In early pro-B cells, heavy-chain gene rearrangement is initiated with D to JH rearrangements. As shown in the top panels, no functional μ protein is expressed, although transcription occurs (red arrow). In late pro-B cells, VH to DJH rearrangement occurs on one chromosome first. If no functional H-chain is produced, VH to DJH rearrangement occurs on the second chromosome. As soon as a productive heavy-chain gene rearrangement takes place, μ chains are expressed by the cell in a complex with two other chains, λ5 and VpreB, which together make up a surrogate light chain. The whole immunoglobulin-like complex is known as the pre-B-cell receptor (center panels). It is associated with two other protein chains, Igα and Igβ, which signal the B cell to halt heavy-chain gene rearrangement; this drives the transition to the large pre-B-cell stage by inducing proliferation. Failure to produce a functional H-chain leading to a pre-B-cell receptor signal leads to cell death. The progeny of large pre-B cells stop dividing and become small pre-B cells, in which light-chain gene rearrangements commence. Vκ–Jκ rearrangement (see Section 5-2) occurs first, and if unsuccessful, Vλ to Jλ rearrangement occurs next. Successful light-chain gene rearrangement results in the production of a light chain that binds the μ chain to form a complete IgM molecule, which is expressed together with Igα and Igβ at the cell surface, as shown in the bottom panels. Signaling via this surface receptor complex is thought to trigger the cessation of light-chain gene rearrangement. Failure to produce a functional L chain leads to cell death. 301 302 Chapter 8: The Development of B and T lymphocytes To produce a complete immunoglobulin heavy chain, the late pro-B cell now proceeds with a rearrangement of a VH gene segment to a DJH sequence. In contrast to D to JH rearrangement, VH to DJH rearrangement occurs first on only one chromosome. A successful rearrangement leads to the production of intact μ heavy chains, after which VH to DJH rearrangement ceases and the cell becomes a pre-B cell. Pro-B cells that do not produce a μ chain are eliminated, as they fail to receive an important survival signal mediated by the pre-B-cell receptor (see Section 8-3). At least 45% of pro-B cells are lost at this stage. In at least two out of three cases, the first VH to DJH rearrangement is nonproductive as each amino acid is encoded by a triplet of nucleotides. When this initial rearrangement is out of frame, rearrangement then occurs on the other chromosome, again with a theoretical two in three chance of failure. A rough estimate of the chance of generating a pre-B cell is thus 55% [1/3 + (2/3 × 1/3) = 0.55]. The actual frequency is somewhat lower, because the V gene segment repertoire contains pseudogenes that can rearrange yet have major defects that prevent the expression of a functional protein. An initial nonproductive rearrangement does not automatically lead to pro-B cell elimination, as it is possible for most loci to undergo successive rearrangements on the same chromosome, and where that fails, the locus on the other chromosome will rearrange. The diversity of the B-cell antigen-receptor repertoire is enhanced at this stage by the enzyme terminal deoxynucleotidyl transferase (TdT). TdT is expressed by the pro-B cell and adds nontemplated nucleotides (N-nucleotides) at the joints between rearranged gene segments (see Section 5-8). In adult humans, it is expressed in pro-B cells during heavy-chain gene rearrangement, but its expression declines at the pre-B-cell stage during light-chain gene rearrangement. This explains why N-nucleotides are found in the V–D and D–J joints of nearly all heavy-chain genes but only in about a quarter of human light-chain joints. N-nucleotides are rarely found in mouse light-chain V–J joints, showing that TdT is switched off slightly earlier in the development of mouse B cells. In fetal development, when the peripheral immune system is first being supplied with T and B lymphocytes, TdT is expressed only at low levels, if at all. 8-3 The pre-B-cell receptor tests for successful production of a complete heavy chain and signals for the transition from the pro-B cell to the pre-B cell stage. The imprecise nature of V(D)J recombination is a double-edged sword. Although it produces increased diversity in the antibody repertoire, it also results in many unsuccessful rearrangements. Pro-B cells therefore need a way of testing whether a potentially functional heavy chain has been produced. They do this by incorporating a functional heavy chain into a receptor that can signal its successful production. This test takes place in the absence of light chains, whose loci have not yet rearranged. Instead, pro-B cells make two invariant ‘surrogate’ proteins that together have a structural resemblance to the light chain and can pair with the μ chain to form the pre-B-cell receptor (pre-BCR) (see Fig. 8.5). The assembly of a pre-B-cell receptor signals to the B cell that a productive rearrangement has been made, and the cell is then considered a pre-B cell. The surrogate chains are encoded by nonrearranging genes separate from the antigen-receptor loci, and their expression is induced by E2A and EBF (see Fig. 8.4). One is called λ5 because of its close resemblance to the C domain of the λ light chain; the other, called VpreB, resembles a light-chain V domain but has an extra region at the amino-terminal end. Pro-B cells and pre-B cells also express the invariant proteins Igα and Igβ, introduced in Chapter 7 as the signaling components of the B-cell receptor complex on mature B cells. As components of the pre-B-cell receptor, Igα and Igβ transduce signals by interacting with intracellular tyrosine kinases through their cytoplasmic tails, just ERRNVPHGLFRVRUJ Development of B lymphocytes. Amino-terminal tails on VpreB and λ5 in adjacent pre-B-cell receptor molecules bind each other and cross-link the receptors, inducing clustering and signaling unique amino termini of VpreB and λ5 VpreB λ5 heavy chain cell membrane ITAMs Igβ Igα Immunobiology | chapter 8 | 08_007 Murphy et al | Ninth edition as they function to transduce signals through the antigen receptor on mature B cells (see Section 7-7). © Garland Science design by blink studio limited Formation of the pre-B-cell receptor and signaling through this receptor provide an important checkpoint that mediates the transition between the pro-B cell and the pre-B cell. In mice that either lack λ5 or have mutant heavy-chain genes that cannot produce the transmembrane domain, the pre-B-cell receptor cannot be formed and B-cell development is blocked after heavy-chain gene rearrangement. In normal B-cell development, the pre-B-cell receptor complex is expressed transiently, perhaps because the production of λ5 mRNA stops as soon as pre-B-cell receptors begin to be formed. Although present at only low levels on the cell surface, the pre-B-cell receptor generates signals required for the transition from pro-B cell to pre-B cell. No antigen or other external ligand seems to be involved in signaling by the receptor. Instead, pre-B-cell receptors are thought to interact with each other, forming dimers or oligomers that generate signals as described in Section 7-16. Dimerization involves ‘unique’ regions in the amino termini of λ5 and VpreB proteins that are not present in other immunoglobulin-like domains and which mediate the cross-linking of adjacent pre-B-cell receptors on the cell surface (Fig. 8.6). PreB-cell receptor signaling requires the scaffold protein BLNK and Bruton’s tyrosine kinase (Btk), an intracellular Tec-family tyrosine kinase (see Section 7-20). In humans and mice, deficiency of BLNK leads to a block in B-cell development at the pro-B-cell stage. In humans, mutations in the BTK gene cause a profound B-lineage-specific immune deficiency, Bruton’s X-linked agamma­ globulinemia (XLA), in which no mature B cells are produced. The block in B-cell development caused by mutations in BTK is almost total, interrupting the transition from pre-B cell to immature B cell. A similar, but less severe, defect called X-linked immunodeficiency, or xid, arises from mutations in the Btk gene in mice. 8-4 Pre-B-cell receptor signaling inhibits further heavy-chain locus rearrangement and enforces allelic exclusion. The signaling generated by pre-B-cell receptor clustering halts further rearrangement of the heavy-chain locus and allows the pro-B cell to become sensitive to IL-7. This induces cell proliferation, initiating the transition to the large ERRNVPHGLFRVRUJ Fig. 8.6 The pre-B-cell receptor initiates signaling through spontaneous dimerization induced by the unique regions of VpreB and λ5. Two surrogate protein chains, VpreB (orange) and λ5 (green), substitute for a light chain and bind to a heavy chain, thus allowing its surface expression. VpreB substitutes for the light-chain V region in this surrogate interaction, while λ5 takes the part of the light-chain constant region. Both VpreB and λ5 contain ‘unique’ amino-terminal regions that are not present in other immunoglobulin-like domains, shown here as unstructured tails extending out from the globular domains. These amino-terminal regions associated with one pre-B-cell receptor can interact with the corresponding regions on the adjacent pre-B-cell receptor, promoting the spontaneous formation of pre-Bcell receptor dimers on the cell surface. Dimerization generates signaling from the pre-B-cell receptor that is dependent on the presence of the ITAM-containing signaling chains Igα and Igβ. The signals cause the inhibition of RAG-1 and RAG-2 expression and the proliferation of the large pre-B cell. Courtesy of Chris Garcia. X-linked Agammaglobulinemia 303 304 Chapter 8: The Development of B and T lymphocytes Igh a/a Igh b/b Igh a/b pre-B cell. Successful rearrangements at both heavy-chain alleles could result in a B cell producing two receptors of different antigen specificities. To prevent this, signaling by the pre-B-cell receptor enforces allelic exclusion, the state in which only one of the two alleles of a gene is expressed in a diploid cell. Allelic exclusion, which occurs at both the heavy-chain locus and the light-chain loci, was discovered nearly 50 years ago and provided one of the original pieces of experimental support for the theory that one lymphocyte expresses one type of antigen receptor (Fig. 8.7). Signaling from the pre-B-cell receptor promotes heavy-chain allelic exclusion in three ways. First, it reduces V(D)J recombinase activity by directly reducing the expression of the RAG-1 and RAG-2 genes. Second, it further reduces levels of RAG-2 by indirectly causing this protein to be targeted for degradation, which occurs when RAG-2 is phosphorylated in response to the entry of the pro-B cell into S phase (the DNA synthesis phase) of the cell cycle. Finally, preB-cell receptor signaling reduces access of the heavy-chain locus to the recombinase machinery, although the precise details of this are not clear. At a later stage of B-cell development, RAG proteins will again be expressed in order to carry out light-chain locus rearrangement, but at that point the heavy-chain locus does not undergo further rearrangement. In the absence of pre-B-cell receptor signaling, allelic exclusion of the heavy-chain locus does not occur. Since a second important role of pre-B-cell receptor signaling is to stimulate proliferative expansion of B-cell precursors with a successful heavy-chain rearrangement, a deficiency in this signal causes a profound reduction in the numbers of pre-B cells and mature B cells that develop. 8-5 Immunobiology | chapter 8 | 08_008 Murphy8.7 et alAllelic | Ninth edition Fig. exclusion in individual © Garland Science design by blink studio limited B cells. Most species have genetic polymorphisms of the constant regions of their immunoglobulin heavy-chain and light-chain genes; these polymorphisms lead to amino acid differences between the encoded proteins. These variants of heavychain or light-chain proteins expressed by different individuals in a species are known as allotypes. In rabbits, for example, all of the B cells in an individual homozygous for the a allele of the immunoglobulin heavy-chain locus (Igha/a) will express immunoglobulin of allotype a, whereas in an individual homozygous for the b allele (Ighb/b) all the B cells make immunoglobulin of allotype b. In a heterozygous animal (Igha/b), which carries the a allele at one of the Igh loci and the b allele at the other, individual B cells can be shown to express surface immunoglobulin of either the a-allotype or the b-allotype, but not both (bottom panel). This allelic exclusion reflects the productive rearrangement of only one of the two Igh alleles in the B cell, because the production of a successfully rearranged immunoglobulin heavy chain forms a pre-Bcell receptor, which signals the cessation of further heavy-chain gene rearrangement. Pre-B cells rearrange the light-chain locus and express cellsurface immunoglobulin. The transition from the pro-B-cell to the large pre-B-cell stage is accompanied by several rounds of cell division, expanding the population of cells with successful in-frame joins by about 30- to 60-fold before they become resting small pre-B cells. A large pre-B cell with a particular rearranged heavy-chain gene therefore gives rise to numerous small pre-B cells. RAG proteins are produced again in the small pre-B cells, and rearrangement of the light-chain locus begins. Each of these cells can make a different rearranged light-chain gene, and so cells with many different antigen specificities are generated from a single pre-B cell, which makes an important contribution to overall B-cell receptor diversity. Light-chain rearrangement also exhibits allelic exclusion. Rearrangements at the light-chain locus generally take place at only one allele at a time, a process regulated by a mechanism not currently understood. The lightchain loci lack D segments, and rearrangement occurs by V to J joining; and if a particular VJ rearrangement fails to produce a functional light chain, repeated rearrangements of unused V and J gene segments at the same allele can occur (Fig. 8.8). Several attempts at productive rearrangement of a lightchain gene can therefore be made on one chromosome before initiating any rearrangements on the second chromosome. This greatly increases the chances of eventually generating an intact light chain, especially as there are two different light-chain loci. As a result, many cells that reach the preB-cell stage succeed in generating progeny that bear intact IgM molecules and can be classified as immature B cells. Figure 8.4 lists some of the proteins involved in V(D)J recombination and shows how their expression is regulated throughout B-cell development. Figure 8.5 summarizes the stages of B-cell development up to the point of assembly of a complete surface immunoglobulin. Developing B cells that fail to assemble a complete surface immunoglobulin undergo apoptosis in the bone marrow, and are eliminated from the B-cell pool. ERRNVPHGLFRVRUJ Development of B lymphocytes. Repeated rearrangements are possible at the light-chain loci Vκn Vκ2 Vκ1 Cκ Jκ1–5 first VJ recombination Vκn Vκ2 Vκ1 Jκ Jκ Jκ Jκ Cκ nonproductive join second VJ recombination Vκn Vκ2 Cκ Jκ Jκ nonproductive join third VJ recombination Vκn Jκ Cκ Immunobiology | chapter 8 | 08_009 As well allelic Murphy et al as | Ninth edition exclusion, light chains also display isotypic exclusion, that design by blinkof studio limited one type of light chain—κ or λ—by an individual B is, theScience expression only © Garland cell. Again, the mechanism regulating this process is not known. In mice and humans, the κ light-chain locus tends to rearrange before the λ locus. This was first deduced from the observation that myeloma cells secreting λ light chains generally have both their κ and λ light-chain genes rearranged, whereas in myelomas secreting κ light chains, generally only the κ genes are rearranged. This order is occasionally reversed, however, and λ gene rearrangement does not absolutely require the previous rearrangement of the κ genes. The ratios of κ-expressing versus λ-expressing mature B cells vary from one extreme to the other in different species. In mice and rats it is 95% κ to 5% λ, in humans it is typically 65%:35%, and in cats it is 5%:95%, the opposite of that in mice. These ratios correlate most strongly with the number of functional Vκ and Vλ gene segments in the genome of the species. They also reflect the kinetics and efficiency of gene segment rearrangements. The κ:λ ratio in the mature lymphocyte population is useful in clinical diagnostics, because an aberrant κ:λ ratio indicates the dominance of one clone and the presence of a lymphoproliferative disorder. 8-6 Immature B cells are tested for autoreactivity before they leave the bone marrow. Once a rearranged light chain has paired with a μ chain, IgM can be expressed on the cell surface (as a surface IgM, or sIgM) and the pre-B cell becomes an immature B cell. At this stage, the antigen receptor is first tested for reactivity to self antigens, or autoreactivity. The elimination or inactivation of autoreactive B cells ensures that the B-cell population as a whole will be tolerant of self antigens. The tolerance produced at this stage of B-cell development is known as central tolerance because it arises in a central lymphoid organ, the bone marrow. However, B cells leaving the bone marrow are not fully mature and require additional maturation steps that take place in peripheral lymphoid organs (see Section 8-8). As we shall see later in the chapter and in Chapter 15, self-reactive B cells that escape central tolerance may still be removed from ERRNVPHGLFRVRUJ Fig. 8.8 Nonproductive light-chain gene rearrangements can be rescued by further rearrangement. The organization of the light-chain loci in mice and humans offers many opportunities for the rescue of pre-B cells that initially make an out-offrame rearrangement. Light-chain rescue is illustrated here at the human κ locus. If the first rearrangement is nonproductive, a 5’ Vκ gene segment can recombine with a 3’ Jκ gene segment to remove the outof-frame join located between them and to replace it with a new rearrangement. In principle, this can happen up to five times on each chromosome, because there are five functional Jκ gene segments in humans. If all rearrangements of κ-chain genes fail to yield a productive light-chain join, λ-chain gene rearrangement may succeed (not shown). 305 306 Chapter 8: The Development of B and T lymphocytes the repertoire after they have left the bone marrow, a process that takes place during the final peripheral stages of B-cell maturation, and is referred to as peripheral tolerance, described in Section 8-7. sIgM associates with Igα and Igβ to form a functional B-cell receptor complex, and the fate of an immature B cell in the bone marrow depends on signals delivered from this receptor complex when it interacts with ligands in the environment. Igα signaling is particularly important in dictating the emigration of B cells from the bone marrow and/or their survival in the periphery: mice that express Igα with a truncated cytoplasmic domain that cannot signal show a fourfold reduction in the number of immature B cells in the marrow, and a hundredfold reduction in the number of peripheral B cells. The release of immature B cells from the bone marrow into the circulation is also dependent on their expression of S1PR1, a G-protein-coupled receptor that binds to the lipid ligand S1P and promotes cell migration towards the high concentrations of S1P that exist in the blood (see Section 8-27). Immature B cells that have no strong reactivity to self antigens continue to mature (Fig. 8.9, first panel). They leave the marrow via sinusoids that enter the central sinus, enter the circulation, and are carried by the venous blood supply to the spleen. If, however, the newly expressed receptor encounters a strongly cross-linking antigen in the bone marrow—that is, if the B cell is strongly self-reactive—development is arrested at this stage. Fig. 8.9 Binding to self molecules in the bone marrow can lead to the death or inactivation of immature B cells. First panels: immature B cells that do not encounter antigen mature normally; they migrate from the bone marrow to the peripheral lymphoid tissues, where they may become mature recirculating B cells bearing both IgM and IgD on their surface. Second panels: when developing B cells express receptors that recognize multivalent ligands, for example, ubiquitous cell-surface self molecules such as those of the MHC, these receptors are deleted from the repertoire. The B cells either undergo receptor editing (see Fig. 8.10), thereby eliminating the self-reactive receptor, or the cells themselves undergo programmed cell death (apoptosis), resulting in clonal deletion. Third panels: immature B cells that bind soluble self antigens able to cross-link the B-cell receptor are rendered unresponsive to the antigen (anergic) and bear little surface IgM. They migrate to the periphery, where they express IgD but remain anergic; if in competition with other B cells in the periphery, anergic B cells fail to receive survival signals and die. Fourth panels: immature B cells whose antigen is inaccessible to them, or which bind monovalent or soluble self antigens with low affinity, do not receive any signal and mature normally. Such cells are potentially self-reactive, however, and are said to be clonally ignorant because their ligand is present but is unable to activate them. Experiments using genetically modified mice that enforce the expression of self-reactive B-cell receptors have shown that there are four possible fates for self-reactive immature B cells (see Fig. 8.9, last three panels). These fates are the production of a new receptor by a process known as receptor editing; cell death by apoptosis, resulting in clonal deletion; the induction of a permanent state of unresponsiveness to antigen, or anergy; and a state of immuno­logical ignorance in which antigen concentrations are too low to stimulate B-cell receptor signaling. The outcome for each self-reactive B cell is dependent on the interaction of the B-cell receptor with the self antigen. Immature B cell (bone marrow) Multivalent self molecule No self reaction Soluble self molecule Low-affinity non-crosslinking self molecule μ+ μ+ μ+ μ+ IgM IgM IgM Migrates to periphery Clonal deletion or receptor editing Migrates to periphery or IgD IgM Mature B cell Apoptosis or generation of non-autoreactive mature B cell Immunobiology | chapter 8 | 08_012 Murphy et al | Ninth edition ERRNVPHGLFRVRUJ © Garland Science design by blink studio limited Migrates to periphery μlow δnormal μ+δ+ IgD IgM μ+δ+ IgD Anergic B cell Mature B cell (clonally ignorant) IgM Development of B lymphocytes. Fig. 8.10 Replacement of light chains by receptor editing can rescue some selfreactive B cells by changing their antigen specificity. When a developing B cell expresses antigen receptors that are strongly cross-linked by multivalent self antigens such as MHC molecules on cell surfaces (top panel), its development is arrested. The cell decreases surface expression of IgM and does not turn off the RAG genes (second panel). Continued synthesis of RAG proteins allows the cell to continue light-chain gene rearrangement. This usually leads to a new productive rearrangement and the expression of a new light chain, which combines with the previous heavy chain to form a new receptor; the process is called receptor editing (third panel). If this new receptor is not self-reactive, the cell is ‘rescued’ and continues normal development, much like a cell that had never reacted with self antigen (bottom right panel). If the cell remains self-reactive, it may be rescued by another cycle of rearrangement; however, if it continues to react strongly with self antigen, it will undergo apoptosis, resulting in clonal deletion from the repertoire of B cells (bottom left panel). Immature B cells that express an autoreactive receptor recognizing a multivalent self antigen can be rescued by further gene rearrangements that replace the autoreactive receptor with a new receptor that is not self-reactive. This mechanism is termed receptor editing (Fig. 8.10). When an immature B cell first produces sIgM, RAG proteins are still being made. If the receptor is not self-reactive, the absence of sIgM cross-linking allows gene rearrangement to cease and B-cell development continues, with RAG proteins eventually disappearing. For an autoreactive receptor, however, an encounter with the self antigen results in strong cross-linking of sIgM; RAG expression continues, and light-chain gene rearrangement can continue, as described in Fig. 8.8. These secondary rearrangements can rescue immature self-reactive B cells by deleting the self-reactive light-chain gene and replacing it with another sequence. If the new light chain is not autoreactive, the B cell continues normal development. If the receptor remains autoreactive, rearrangement continues until a non-autoreactive receptor is produced or until no additional light-chain V and J gene segments are available for recombination. The importance of receptor editing as a mechanism of tolerance is well established, as defects in this process contribute to the human autoimmune diseases systemic lupus erythematosus and rheumatoid arthritis, two diseases characterized by high levels of autoreactive antibodies (see Chapter 15). It was originally thought that the successful production of a heavy chain and a light chain caused the almost instantaneous shutdown of light-chain locus rearrangement and that this ensured both allelic and isotypic exclusion. The unexpected ability of self-reactive B cells to continue to rearrange their lightchain genes, even after having made a productive rearrangement, suggests an alternative mechanism of allelic exclusion, where the fall in the level of RAG proteins that follows a successful non-autoreactive rearrangement could be the principal means by which light-chain rearrangement is terminated. It is now apparent that allelic exclusion is not absolute, as there are rare B cells that express two different light chains. Cells that remain autoreactive when receptor editing efforts fail to generate a non-autoreactive receptor undergo a process known as clonal deletion, in which they are subjected to cell death by apoptosis to eliminate their specific autoreactivity from the repertoire. Early experiments using transgenic mice expressing both chains of an immunoglobulin specific for H-2Kb MHC class I molecules, in which nearly all developing B cells expressed the anti-MHC immunoglobulin as sIgM, suggested that clonal deletion was a predominant mechanism of B-cell tolerance. These studies found that transgenic mice not expressing H-2Kb had normal numbers of B cells, all bearing the transgene-encoded anti-H-2Kb receptors. However, in mice expressing both H-2Kb and the immunoglobulin transgenes, B-cell development was blocked. Normal numbers of pre-B cells and immature B cells were found, but B cells expressing the anti-H-2Kb immunoglobulin as sIgM never matured to populate the spleen and lymph nodes; instead, most of these immature B cells died in the bone marrow by apoptosis. However, more recent studies, using ERRNVPHGLFRVRUJ Strong ligation of IgM by self antigen IgM Arrest of B-cell development and continued light-chain rearrangement: low cell-surface IgM A new receptor specificity is now expressed If the new receptor is still self-reactive, the B cell undergoes apoptosis If the new receptor is no longer selfreactive, the immature B cell migrates to the periphery and matures Immunobiology | chapter 8 | 08_013 Murphy et al | Ninth edition © Garland Science design by blink studio limited Rheumatoid Arthritis Systemic Lupus Erythematosus 307 308 Chapter 8: The Development of B and T lymphocytes mice bearing transgenes for autoantibody heavy and light chains that have been placed within the immunoglobulin loci by homologous recombination (see Appendix I, Section A-35, for details of this method), indicate that receptor editing, rather than clonal deletion, is the more likely outcome for immature autoreactive B cells. We have so far discussed the fate of newly formed B cells that undergo multi­ valent cross-linking of their sIgM. Immature B cells that encounter more weakly cross-linking self antigens of low valence, such as small soluble proteins, respond differently. In this situation, some self-reactive B cells are inactivated and enter a state of permanent unresponsiveness, or anergy, but do not immediately die (see Fig. 8.9). Anergic B cells cannot be activated by their specific antigen even with help from antigen-specific T cells. Again, this phenomenon was elucidated using transgenic mice. Hen egg-white lysozyme (HEL) was expressed in soluble form from a transgene in mice that were also transgenic for high-affinity anti-HEL immunoglobulin. The HEL-specific B cells matured and emigrated from the bone marrow, but could not respond to antigen. Furthermore, the migration of anergic B cells is impaired, as the cells are detained in the T-cell areas of peripheral lymphoid tissues and are excluded from lymphoid follicles, thereby reducing their life-span and their ability to compete with immunocompetent B cells (described further in Section 8-8). Under normal circumstances, where few self-reactive anergic B cells successfully mature, these cells die relatively quickly. This mechanism ensures that the long-lived pool of peripheral B cells is purged of potentially self-reactive cells. The fourth potential fate of self-reactive immature B cells is that nothing happens to them; they remain in a state of immunological ignorance of their self antigen (see Fig. 8.9). Immunologically ignorant cells have affinity for a self antigen but for various reasons do not sense and respond to it. The antigen may not be accessible to developing B cells in the bone marrow or spleen, or may be in low concentration, or may bind so weakly to the B-cell receptor that it does not generate an activating signal. Because some ignorant cells can be (and in fact are) activated under certain conditions such as inflammation or when the self antigen becomes available or reaches an unusually high concentration, they should not be considered inert, and they are fundamentally different from cells with non-autoreactive receptors that could never be activated by self antigens. The fact that central tolerance is not perfect and some self-reactive B cells are allowed to mature reflects the balance that the immune system strikes between purging all self-reactivity and maintaining the ability to respond to pathogens. If the elimination of self-reactive cells were too efficient, the receptor repertoire might become too limited and thus unable to recognize a wide variety of pathogens. Some autoimmune disease is the price of this balance: we shall see in Chapter 15 that ignorant self-reactive lymphocytes can be activated and cause disease under certain circumstances. Normally, however, ignorant B cells are held in check by a lack of T-cell help, the continued inaccessibility of the self antigen, or the tolerance that can be induced in mature B cells following their emigration from the bone marrow, which is described below. 8-7 Lymphocytes that encounter sufficient quantities of self antigens for the first time in the periphery are eliminated or inactivated. While large numbers of autoreactive B cells are purged from the population of new lymphocytes in the bone marrow, only lymphocytes specific for autoantigens that are expressed in or can reach this organ are affected. Some antigens, like the thyroid product thyroglobulin, are highly tissue specific, or are compartmentalized so that little if any is available in the circulation. Therefore, newly emigrated self-reactive B cells that encounter their specific autoantigen ERRNVPHGLFRVRUJ Development of B lymphocytes. Immature B cell (spleen) Multivalent self molecule Soluble self molecule Low-affinity non-crosslinking self molecule No self reaction IgM μ+ μ+ μ+ IgM IgM IgM μlow δnormal μ+δ+ IgD Anergic B cell Apoptosis Apoptosis IgD μ+δ+ IgM Mature B cell (clonally ignorant) IgD IgM Mature B cell Immunobiology | chapter 8 | 08_100 Murphy et alfirst | Ninthtime edition in the periphery must be eliminated or inactivated also. This for the © Garland Science design by blink studio limited tolerance mechanism, which acts on newly emigrated B cells that are still immature, is known as peripheral tolerance. Like self-reactive lymphocytes in the central lymphoid organs, lymphocytes that encounter self antigens de novo in the periphery can have several fates: deletion, anergy, or survival (Fig. 8.11). In the absence of an infection, newly emigrated B cells that encounter a strongly cross-linking antigen in the periphery will undergo clonal deletion. This was elegantly shown in studies of B cells expressing B-cell receptors specific for H-2Kb MHC class I molecules. These B cells are deleted even when, in transgenic animals, the expression of the H-2Kb molecule is restricted to the liver by the use of a liver-specific gene promoter. There is no receptor editing: B cells that encounter strongly cross-linking antigens in the periphery undergo apoptosis directly, unlike their counterparts in the bone marrow, which attempt further receptor rearrangements. This difference may be due to the fact that the B cells in the periphery are somewhat more mature and can no longer rearrange their light-chain loci. As with immature B cells in the bone marrow, newly developed peripheral B cells that encounter and bind an abundant soluble antigen become unresponsive. This was demonstrated in mice by placing the HEL transgene under the control of an inducible promoter that can be regulated by changes in the diet. It is thus possible to induce the production of lysozyme at any time and thereby study its effects on HEL-specific B cells at different stages of maturation. These experiments have shown that both pe