Frixos Paraskevas
Ontogeny
Pluripotential stem cells give rise to all hematopoietic lineages, including B and T lymphocytes. These cells undergo asymmetric division; as a result, one daughter cell follows a pathway of differentiation, whereas the other remains a self-renewing stem cell. These stem cells differentiate into various lineages depending on their interaction with the appropriate stromal cells as well as growth and differentiation factors. Stem cell differentiation is guided by the type of stromal elements to which the stem cells are exposed. Stem cells placed into the thymic microenvironment develop into T cells, whereas those in contact with bone marrow–derived stromal cells develop into B lymphocytes or myeloid cells. B-cell development and differentiation can be divided into two broad periods: the first period, from stem cell to immature immunoglobulin (Ig) M+ B cell, is antigen-independent; the second period, from immature B cell to plasma cell, is antigen-dependent. The first period can be further subdivided into two stages: from stem cell to progenitor B (pro-B) cell, and from pro-B cell to immature Ig+ B cell.
B-cell differentiation has been studied in animals and humans on cells obtained from different stages of development (embryonic vs. adult) as well as from cells from patients with lymphoproliferative disorders, which, by some, are considered “frozen windows” of normal differentiation. This approach, however, is not accepted by all, because some evidence suggests the existence of some degree of asynchronous maturation in acute leukemias, with phenotypes not found normally (1).
Stem Cell to Progenitor B (Pro-B) Cell
See Figure 15.1.
Transcriptional Regulation
Hematopoiesis is a highly complex process coordinated by several genes that orchestrate through their products multiple cell interactions and the release of growth and differentiation factors (2). During this process, more than eight specific cell lineages arise from pluripotent stem cells. Their differentiation is guided through a well-defined hierarchical sequence to mature functional cells (3).
Lymphopoiesis during fetal life takes place in the yolk sac and liver, but after birth, like the rest of definitive hematopoiesis, it takes place in the bone marrow (4). “Master” genes regulate differentiation of lineages through transcription factors, which activate target genes, progressively narrowing their differentiation potential to specific lineages (3). Multiple genes, through the transcription factors they induce, coordinate the various stages of lymphocytic development (5,6,7,8). Myeloid and lymphoid lineages originate from a common progenitor, which gives rise to the common myeloid/erythroid progenitor and the common lymphocytic progenitor (for B, T, NK, and DC lineages).
Normal development of lymphopoiesis depends on the Ikaros family of transcription factors, which selectively regulate lymphocytic development. Ikaros is a zinc-finger transcription factor expressed in B and T cells at different stages of differentiation (except plasma cells) (9). Ikaros generates eight protein isoforms by alternative splicing, and some of them bind through an N- terminal zinc-finger motif to DNA sequences that contain GGGA core motifs (10). Ikaros-/- mice have a complete block of B-cell differentiation, with lack of pro-B and precursor B cells (pre-B cells) in fetal liver or bone marrow. Ikaros is localized in the nucleus of most primitive hematopoietic stem cell subsets, particularly at two stages: (a) in long-term self-renewing stem cells and (b) in non–self-renewing multipotent progenitors able to differentiate into lymphoid-committed progenitors (11). The isoforms detected in hematopoietic stem cells differ from those detected in lymphoid progenitors, but all of them share two C-terminal zinc fingers that mediate their self-association, forming multiple heteromeric complexes. Other members of the Ikaros family (i.e., Aiolos and Helios) encode transcription factors that form multimeric complexes with Ikaros (12). The Ikaros gene is required not only for early stages of lymphocytic differentiation but also for late stages, especially in T-cell maturation. Ikaros regulates gene expression by recruiting repressor complexes or what is known as chromatin-remodeling machines, an unconventional function for transcription factors. Ikaros is also needed for the maintenance of B cells by regulating BCR signaling (13). The transcription factor PU.1 acts at the multipotent level of myeloid-lymphoid progenitors. Expression of PU.1 maintains the hematopoietic progenitor pool by supporting the generation of the earliest lymphoid and myeloid progenitors (14). It regulates early B-cell development by activating the IL-7Rα gene, rendering B-cell progenitors responsive to appropriate differentiation signals and thus promoting differentiation to the pro-B-cell stage (15). Among the three main B-cell populations, B1, B2 (follicular), and marginal-zone B cells, PV-1 directs differentiation toward the B2 subpopulation (16). PV.1 enhances the activity of other transcription factors, i.e., the interferon (IFN)-regulatory factor. The E2A gene is highly important for lymphocytic differentiation. It encodes two proteins, E12 and E47 of the basic helix–loop–helix family, by differential splicing. The proteins encoded by E2A bind to DNA as homodimer, uniquely in B cells (17), and are essential for coordination of Ig gene rearragements (18); therefore, in their absence, B-cell differentiation is blocked before entrance into the pro-B-cell stage. The basic region mediates DNA binding, whereas the HLH domain is required for protein dimerization. The target genes for E12 are the early B-cell factor (EBF) and Pax-5 (19). E12 acts on a progenitor cell, directing differentiation along B lineage while blocking myeloid differentiation.
Another important gene for lymphoid development is Pax5, which has multiple isoforms generated by alternative splicing, one of them known as the B-lineage–specific activator protein (BSAP). Pax-5 is exclusively expressed from pro-B- to the mature B-cell stage, but not in plasma cells (20). The target genes for BSAP are CD19, VpreB1, λ5, and several intronic sites of CH gene. Pax5 is needed for V(D)J heavy-chain Ig gene rearrangements, and the precise block is after the first DJH rearrangement (21). As a result, Pax5 mutation does not block recombinase induction per se and allows a significant pro-B-cell population to be formed. The target for BSAP is the adaptor protein BLNK (B-cell linker protein) (SLP-65), which acts as scaffolding in linking Syk tyrosine kinase with downstream signaling proteins (i.e., PLCγ2, Bruton tyrosine kinase [Btk], etc.) (22). BSAP regulates BLNK gene expression, a checkpoint in the pro-B to pre-B transition and mediates the constitutive signaling of the pre-B-cell receptor (pre-BCR) in cell proliferation, growth-factor responsiveness, and V(D)J recombination. BSAP also diverts differentiation of a common myeloid/ lymphoid progenitor to lymphocytic differentiation as a result of its ability to suppress the response of the progenitor to myeloid growth factors (23).Thus, genes inappropriately expressed at the pro-B-cell stage are repressed by Pax-5 (24).
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Figure 15.1. B-cell differentiation. The first stage of B-cell development takes place in the bone marrow. Normal development of lymphopoiesis depends on the Ikaros family of transcription factors, which form multimeric complexes with other members, i.e., Aiolos and Helios. These complexes are known as chromatin remodeling machines, and their targets induce a second wave of transcription factors, i.e., PU.1, E2A, EBF, PAX5, etc. Their targets are a third set of genes, forming BSAP that acts on the adaptor protein BLNK (SLP-65). Targets of BSAP are the genes for CD19, VpreB, and λ5, i.e., markers distinctly of the B-cell lineage. BLNK regulates the transition from pro-B to pre-B, whereas PAX5 is needed for V(D)J recombination. This stage of development is independent of exposure to antigen and is followed by an antigen-dependent stage that unravels in the peripheral lymphoid organs. Antigen receptors, i.e., Igs, expressed on their surface and following interaction with antigen and “helper” T cells, trigger B-cell differentiation (plasma cells) and antibody secretion. Class switching generates distinct classes of antibodies suitable to dispose antigens in the various microenvironments, i.e., inside the body as well as on body surfaces. |
The early B cell factor (EBF) is a transcription factor that is essential for early B-lymphocyte development in regulation of the expression of Igα (mb-1) and Igβ (B29) coreceptors. The EBF-deficient mice lack Igα and Igβ transcripts in pro-B cells (25), and isolated human EBF factor has been shown to bind to Igα and Igβ promoters (26). EBF activates transcription of B-cell–specific genes and initiates necessary events such as gene activation of other transcriptional regulators (i.e., Pax5) that direct the early stages of B-cell lymphopoiesis (27). Mutations of E2A and EBF combined arrest B-cell development at a stage earlier than that of Pax5, because recombinase expression is blocked, there is no expression of pre-BCR genes, λ5, and VpreB, and, consequently, no formation of the pre-BCR (27). The three transcription factors Pax5, E2A, and EBF form a cross-regulatory network; i.e., E2A is essential for Pax5 expression and normal EBF expression, whereas EBF is needed for Pax5 and E47 expression, which is a product of the E2A gene, the most potent B-cell regulator.
Collectively, the targets of Ikaros are the genes of the transcription factors E2A, PU.1, EBF, and Pax5, all of which regulate stages of B-cell differentiation before the expression of Ig genes. It has been shown, however, that the E2A gene plays a central role for some aspects in subsequent stages of B-cell lymphopoiesis (28). E2A proteins are required for the interleukin (IL)-7–dependent expansion of pro-B cells and their progression to the pre-B-cell stage and, even later, for the regulation of rearrangements of the Igκ genes and heavy-chain isotype switching (29,30,31). E2A encodes proteins that enhance hypermutation by recruiting AID to the Ig loci (29) and is essential in promoting pre-B- and B-cell survival (30).
Cell Interactions in Early B-Cell Development
Study of B-cell differentiation has become feasible with the development of long-term bone marrow culture techniques. This approach has helped to define the cells that are essential for the development of B cells and the factors that support B-cell growth and differentiation. The bone marrow stroma makes a critical contribution to hematopoietic differentiation, and the term stroma (Greek for mattress) is used in a collective sense to include a variety of cells, such as adventitial reticular cells, adipocytes, fibro-blasts, and endothelial cells of the sinuses (32). The stroma also includes extracellular matrix, composed of various fibrous proteins, glycoproteins (gps), and heparan sulfate, effective in binding cytokines. Granulocyte-macrophage colony-stimulating factor can bind to marrow stromal glycosaminoglycans and be presented to hematopoietic stem cells. Stromal cells have been isolated from adult bone marrow (33) or fetal bone marrow after removal of adherent cells (i.e., macrophages, endothelial cells). These cells have the morphology of an adventitial/reticular/fibroblast cell, which expresses several adhesion molecules. They support, in cultures, differentiation of fetal bone marrow cells (CD34+, CD19-) (34) toward B-cell lineage (i.e., with loss of CD34 and acquisition of CD19 and the VpreB protein).
Several adhesion molecules have been implicated in the mediation of interaction between B-cell progenitors and stromal cells; critical among them are vascular cell adhesion molecule-1 (VCAM-1) on stromal cells and very late antigen-4 (VLA-4) on B-cell progenitors (33,35). CD34 is expressed not only on stem cells (36), but also on stromal cell precursors (37) and endothelial cells (38), thus mediating their interactions with its counterreceptor, L-selectin, which is expressed on progenitor cells.
Among cytokines that could mediate growth and differentiation of lymphocytes in the bone marrow, IL-7 attracted attention because it was shown in experiments in mice that it plays a critical role in B-cell development (39). IL-7 acts in an early stage of common lymphoid progenitors (CLP, B/T/NK) and maintains B-cell differentiation programs open (40). In the absence of IL-7, T/NK differentiation proceeds normally, but B-cell differentiation is arrested. IL-7 modulates EBF expression, which activates target genes that are specific for B-cell differentiation programs (40). In mice that lack IL-7R, the recombination process comes to a halt, because the IL-7R normally targets VH segments to the recombinase complex; furthermore, Pax5, which normally stimulates recombination, is silenced (41). Although the block of IL-7 deficiency is after the pro-B cells, these cells have certain abnormalities, such as failure to up-regulate terminal deoxynucleotidyl transferase (TdT) and the high-affinity IL-7R α chain (42). The net result of IL-7 deficiency is the lack of expression of μ chain and the pre-BCR, events that normally promote the advancement from pro-B-cell to pre-B-cell stage. The stimulation of proliferation and μ-chain expression follows distinct signaling pathways, because mutation Y449F in the cytoplasmic region of IL-7Rα abrogates proliferation induced by IL-7, but it does not block μ-chain expression (43).
A cytokine isolated from thymic stroma, thymic stroma lymphopoietin (TSLP) (see Chapter 14), binds to the IL-7Rα receptor, and it is likely that, in the absence of IL-7, thymic stroma lymphopoietin promotes continuation of B-cell differentiation in the absence of IL-7, with development of IgM+ B cells from IgM- precursors (44).
However, in human X-linked severe combined immunodeficiency (X-SCID), which is a result of mutations of the γc chain, a common subunit for several cytokine receptors (i.e., IL-2, IL-4, IL-7, IL-9, and IL-15), the patients lack T and natural killer (NK) cells but have normal or even elevated B cells (45). Likewise, patients with mutations of the IL-7Rα chain have normal numbers of B cells (46).
IL-7 exerts some change in gene expression, especially in concert with other growth factors or stromal cells (47). It synergizes with flt-3 ligand and induces strong expansion of fetal B cells in vitro (48). Furthermore, IL-7R has been detected on human B-cell progenitors, which lack expression of CD19 and have a pro-B-cell phenotype as well as clonogenic capacity (49). These cells are also CD34+, have messenger RNAs (mRNAs) for Igβ, RAG-1, and Pax5, and are TdT+. In addition, IL-7Rα-/CD19-/CD34+ cells do not differentiate in short-term cultures into pro-B cells. Therefore, IL-7Rα expression defines an entry into a stage characterized by up-regulation of multiple B lymphoid–associated markers.
Single adult human CD34+/Lin-/CD38- cells in culture over a murine fetal liver–adherent cell line (AFT024) supplied with IL-7, flt-3 ligand, and IL-3 differentiate into B cells, NK cells, myeloid cells, and dendritic cells (50).
In summary, B-lineage development, as in other hematopoietic cells, is a complex, interconnected network of genetic programs. At appropriate times some will turn off, while others are maintained open and provide lineage commitment and specification (51,52).
Expression of the CXCR4 chemokine receptor has been detected in CD34+ hematopoietic progenitors (53), and a cell committed to B-cell development was positive for the chemokine receptor CXCR4 (being the fourth chemokine receptor to be cloned) (54). The ligand for CXCR4 is the stromal cell–derived factor-1/pre-B-cell growth-stimulating factor (SDF-1/PBSF) (55). CXCR4, also known as fusin, together with CCR5, acts as a coreceptor for human immunodeficiency virus (HIV) to infect host cells, and SDF-1 blocks the entry of HIV to T cells (54). CD34+, CXCR4-bone marrow cells can generate myeloid and lymphoid progenitors, but the CD34+, CXCR+ cells, although they lack myeloid, erythroid, megakaryocytic, and mixed colony-forming potential, give rise to B- and T-lymphoid progenitors. CXCR4 is expressed earlier than IL-7R and TdT; therefore, it defines a B- lineage–committed progenitor better and more accurately than other commonly used markers.
Pro-B Cell to Immunoglobulin+ B Cell
Molecular definition of the stages of early B-cell development started with the demonstration of cells that display in the cytoplasm μH chains without L chains and no mature IgM molecules on the cell surface that were called pre-B cells (56). Further definition followed two different avenues: phenotypic markers or Ig gene rearrangement status (57). Single-cell polymerase chain reaction analysis, however, allowed the stages of B-cell development to be ordered better by the sequence of the Ig gene rearrangements than by phenotype.
Ig gene rearrangements are the most important indication of B-lineage commitment and the transition from stem cells to the pro-B-cell stage (Fig. 15.1). With the continuation of Ig gene rearrangements, the pro-B cell becomes a pre-B cell, which, sometimes, is divided into pre-BI and pre-BII stages. When the Ig gene rearrangements reach their final stage and IgM is expressed on the cell surface, differentiation reaches the end of the first period (i.e., the immature B lymphocyte). During the pro-B-cell and pre-B-cell stages, three sets of genes prepare for the expression of the complete Ig molecule on the B-cell surface. One set includes RAG-1 and RAG-2; the second set, the surrogate light chain (SLC)genes, composed of the products of the two genes VpreB and λ5; and the third set consists of the genes CD79A (mb1) and CD79B (B29). The products of the last two genes, Igα and Igβ, or CD79a/CD79b, form the coreceptor, which supports the expression of IgM on the surface of the B cell. Throughout these stages, the expression of IgM, as well as B-cell-receptor repertoire and allelic exclusion (i.e., expression of only one of the two alleles), becomes possible.
Rearrangements of the Ig genes start with the joining of a D to JH segment in the pro-B cells. Pro-B cells proliferate in contact with stromal elements and express the SLC linked to a gp30 protein, which sometimes is referred to as the surrogate heavy chain, to form the pro-B-cell receptor (58,59,60). Expression of SLC is easily detected on mouse pro-B cells but is more difficult to detect on human pro-B cells (59,60). The SLC consists of two noncovalently associated proteins, VpreB and λ5 (Fig. 15.2), encoded by genes located on chromosome 22, where the λ-chain genes are also located (61,62). There is only one VpreB gene in humans.
VpreB and λ5 have several unusual features that distinguish them from conventional light chains. The VpreB protein is composed of 102 amino acids with homology to a conventional V domain, and both have atypical sequences that target them to the endoplasmic reticulum (ER) (63). However, whereas the conventional V domain has a total of nine β strands, including a sequence encoded by a J segment, the VpreB has no sequence similar to the J segment and is shorter by one strand—the β7 strand. The C-terminal portion of VpreB has no homology to any other known protein. The N-terminal end of the λ5 protein is unique, with only marginal sequence homologies to conventional domains. The C-terminal end of λ5 has an Ig-like fold, homologous to the conventional λ-chain C domain. This is followed by a sequence with marginal homology to the Ig structures and an extra β strand that is homologous to the J region of the conventional Vλ domains. The unique regions of the VpreB and λ5 chains do not contribute to the assembly of the SLC, which is mediated by the extra β7 strand of λ5, because deletion of this strand abolishes formation of SLC (63). The unique region of the λ5 chain, however, acts as an intramolecular chaperon by preventing the folding of the protein when it is expressed in the absence of VpreB. This extra β strand complements the missing VpreB β strand. Thus, it appears that the Ig domains in each protein split between the two of them the control of the folding and the assembly of SLC (66). The assembled SLC associates with the μ chain to form the pre-BCR. In the meantime, the CH1 domain of the μH chain is protected from improper folding by the hsp70 protein BiP, which retains the μH chain in the ER (64). Protection from improper folding of CH1 is taken over by the Cλ5 protein as long as it is associated with the VpreB as SLC (65). SLC escorts the μH chain to the surface of the cell. However, not all μH chains can pair with the SLC, because some V segments have structural features that prevent their association. At this point, the RAG-1, RAG, and TdT genes are turned off, securing allelic exclusion (66).
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Figure 15.2. The hypothetical structure of the pre-B-cell receptor. The pre-B-cell receptor is composed of the μ chain and a surrogate light chain that consists of two components, the λ5 and the VpreB. The μ chain is linked by disulfide bond to the λ5 component, whereas the VpreB is noncovalently attached. It is not yet clear whether surrogate light chain can be expressed on the surface without μ chain. |
In the pre-BCR, the λ5 chain represents the equivalent of the C domain of the conventional L chain of BCR, a reasonable assumption considering that λ5 has 85% homology with the Cλ domain. The VpreB, on the other hand, is the equivalent to the VL domain (67).
SLC is linked to the μH chain through the first constant domain (Cμ1) by a disulfide bond (68). The role of the SLC in the formation of the pre-BCR is dual. The C domain of λ5 protein interacts with Cμ1 (which was bound by BiP). The second interaction involves VpreB (and part of λ5) with the VH domain of the μH chain (69). This interaction is likely to be the first step in the selection of the B-cell repertoire (68). The pre-BCR in contact with unknown ligand on stromal cells induces asymmetric divisions of pre-B cells (70). One of the two cells retains contact with stroma and continues to divide (large pre-BI cell), whereas the second cell loses contact and differentiates to pre-BII-cell stage (small cells). The mechanism of signaling by the pre-BCR is still debated—i.e., whether extracellular signals are necessary or not. Stromal heparin sulfate (71) or galectin-1 (72) have been considered pre-BCR ligands. The SLC (λ5 and VpreB) may mediate constitutive receptor aggregation signaling and internalization, and the transition from pro-B cells to pre-B cells is driven by the Ig-α and Ig-β chain ITAMS fused to the Lck myristolyation/polmitoylation sequence (73). Pre-B-cell signals induce proliferative activity of pre-B cells, inducing allelic exclusion of the heavy-chain locus and activation of the light-chain loci preparing for V(D)J recombination (74).
With the pre-B cell ceasing to divide, the rearrangement machinery is turned on again. L-chain genes start to rearrange and, if the rearrangement is productive, an L chain is synthesized to form a complete IgM molecule, expressed on the cell surface. This marks the transition to the immature B-cell stage. The strong proliferative capacity of the pre-B cells was analyzed in Pax5-/- mice. Pre-B cells from these animals have an extensive renewal capacity, multipotency, and ability to reconstitute recipient animals with lymphoid and myeloid lineages (75). Signaling is transmitted by the Igα/Igβ partners of the pre-BCR, which is linked to several protein tyrosine kinases (i.e., Bruton’s tyrosine kinase [BTK], Syk, lyn, BLNK, and phosphoinositide 3-kinase [PI3K]) (76). Ligand binding causes aggregation of the pre-BCR, resulting in endocytosis of pre-BCR, loss of cell-surface expression, and accumulation of pre-BCR within membrane lipid microdomains.
From its location in lipid rafts, the pre-BCR initiates signaling and achieves two important outcomes for the subsequent fate of the differentiating B cell: (a) it activates MAP kinases to block apoptosis, which is usually triggered by the engagement of CD24 (77); and (b) it down-regulates RAG-1/RAG-2 expression to turn V(D)J recombination off so that the second μH allele closes (67) and allelic exclusion is accomplished (78).
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Figure 15.3. Human immunoglobulin heavy-chain gene locus. The heavy-chain gene locus contains the μ and δ genes close to the JH genes, whereas the remaining heavy-chain genes are farther downstream in two clusters from duplications. See text. |
The RAG-1/RAG-2 proteins are still expressed at the active proliferating pre-BI stage but are silenced during the quiescent pre-BII stage. Because the cell has achieved its objectives (i.e., survival and allelic exclusion), it reactivates the V(D)J recombination machinery to allow rearrangements of the conventional κ/λ chains, to be able to form the complete IgM molecule and thus enter the immature B-cell stage.
The nontranscribed allele is in close association with heterochromatin and the transcription factor Ikaros, both markers of transcriptionally silent genes (79,80). The importance of pre-BCR signaling for progression from pro-B-cell to pre-B-cell stages and beyond is emphasized by the fact that deficiencies of any component of the pre-BCR or downstream signaling molecules blocks developmental progression of B cells at the pro-B or pre-B stage (81). Targeting the genes for VpreB (82), λ5 (83), or the exons of the μ chain (84) blocks B-cell differentiation at the pro-B- to pre- B-cell stage in mice, although the VpreB expression in λ5-deficient mice is normal (85). An 11-year-old boy with λ5 deficiency had virtually no B cells and was agammaglobulinemic (86).
The proteins of the SLC are not detected on the surface or in the cytoplasm of the majority of B cells in the bone marrow or circulation, except in a subpopulation of self-reactive B cells found in germinal centers (87) and circulation (88). VpreB+ B cells with RAG mRNA have also been described in normal human tonsils and the joints of patients with rheumatoid arthritis (89,90).The pre-B cell signaling that terminates pre-B cell expansion and induces Ig L-chain rearrangement is transmitted by Bruton’s tyrosine kinase and the SLP-65 (Src homology 2 domain, containing a leukocyte-specific phosphoprotein of 65 kDa) (91). The VpreB and λ5 proteins of the pre-BCR are invariant, but each contains unique evolutionarily conserved non-Ig sequences (unique regions or URs) attached to the Ig sequences (92). These URs interrupt the SLC complex and may be considered equivalent to the CDR3 of the Ig L chain, which participates in antigen binding (93).
The transcription factors that are most active and are specifically up-regulated at this stage are E2A, EBF, Pax5, and IRF-4 (94). The pre-BCR not only switches Ig genes on, it appears to select preferentially those that provide μH chains of a higher quality, i.e., with a higher potential to assemble with Ig L chains (95).
In conclusion, the pre-BCR plays a major role in the expansion of pre-B cells, allelic exclusion, repertoire selection, activation of V(D)J recombination, and developmental progression to IgM+ B cells (74).
Immunoglobulin Genes
Among the first successes of recombinant DNA investigation was the characterization of the Ig genes. They exist in three loci—one each for the heavy, κ, and λ chains. For human Ig, these loci are located on chromosomes 14 (96), 2 (97), and 22 (98,99), respectively. Separate genes encode the V and C domains of the Ig molecule. The human heavy-chain genes are not completely linked, with the Cμ gene being closest to the V-D-J segments. At the 3′ end of the H-chain locus, there are two copies of a γ-γ-∊-α unit (Fig. 15.3). One of the duplicated ∊ sequences is a pseudogene (Ψ∊) in which the CH1 and CH2 domains have been deleted. The genome contains a third closely homologous ∊-related sequence: a “processed”pseudogene found on chromosome 9. These kinds of pseudogenes probably are products of a reverse transcription from RNA and then are inserted at locations in the genome that are unrelated to the original locus.
A γ-related pseudogene lacking the switch region is present between the two γ-γ-∊-α duplications. The recombination events occur in a region on the 5′ side of the CH coding sequences. This region contains repetitive DNA sequences and has been known as the switch region (S) or sequence. Within the switch regions occur the recombination breakpoints during isotype switch (see Chapter 17). Usually, the stimuli for switch recombination, like IL-4 and CD40, promote transcription across the S regions to produce switch junctions, one of which is retained in the chromosome and the other of which is found in a circular DNA (see Chapter 17).
VH-D-JH Genes
The heavy chain of the IgM molecule is encoded by four sets of DNA: variable (VH), diversity (D), joining (JH), and the constant (Cμ) genes.
VH Genes
A VH gene encodes the first 95 or 96 amino acids of a V domain. The exact number of VH genes is not known but is estimated to be 100 to 200. From the available VH genes, only a portion of them are functional, and an even smaller portion is available for rearrangement (100,101). The VHgenes constitute seven families based on their homology at the DNA level and are known as VHI to VHVII. Most VH genes are polymorphic, but the degree of variation varies, usually being small (102). The VH segment encodes the hydrophobic leader sequence, the three framework regions, and the two complementarity-determining regions (CDR).
D Genes
The term D gene or segment was proposed to indicate the “diversity” of an antibody at positions after amino acid 99 to the beginning of the J segment (103), which spans the third CDR. The D segments are located in chromosome band 14q32, with the major locus between VH and JH loci and several D segments interspersed within the VH segments. The segments are sandwiched between signal sequences (see later).
The CDR3 is encoded by the 3′ end of the VH segment, the D gene segment, and the D-JH junctional area. There are approximately 30 D segments grouped in seven families, and each one spans approximately 70 kb, with a promoter on its 5′ end that allows initiation of transcription when Ig genes at the pre-B-cell stage start rearrangements. The first rearrangement produces a DJH complex, which, together with the μ gene, encodes a protein known as Dμ protein.
JH Genes
There are six functional JH genes and three pseudogenes, each JH gene encoding the 3′ end of CDR3 and the fourth framework region.
V(D)J Recombination
Formation of Coding and Signal Joints
Immediately next to the V, D, and J genes are conserved sequences of seven nucleotides (heptamers), which are attached to the 3′ side of the V segments, the 5′ side of the J segments, and both sides of the D segments (Fig. 15.4). The heptamer is followed by 12 or 23 nonconserved base pairs (bp) (spacers), followed by another sequence of 9 bp (nonamer), which may diverge from the consensus sequence. This noncoding sequence is known as the recombination signal sequence (RSS) and, in its consensus form, is as follows: 5′-coding sequence–CACAGTG–12 or 23 spacer–ACAAAAACC-3. The 12-bp spacer corresponds to one turn of the DNA α helix, whereas the 23-bp spacer corresponds to two turns. This way, the recombining segments are juxtaposed on the same side of the DNA helix, so that they can be recognized by the enzymes of the recombination machinery (104,105,106). Joining of the various segments is limited between an RSS with a 12-bp spacer and one with 23 bp (107) (the 12/23 rule). Recombination follows strictly the 12/23 rule of spacers, which prevents inappropriate recombination (i.e., a VH segment joining directly to a JH segment, as both of them have a 23-bp spacer between their heptamers and nonamers) (107). The pattern of the RSS at each locus is uniform. For example, in the Ig locus, all Vk segments have next to their heptamers a 12-bp spacer, whereas all Jk segments have a 23-bp spacer. This prevents accidental joining of two Vk or Jk segments. The RSSs are the only sequences that are required for recombination, whereas the coding sequences (V, D, or J) can be replaced by other DNA, and joining still occurs if the 12/23 rule is satisfied.
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Figure 15.4. Recombination signal sequences (RSS) and heptamer/nonamer spacing sequences. The coding sequences of the immunoglobulin genes are flanked by a 7-nucleotide sequence (heptamer), which is followed by either 12 or 23 nucleotides, which in turn are followed by a 9-nucleotide sequence (nonamer) and again by a 23- or 12-nucleotide spacer. This order allows rearrangements only between a segment in which the 7/9 sequences are separated by a 12mer spacer and another segment in which they are separated by a 23mer spacer. This is known as the 12/23 rule. |
During recombination, two new structures are formed: the joining of the coding sequences (coding joint), which is imprecise, and the joining of the RSS (signal joint), which is precise.
The V(D)J recombination (Fig. 15.5) is mediated by two enzymes known as RAG proteins (108). RAG-1 and RAG-2 are not related to other proteins, and RAG-2 has no known relatives. Their expression is normally limited to immature B and T cells and continues after the expression of surface antigen receptor but then ceases when the receptor is cross-linked. Deficiency of RAG proteins in mice (109,110) or humans (111) results in SCID. The RAG-2 is divided into an N-terminal “core” domain and a C-terminal non-core domain. The core domain is necessary and sufficient for carrying out the V(D)J recombination. The C terminus of RAG-2 contains a PHD finger motif, usually present in chromatin-associated proteins, that bends zinc. Mutations of RAG-2 at this site result in immunodeficiencies. The PHD finger of RAG-2 modulates V(D)J recombination (112) (see later).
The RAG-1/RAG-2 proteins, as a tetramer, initiate the V(D)J recombination, which proceeds in three steps (113,114,115,116). The RAG-1 binds to the nonamer, which acts as an “anchoring” platform while the heptamer stabilizes the complex in the presence of RAG-2. The recognition by RAG proteins of the RSS is assisted by sequence-nonspecific DNA-binding proteins, HMG1/2 (high-motility group), which enhance binding and cleavage. In the RAG1/RAG2 complex, the RAG2 C terminus probably interacts directly to histones and stabilizes the RAG1/RAG2 complex. This interaction may bring the recombinase to specific recombination signal sequences or may be required for the postcleavage phase of the recombination. Mg2+ is the divalent metal ion required in the interaction.
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Figure 15.5. V(D)J recombination. Shown in this figure is the recombination of two hypothetical segments, D27 and J5. The recombination begins with recombination-activating gene-1 (RAG-1) and RAG-2 recognizing the heptamers and monamers (see text) (Panel 1). They cut out one strand at the junction between the heptamer and the coding sequence. The other strand is severed by a nucleophile attack from the cut end (Panel 2). The two blunt ends of the heptamers form the signal joint (Panel 3), while a hairpin seals the cut coding ends (Panel 4). Hairpin is nicked open either by the RAG proteins or by a ubiquitous DNA double-strand repair complex (Panels 5 and 6). The opening is usually asymmetric and creates “overhangs,” with the nucleotides at the end of the “overhang” being complementary because they derive from the complementary opposite strand. These are known as palindromic (P) nucleotides (Panels 5 and 6) (from the Greek palindromicos, meaning moving forward and backward in succession). The double DNA strand breaks of the coding joint receive nucleotides from the function of TdT (nongermline nucleotides) known as N-nucleotides (Panel 7). Improperly paired nucleotides are removed (Panel 8), and the coding joint is completed with the addition of missing nucleotides (Panel 9). The P- and N-nucleotides added to the coding joint constitute the junctional diversity. |
HMGB proteins bind to DNA through two DNA-binding domains known as HMG-boxA and -B (118). Each consists of 80 amino acids interacting with the minor groove of the DNA. Box A bends distorted DNA structures, whereas box B severely bends linear DNA sequences. Mutations in HMGB proteins block synoptic complex formation. Another possibility is that HMGB proteins recognize distorted DNA structures induced initially by RAG1/ RAG2 binding and, following their binding, stabilize the formation of the synoptic complex and thus promote the DNA cleavage (119). The recognition by RAG proteins of the RSS is assisted by sequence-nonspecific DNA-binding proteins, HMG 1/2 (high-motility group), which enhance binding and cleavage.
During the first step of V(D)J recombination, the RSSs are recognized by the recombinase and are brought in juxtaposition, forming the synaptic complex, which is composed of a dimmer of RAG-2 and at least a trimer of RAG1 (120). In the second step, the “recombinase” nicks the phosphoester bond between the last nucleotide of the coding sequence and the first nucleotide of the RSS (Fig. 15.5, Panel 1). The precise mechanism of the hydrolysis of the phosphate ester bond is not known. The nicking reaction requires the physiologic divalent cation Mg2+. Cleavage leaves blunt phosphorylated signal ends and hairpin sealed coding ends (Fig. 15.5, Panels 2 and 3). The hairpin in the coding ends is formed between the 3′-OH of the coding sequence (top strand) and the central phosphor atom of the phosphate group between the coding sequence and the RSS of the opposite strand (the lower strand in Fig. 15.5). Formation of the hairpin requires significant bending of one or both DNA strands (Fig. 15.5, Panel 4). In the third step, the hairpin must be nicked open, so that the two coding ends form the coding joint. Opening of the hairpin may be done by a DNA repair complex (121) or RAG proteins themselves (122). Because nicking may not be exactly in the center of the hairpin, the opening creates an overhang in one strand formed by the nucleotides from the other strand (Fig. 15.5, Panels 5 and 6). These are known as P-nucleotides and are coming from the opposite or complementary end of the hairpin or palindrome (from the Greek palindromic, meaning movement forward and backward successively [i.e., the piston of an engine]). As a result, they have an inverse complementary relationship with the adjacent coding end. Most of the coding ends detected in normal lymphoid precursors have 3′ overhangs (123). TdT adds nucleotides known as N-nucleotides (nongermline) to the strand with the missing nucleotides (Fig. 15.5, Panels 7 and 8). Disruption of TdT drastically lowers junctional insertions. In fetal and neonatal periods, the N insertions are low or absent as a result of the developmental regulation of TdT. The alterations in the coding joint as a result of P- and N-nucleotides are the basis for “junctional diversity” (Fig. 15.5, Panel 9). The RAG proteins continue to be bound to the signal and coding ends in a four-end complex, known as cleaved signal complex, at least while the coding ends are still processed.
The RSSs are highly conserved among vertebrates from sharks to humans. In the RSS, the three heptamer nucleotides closest to the recombination site are the most important, whereas mutations at other heptamer positions still allow recombination. Variations in the position of RSS may influence the use of gene segments, Igκ vs. Igλ.
The RSS joint is assumed to be lost, being converted to reactive, broken DNA, to be disposed of in the next cell division: This is known as deletional recombination. However, the existence of inverted segments of DNA in these loci indicates that, sometimes, signal joints are retained in the chromosome (invertional recombination), a mechanism that restores chromosomal integrity.
The fate of the signal joint has taken an unexpected direction as a result of findings indicating that the RAG proteins perform genetic transposition (i.e., act as transposases) (124,125). As the name indicates, transposases transpose or insert pieces of DNA in a new location. The transposase MuA of the bacteriophage Mu is the best characterized of these types of enzymes. It consists of a tetramer in which two of the subunits nick the two ends of the Mu genome and the other two catalyze the transfer of the ends into the target DNA. These reactions are important in the transmission of drug resistance among bacteria and integration of retroviruses, such as HIV and others, into the genome. In fact, the MuA active site exhibits a striking similarity to the HIV integrase. It is interesting that the RAG-1/2 also act as a tetramers in V(D)J recombination and resemble the HIV integrase responsible for inserting DNA copies of the viral genome into the cellular chromosomes. There are striking similarities between the mechanisms of transposition and V(D)J recombination, suggesting that the RAG proteins may be members of the retroviral integrase superfamily (126). Hairpins during V(D)J recombination are the targets of the RAG-mediated transposition. Notable differences, however, exist in the nick-cleavage mechanisms between the two (127). Transposition of RSS in various positions, targeted to DNA, results in branched molecules (128,129). The high Mg2+ concentration prevailing in mammals, however, results in removal of transposed DNA by disintegration. No transposition in vivo has been reported as yet, probably because it is suppressed by GTP and the C terminus of the RAG-2 protein (130,131). Active transposition events are detrimental to the host because of the mutagenic potential of genomic rearrangements.
As a result of these recent insights into the nature of V(D)J recombination and the function of RAG-1/2, the question of the fate of the signal joint becomes very important. Signal joints have been found to be cleaved quite efficiently (132). In vitro experiments showed that transposition is targeted not to a particular DNA sequence, but to structural features, such as hairpins, inverted repeats, supercoiling of DNA forming cruciform structures, and generally distorted DNA structures (133,134). In vitro experiments demonstrating transposition were performed with only the core part of RAG-1/2; it is conceivable that other parts of the molecule exert an inhibitory effect on this potential function of RAG-1/2. Although the in vitro experimental evidence suggests that there is a bias toward transposition, there is no evidence that this occurs in vivo, as it severely compromises genomic stability in the lymphocytes. On the other hand, lymphoid malignancies are associated with chromosomal translocations, many of which involve the Ig on T-cell receptor (TCR) loci and potentially may be mediated by the V(D)J recombinase (135).
DNA Repair Mechanisms
Cleavage of DNA is always potentially dangerous, and although the recombinase function is essential for the integrity and normal function of the immune system, it is, at the same time, perilous. The V(D)J recombination as well as class-switch recombination are necessary processes, but they demand double-strand DNA breakage. As vital as these two processes are for survival, the DNA double-strand break (DSB) is a threat to survival, and it does occur in viral infections and malignancies. If genetic integrity is to be maintained, the DNA breaks need to be repaired (136). Nature has developed a highly complex and obviously efficient repair mechanism. Double-strand breaks generate an “alarm” mechanism, based on the transduction of signals from “sensors” to “transducers” and eventually to “effectors” for the DNA repair job. An important “sensor” is the MRN complex consisting of a nuclease (Mre-11), chromosome protein (Rad50), with a function concerned to chromosomal structural maintenance, and the protein Nbs-1 (137,138,139). Rad50 and Nbs1 are recruited rapidly to DSB sites to process the broken ends. MRN transduces the early signals to the main transducer, a kinase, known as ATM (Ataxia Telangiectasia Mutated), which rapidly phosphorylates various substrates needed in the repair pathway. ATM belongs to a family of proteins known as PI3-K-like protein kinases (PIKK), with five members, another being the catalytic subunit of the DNA-PK kinase. Four of the PIKKs are involved in the DNA-damage response: DNA-PK, ATM, ATR, and hSMG-1. ATM is first to be recruited to DSBs, where it is activated, probably as a result of changes in chromatin configuration, by MRN and then phsophorylates and activates several DNA repair and cell-cycle checkpoint proteins, i.e., the histone λH2AX and NBS1, which are required for recruiting other ATM targets.
ATM initiates a pathway that activates NF-κB (associated with cellular survival) as well as phosphorylates BID, which plays an anti-apoptotic role.
The DNA-PK (DNA-dependent protein kinase) complex consists of a catalytic subunit (DNA-Pkcs) and a DNA-binding complex called Ku, which binds altered DNA structures such as double-strand breaks, nicks, or hairpin loops. Recombination of broken DNA strands occurs either between strands that have long stretches of homology (homologous recombination) or between DNA strand breaks without relying on the presence of considerable homology between them. This recombination is known as nonhomologous end joining (NHEJ) (140). Mutations in this system result in the accumulation of V(D)J-specific double-strand breaks, indicating a defective repair mechanism, which causes SCID.
A new gene has recently been added to those already known that regulate double-strand DNA break repairs. It has been named Artemis (141), after the Greek goddess who was the protector of small children and animals (142). It belongs to the superfamily of metallo-β-lactamase enzymes and is associated with the DNA-PK complex (143). It is involved in the nonhomologous end-joining pathway, a DNA repair process used by eukaryotic cells (144,145), as well as in the repair of broken DNA ends from the V(D)J recombinase activity.
The repair requires that the hairpin in the coding joint is opened by the DNA-PK–Artemis protein complex (143,146). In such a complex, Artemis acquires endonuclease activity, which it does not possess by itself (146). Artemis has been shown to be mutated in patients with SCID expressing radiosensitivity (141). Patients with hypomorphic mutations in Artemis have not only immunodeficiency, but also predisposition to lymphoma (146).
RAG-1 and RAG-2 Proteins
The evolutionary origin of the RAG proteins has been controversial. Up to now it was accepted that the RAG genes originated by a horizontal gene transfer of a transposon, i.e., a mobile DNA element, and therefore are related to transposases, as discussed earlier. Recently, however, two genes in sea urchins (echinodermata) were detected that bear structural similarities to the RAG proteins (147). Echinodermata are an earlier evolutionary stage of chordates, to which vertebrates and humans belong. It is therefore possible that RAG proteins may have arisen very early in evolution and acquired their present-day function early in vertebrae evolution.
Both RAG proteins are required for V(D)J recombination, because lack of function of either one leads to SCID (148,149). A region that retains the recombinase activity is known as core and has been used for studies in vitro. For RAG-1, the core is located in the sequence 384–1,008 from a total of 1,040 amino acids and for RAG-2 in amino acids 1–383 of 527. The core of RAG-2 contains six repeats, each consisting of an antiparallel β sheet formed by four β strands. The repeats are arranged in a circular formation like blades of a propeller (149), a structure that is known to mediate protein–protein interactions. The C-terminal quarter of RAG-2 consists of a plant homeodomain fold, which is found in proteins with chromatin-binding properties (150).
Mutational analysis of the RAG proteins has provided some clues about structure–function relations. The catalytic properties of the proteins have similarities with members of the retroviral integrase superfamily (135) and require divalent metal ions for their function—a requirement that is characteristic of some nucleases, the functions of which depend on acidic amino acids. Indeed, in RAG-1, several acidic amino acids are critical for both nicking and hairpin formation, without affecting the DNA binding (151). For RAG-1, the N terminus is important for activity (152), and the binding of RAG-1 directly to DNA is supported by basic residues of RAG-2 (153). Deletion of the C-terminal region of RAG-2 results in a reduction of the number of B and T cells (154). The plant homeodomain of RAG-2 regulates differential access to sites of recombination, and although it is dispensable for D/JH recombination, it is essential for the VH/DJH step (155).
Somatic Hypermutation
The primary importance of somatic hypermutation (SHM) is thought to be related to affinity maturation, i.e., the increase of the affinity of antibodies following repeated antigenic stimulations (156). However, a wider scope of hypermutation is to provide better defence against hypermutating microorganisms. Many pathogens evade the immune system with variation of the antigens of their coat (“antigenic variation”), a mechanism employed by Trypanosoma, Neisseria, influenza virus, and HIV. Accordingly, SHM is the adaptation of the B cell in response to hypermutating microorganisms (157). SHM introduces mutations in the V gene at the rate of ∼103 mutations/base pair/cell division, i.e., 106-fold higher than spontaneous mutations of somatic cells.
At this rate, genomic integrity can be maintained only if the SHM specifically targets the Ig genes. However, it has been suggested that SHM is not a mechanism specific for the Ig genes but it may have targets other than the Ig genes. Perhaps the apparent preference for the Ig genes may be due to their higher density of certain “hot spots” (158). Somatic hypermutation appears to target preferentially certain hot spots, such as the short DNA motif DGYW, where D denotes adenosine (A), guanosine (G), or erythmidine (T); Y denotes cytidine (C), and W denotes A or T. Another motif is WRCH, where R denotes A or G and H denotes T, C, or A. Mutations from SHM have been detected in proto-oncogenes in diffuse large B-cell lymphomas.
Human Light-Chain Gene Loci
Recombination takes place between one RSS with 12-bp spacer (12-signal) and one with a 23-bp spacer (23-signal). This is the so-called 12/23 rule (see earlier). In the presence of Mu+, a single signal sequence supports double-strand cleavage, whereas Mg+ requires two signal sequences (159).
The two light-chain isotypes, κ and λ, comprise approximately 60% and 40% of all Igs, respectively. They consist of a V domain and a C domain of approximately 107 residues in length. The V domain is encoded by the V and J segments, the former encoding the first 95 to 96 residues and the latter encoding the remaining 12 to 13. There is a single Cκ gene, and there are several Cλ genes.
The κ locus contains approximately 76 Vκ segments grouped into six families and five Jκ segments but no D segments.
The λ locus contains 10 families of Vλ segments, a high number of pseudogenes, and several Cλ genes, each preceded by a single Jλ segment.
Heavy and Light Chains Getting Together
One of the consequences of the imprecision of the V(D)J recombination is a change in the reading frame at the junction between the two gene segments. When the segments join out of phase so that the triplet reading frame for translation is not preserved, the rearrangement results in V(D)J combinations with numerous stop codons that interrupt the translation. Such recombination events are known as nonproductive rearrangements. When gene segments are joined in phase, the reading frame is maintained, and the rearrangement is productive. If the junction lies within a codon, the resulting amino acid is encoded by nucleotides from both gene segments involved. The identity of the amino acid depends on the exact position of the joint and on the sequences of the individual gene segments. It is estimated that only one in three recombination attempts are productive, but the imprecision in the joints between variable gene segments increases their diversity by at least 100-fold.
The Ig gene rearrangements occur in an ordered fashion. They show preference for using certain gene segments, result in allelic exclusion, and follow a certain sequence in the order of rearrangement of the various loci. This indicates that there are regulatory mechanisms underlying the process of rearrangement. The IgH heavy-chain gene is rearranged first and results in the formation of a complete V gene from three individual segment clusters. One of the D segments joins one of the JH segments, but the D-to-JH rearrangement does not take place exclusively in B cells because it has also been found sometimes in T cells. In the next step, which is regulated by the Dμ protein, a V segment joins the DJH complex. Dμ protein is encoded by the DJH complex and the μ–heavy-chain constant gene—that is, it is a μ chain that lacks a VH segment. Dμ protein can be expressed on the membrane of the cell with an SLC. Expression of Dμ protein prevents further VH-to-DJH rearrangement, or rearrangements are diverted to another pathway such as κ-chain rearrangements, which are accelerated. Productive, complete μ-chain rearrangement inhibits further rearrangements in the opposite allele (allelic exclusion) (160). Completion of the μ-chain rearrangement is followed by rearrangements of the κ chain, and if both κ alleles fail to rearrange productively, the λ-chain gene is rearranged last (Fig. 15.6). However, this hierarchical order of Ig gene rearrangements has been challenged because examination of B cells at different stages of early development showed that the heavy- and light-chain genes rearrange independently (161). At the end of its early developmental stage, the B cell emerges with the expression on its surface of a unique antigen receptor consisting of IgM accompanied by two accessory molecules, Igα and Igβ (162).
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Figure 15.6. Heavy immunoglobulin (Ig) M chain–kappa light chain rearrangement. With the completion of the V(D)J recombination of the μ chain and the formation of a complete μH chain, the LK chain rearranges, and, if successful, it forms the complete IgMK molecule. If both κ alleles rearrange unsuccessfully, the λ gene rearranges. If successful, an IgMλ molecule is formed. If both λ genes cannot rearrange successfully, the cell dies by apoptosis. |
Regulation of V(D)J Recombination
For the recombinase to initiate rearrangements, the gene must be accessible—that is, the locus must be able to act as a template for the recombinase. In cells that are competent to rearrange, it was found that JK and VH genes have already been transcribed, but these RNAs (called germline or sterile transcripts) are incapable of encoding the protein. There is correlation between transcription and gene rearrangement. Several possibilities may exist to explain this finding. Because inactive genes are not accessible to the recombinase, it is possible that the altered structure of the chromatin allows the recombinase to recognize the RSSs. Alternatively, enhancers establish altered chromatin regions where both the recombinase and the transcription machinery have access. This is known as the accessibility hypothesis (105,163).
Enhancers play an important role, but promoters and silencers also function in gene accessibility. Enhancers and promoters function as accessibility control elements (ACEs) to regulate V(D)J recombination. Transcription factors are recruited to ACEs and alter accessibility to the recombinase. The Ig heavy-chain enhancer (Eμ) is located within the intron between the JH segments and the Cμ gene and is associated with matrix attachment regions. Targeted mutations of Eμ have resulted in a dramatic decrease in JH recombination (164). There are three additional enhancers within the Ig heavy-chain locus, and one of them (DQ52) seems to compete more efficiently for conferring accessibility to the JHregion. Interaction between DQ52 and Eμ is likely responsible for the ordered rearrangement (i.e., D to JH followed by VH to DJH) (165). The κ–light-chain intronic enhancer is active only in mature B cells and plasmacytomas, and its stage restriction is dictated by a single motif that binds the transcription factor nuclear factor-κB (NF-κB), which recognizes a 10-bp motif termed κB. NF-κB consists of two subunits, with the smaller one containing the DNA-binding function.
Enhancers lie also in the vicinity of the Cα gene and two others in the κ locus. The one between Jκ and Cκ genes becomes transcriptionally active during pre-B- to B-cell transition (166). The mechanism by which promoters regulate accessibility is not known. The matrix attachment regions are regions rich in AT, bind nuclear matrix, and demarcate regions of chromatin that undergo base unpairing and other regions that mediate binding of topoisomerase II. This regional division of chromatin may also be regulated from a distance by trans-acting factors, which bind to specific motifs within the enhancer and promoter elements. For example, disruption of E2A and EBF transcription factors results in block of B-cell development at a stage before Ig heavy-chain gene rearrangements. Despite the promiscuity and redundancy of regulatory elements, transcription is still tissue lineage- and stage-specific.
V-Gene Repertoire
Certain aspects of the study of the Ig V gene repertoire and the Ig gene rearrangement have found wide applications in clinical medicine (167). The total number of V genes defines the available repertoire. However, not all genes are equally expressed; as a result, the expressed repertoire is usually biased, because some V genes are expressed in significantly higher frequency than would be expected if all had an equal chance for rearrangement. This biased expression affects all gene segments—V, D, and J. For example, the JH4 segment (one of the existing six), on the basis of equal opportunity with the other five segments, should be detected in 17% of B cells, yet it is found in 32% of B clones from fetal liver and in 42% of pre-B-cell acute lymphocytic leukemia (ALL). The bias of the expressed repertoire shows striking association with certain diseases. For example, a member of the VH4 family, VH4-21, is found in cold agglutinins (168). Preferential expression of certain VHsegments is also detected in ALL clones. In ALL, the frequency of N-nucleotide additions differs in children <3 years of age (12.5%), as compared with children with ALL >3 years of age (89%) (169). Because in fetal B lymphocytes the frequency of N-nucleotide additions is low, it is suggested that the transforming event in the younger age group probably occurred during fetal life.
In chronic lymphocytic leukemia (CLL), the use of V genes is also restricted; the same is true in non-Hodgkin lymphomas. It is interesting that in certain cases of follicular lymphoma, the V genes have not only undergone somatic mutations, but also continue to do so during the course of the disease, suggesting that the malignant clone is responsive to an antigen (170,171). Somatic mutations have also been detected in multiple myeloma, as expected, because the malignancy derives from an advanced stage of differentiation of B cells (172).
Defects in V(D)J recombination have been identified in human diseases (173). For example, loss of RAG-1/RAG-2 function results in SCID. In certain autosomal recessive diseases, hypersensitivity to DNA-damaging agents results in chromosomal breaks and rearrangements with an increased incidence for development of leukemia and other malignancies. They have been classified as DNA repair disorders and include AT, Fanconi anemia, xeroderma pigmentosum, and Bloom syndrome (see later). V(D)J recombination with artificial substrates is normal in these patients.
In mice, an autosomal recessive mutation, the SCID mutation, results in absence of B and T cells as a result of impairment of the V(D)J recombination. Both lymphoid and nonlymphoid SCID cells are hypersensitive to killing by ionizing radiation because of a DNA double-strand base-repair defect. It is rather important that a human gene on chromosome 8q11 has been identified that restores V(D)J recombination, double-strand base repairs, and normal resistance to irradiation in SCID cells (174,175).
Pathology of V(D)J Recombination
V(D)J recombination is absolutely necessary for immune diversity and survival. However, this objective can be achieved only through DSBs that threaten genomic stability. The complexity of the recombinase function is matched by complex DNA repair machinery. Disorders related to V(D)J recombination may be considered in two categories: those related to the first step (cleavage) and those related to the second step (cleavage repair) (176).
The Mutation of Severe Combined Immunodeficiency
An experiment of nature in mice has revealed the two fundamental processes operating in V(D)J recombination, i.e., double-strand breaks during the gene rearrangements, followed by DNA repair. Bosma reported the SCID mutation in mice (177), associated with severe combined immune deficiency, lack of mature lymphocytes, and agammaglobulinemia. Productive rearrangements occur only at a low level, and coding joints are absent, whereas signal joints are normal. SCID T cells have long P-nucleotide sequences and accumulate hairpins at the TCRδ coding ends. In addition to the lack of rearrangements, there is an inability to repair damage from irradiation. The SCID mutation is located on mouse chromosome 16; because human chromosome 8 can complement both mouse SCID defects, it has been assumed that the SCID defect for humans should be in chromosome 8. However, human chromosome 8 is not associated with any known immunodeficiency or DNA-repair diseases. The gene for the DNA polymerase 8, however, is in chromosome 8p11-12. Mutated RAG genes have been detected in nine patients from seven families (178). Recombination activity was undetectable with mutations in the RAG core domains, and in four patients there was a complete B-cell differentiation arrest between the pre-BI and pre-BII stages.
V(D)J Recombination Defects: DNA Cleavage
Two types of RAG-1 and RAG-2 gene inactivations have been identified in mice. The first type results in the inactivation of the recombinase so that V(D)J recombinations cannot be initiated (109,110). The mice have no mature T and B lymphocytes, but there is an increase of immature lymphocytes in the lymphoid organs. The second type of mutation consists of a single conservative amino acid substitution in both proteins. It does not affect the initiation of V(D)J recombination but severely impairs the coding and signal joint formation (179,180). The mutant enzymes are defective in hairpin opening in vitro and the formation of the coding joint in vivo. This evidence indicates that the RAG proteins, in addition to their cleavage function, are important for the formation of the joints; therefore, they must remain associated with the postcleavage complex to achieve opening of the hairpin.
No human syndrome has yet been identified that is comparable to the mouse SCID mutation. Approximately 20% of SCID patients lack both T and B lymphocytes and have normal NK cell counts (181). Although some of these patients have RAG-1 or RAG-2 gene mutations (182), others are characterized by increased radiosensitivity of their bone marrow cells and fibroblasts (RS-SCID) (183).
The predicted RAG-2 structure consists of two globular domains separated by a hinge of approximately 60 amino acids. The largest domain (350 residues) includes the previously described core region of RAG-2, which is enzymatically active. This domain forms a β propeller with six blades arranged in a circle from a center. Each blade is a β sheet consisting of four β strands. The β-propeller fold (present in some integrins and other proteins) is known to mediate protein–protein interactions. Six out of seven mutations so far described in humans are clustered on one side of the propeller in regions exposed to the solvent (184). This side is involved in RAG-1–RAG-2 interactions, which are absolutely critical for functional activity. Two of these mutants (C41W and M285R) have been found to reduce the interaction between RAG-1 and RAG-2 in vitro, and no DNA-binding activity of the complex was detected (185). This is consistent with the hypothesis that RAG-2 stabilizes the RAG-1/RAG-2 complex (186).
Omenn Syndrome (OMIM No. 603554)
Omenn syndrome (OS) is an inherited disorder characterized by absence of circulating B cells and infiltration of the skin and intestine by activated oligoclonal T lymphocytes (HLA-D+). There is eosinophilia and elevated serum IgE (187,188). The patient has diffuse erythrodermia, lymphadenopathy, hepatosplenomegaly, protracted diarrhea, and failure to thrive. The activated T cells secrete Th2 types of cytokines (189), highly restricted in their TCR repertoire (190). They infiltrate skin, gut, liver, and spleen, causing a graft-versus-host–like disease (191).
Inherited mutations in RAG-1 and RAG-2 genes have been reported (192,193) in a series of 20 patients with Omenn syndrome resulting in partial V(D)J recombinase activity (194). Because similar mutations have been detected in patients with T/B SCID, it has been suggested that an additional factor exists in OS to account for the different clinical picture, such as an autoantigen or an external antigen that drives T-cell activation. In an interesting mutation in a 5-week-old girl with Omenn-like SCID, the RAG-1 gene had a deletion of a nucleotide (631 delT) (193); as a result, the N-terminal region of RAG-1 protein was deleted by a premature stop codon. The patient had a high number of T cells with almost a polyclonal TCR gene rearrangement, but there were no B cells and hardly detectable Ig gene rearrangements. The truncated RAG-1 protein apparently could still support TCR, but not Ig gene rearrangements. This suggests that the N terminus of RAG-1 is specifically involved in Ig V(D)J rearrangements. A case of OS was reported with a mutation in the Artemis gene (195). The maternal allele was mutated, and as a result, the Artemis protein was not functional as endonuclease and was lacking hairpin opening activity in a V(D)J recombination assay.
In a cohort of nine patients with OS, only two of the nine had RAG mutations (196). All patients expressed restricted TCRVB repertoire, which might have occurred in response to infections. The cause of OS in the seven patients remained unknown.
V(D)J Recombination Defects: DNA Repair
A DSB is one of the most significant lesions that threaten cell integrity, but the cells have developed exquisite repair machinery to maintain genomic stability. Immune diversity, another function vital to the survival of an organism, has to cross the dangerous path of DSBs (197). The body uses the DSB machinery to repair the “damage” caused by the recombinase (198). The major mechanism for repair of DNA DSBs in mammalian cells is nonhomologous end joining. Five proteins operate in nonhomologous end joining; three of them form the DNA-PK complex (Ku70, Ku80, and DNA-PK), the XRCC4, and the DNA ligase IV (199). The Ku protein is an autoantigen that induces autoantibodies in patients with scleroderma, polymyositis, or systemic lupus erythematosus. Two proteins, Ku70 and Ku82, form a stable heterodimer, which binds DNA ends regardless of sequence composition. The heterodimer forms a complex with a third component—the DNA-PK. The DNA-PK complex phosphorylates many DNA-binding proteins, including transcription factors c-Jun and p53. However, the most efficiently phosphorylated substrates are those bound close to DSBs, including the Ku components of the DNA-PK complex. It appears that the main function of the DNP-PK complex is regulation of the nonhomologous end-joining process. Mice that are defective in Ku proteins have SCID (200,201); in general, defects in the DSB repair machinery are associated with immunodeficiencies (202).
Ataxia Telangiectasia (OMIM No. 208900)
AT is an autosomal recessive disorder characterized by immuno-deficiency, progressive cerebellar ataxia, oculocutaneous telangi-ectasias, clinical radiosensitivity, chromosomal instability, and elevated risk for development of lymphoid malignancies. AT is due to deficiency of a protein kinase that mediates repair to double-strand breaks generated during metabolic processes or from DNA-damaging agents (203). One of the hallmarks of AT is the generation of aberrant rearrangements during V(D)J recombination, such as translocations, large chromosomal deletions, and inversions. A protein kinase known as ATM (Ataxia Telangiectasia Mutated) mediates repairs of DSB by phosphorylating and activating several repair proteins (137,139).
Immunodeficiency affects both T and B cells, and there is a decrease of serum IgA. The AT cells display γ-irradiation sensitivity and cell-cycle checkpoint control (i.e., inability to arrest at the G1-S- and S-phase checkpoints and, at the time of irradiation, an impaired G2-M arrest). AT cells also have a DNA-repair defect (204). They can rejoin DNA BSB efficiently, but the defect is localized on a protein member of the PI3K family of kinases with serine threonine protein kinase activity (205), which phosphorylates a number of proteins involved in mechanisms of damage repair (i.e., p53 and Chk1) (206,207). The majority of patients have frameshift mutations, which inactivate the gene.
Nijmgen Breakage Syndrome (OMIM No. 251260)
Nijmgen breakage syndrome is a rare autosomal disorder with clinical features overlapping with those of AT. The patients have defective humoral and cellular immunity, radiosensitivity, chromosomal instability, and predisposition to cancer (207). The patients have recurrent bacterial sinopulmonary infections, hypogammaglobulinemia, and impaired antibody responses to antigens. The cells from the patients have defects at some checkpoints of the cell cycle (208). The defective protein is nibrin or p95 (NBS1), which shows homology with the protein Xrs2, involved in DNA repair response in yeast.
Bloom Syndrome (OMIM No. 210900)
Bloom syndrome (BS) is a rare autosomal recessive disorder with immunodeficiency, genomic instability, and predisposition to cancer. It presents with a variable clinical picture—respiratory infections, chronic lung disease, and low IgM levels. Affected individuals show sun sensitivity in the face and infertility (209). BS is caused by mutations in the BLM gene, located in chromosome 15 at 15q26.1, which encodes the BLM protein (210), a member of the helicase subfamily. It displays ATP- and Mg2+-dependent 3′-5′-DNA helicase activity. It is a member of the BASC (BRCA-1–associated genome surveillance complex) family, which includes ATM, NBS1 (defective in Nijmegen syndrome), MRE11, and other proteins that have been identified in breast and colorectal cancers. BLM accumulates at damaged chromosomal sites following ionizing radiation and is phosphorylated by ATM. It accumulates in stalled replication forks and plays a crucial role in control of chromosomal replication.
Defects in Ligases
There are multiple DNA ligases in higher organisms. A point mutational change in DNA ligase I, which does not abolish the activity of the enzyme, was found in an individual who experienced recurrent sinopulmonary infections leading to bronchiectasis (211). The LIG4 syndrome is associated with defects in DNA ligase IV, which functions in NHEJ. The patients display pancytopenia and microcephaly. The V(D)J recombination shows mildly impaired fidelity, which may result in oncogenesis (176). A severe form with a SCID presentation, bacterial, and viral infections evolving into an Epstein-Barr virus (EBV)–induced lymphoma has been reported (211).
Xeroderma Pigmentosum (OMIM No. 278700)
Xeroderma pigmentosum is a rare disorder associated with sun sensitivity, high risk of cutaneous malignancy in sun-exposed areas, and immunodeficiency in some patients (212). Some aspects of immunosuppression associated with xeroderma pigmentosum are due to defects in the DNA-repair machinery. The patients have impaired NK cell cytotoxicity.
Genetic Defects of Early B-Cell Development
Cell Signaling during B-Cell Development
After the VH-to-DJH rearrangement of the H-chain gene, the cytoplasmic μchain pairs with the SLC and traffics to the cell surface in association with the signal-transducing chains Igα and Igβ to form the pre-B-cell complex. Signaling for the transition from pre-B cells to Ig+ B cells requires the immunoreceptor tyrosine-based activation motif (ITAM) motifs of the cytoplasmic domains of the Igα and Igβ chains, because inactivation of ITAMs by mutation blocks the transition to the Ig+ B-cell stage (213). Deficiency of Igβ chain by gene targeting abolishes formation of the Igα/Igβ dimer and blocks assembly of the SLC. As a result, differentiation of B cells is arrested at the pro-B-cell stage (214).
Interactions of pro-B and pre-B cells with stromal elements are necessary for progression through the early stages of B-cell development, but a ligand for the pre-BCR has not yet been identified, and it is conceivable that the receptor may be signaling constitutively. It is apparent that the presence of ITAM motifs and the concentration of signaling molecules around them are the critical factors in determining progression through the early stages of B-cell development. Pro-B cells are able to become pre-B cells in the absence of the μH chain as long as the Igα/Igβ chains are aggregated and thus can recruit sufficient numbers of signaling molecules around their ITAMs (215). Furthermore, signaling by other molecules unrelated to pre-BCR, such as EBV latent protein 2A (LMP2A), can drive pro-B- to pre-B-cell transition. LMP2A protein spanning the membrane 12 times, with ITAM motifs in its cytoplasmic region, constitutively possesses its own signaling activity in nontransformed cells (216). Phosphorylation of these motifs recruits Syk and Src kinases and thus bypasses the normal checkpoints of B-cell development. Surface Ig (sIg)–negative B cells with latent EBV infection are released in the periphery to colonize peripheral lymphoid organs.
Of all the signaling molecules assembled around the cytoplasmic tails of the pre-BCR complex, the Syk kinase is important for the transition from pro-B- to pre-B-cell. The ITAMs of Igα and Igβ chains act as docking sites for Syk kinase and trigger its activation (217). An important function for signaling is provided by the adaptor protein BLNK, which acts as a scaffolding to link Btk (and other molecules) to downstream signaling molecules. In BLNK-/- mice, the transition from pro-B- to pre-B-cell stage is blocked (218), and a similar defect has been detected in a patient with deficiency of BLNK and block of B-cell development (219). Syk kinase deficiency is more effective than BLNK deficiency in arresting B-cell development.
Signaling Defects
Failure of B-cell development may result from defects in signaling through the pre-BCR. These experiments of nature have helped in delineating the molecular mechanisms of early B-cell development (81). Immune deficiencies resulting from arrest of B-cell development are usually associated clinically with recurrent bacterial infections, laboratory findings of markedly reduced numbers of B cells, and hypogammaglobulinemia. Of all the patients, 85% have mutations in Btk, whereas the remaining 15% constitute a heterogeneous group with defects of various signaling molecules.
Defects of λ5
The SLC is formed from two components: the VpreB and the λ5. In humans, there is one VpreB gene and three λ5 genes (220). A boy with mutations in both alleles of the gene λ5/14-1 has been described with recurrent infections, hypogammaglobulinemia, and lack of B cells detected in early age. The patient had three base-pair changes in a single allele and alterations within exon 3. Changes in codons 131 and 140 were silent, whereas that in codon 142 resulted in the replacement of proline, which in this place is highly conserved, to leucine (P142L) (86). The boy at 9 years of age had 0.1% CD19+ cells in the blood, but these cells were of the mature phenotype. In contrast to this patient, mice that are deficient in λ5 have a leaky block at the pro-B-cell stage and still maintain 10 to 20% of the B cells.
Defects of Immunoglobulin α(CD79a)
While screening several patients with defects of B-cell development, one 2-year-old girl was identified with an A-to-G substitution in the splice-acceptor site preceding exon 3 of Igα (221), resulting in Igα transcripts that were aberrant, because most of them had no exon 3. The patient showed failure to thrive, had diarrhea in the first month of life, and, later, had bronchitis and neutropenia. She had severe hypogammaglobulinemia and absent B cells. The block of B-cell development was at the pro-B-cell stage.
Defects of μ Heavy Chain
Defects in μH have been associated with agammaglobulinemia in individuals from two families. The defects consist of a large deletion of a 75- to 100-kb segment, including D, JH, and μ genes, or a base-pair substitution in the alternative splice site, with inhibition of the synthesis of the membrane form of Ig (222). μH mutations with agammaglobulinemia should be distinguished from X-linked agammaglobulinemia, because the disease from μH mutations can occur in females. Another report described a female patient with a cytosine insertion at the beginning of the CH1 exon of the μ gene, which resulted in premature codon and absence of the μH chain (223).
Defects of B-Cell Linker Protein
The gene for human BLNK is located on the long arm of chromosome 10, at 10q23.22. BLNK is an adapter protein, which, after phosphorylation by Syk, recruits several signaling molecules (i.e., PLCγ, Vav, Cbl, and Btk). A 20-year-old man with absent B cells and hypogammaglobulinemia was found to have two base-pair alterations: One of them did not change the amino acid (proline), but the second one, which was an A-to-T substitution, affected the +3 position of the splice-donor site for intron 1 approximately 20 bp downstream from the first alteration. The second defect resulted in a marked decrease of the BLNK transcripts and BLNK protein (219). The patient had recurrent infections, undetectable serum Ig, and, at the age of 20 years, <0.01% of CD19+ cells in the blood. Mice with BLNK deficiency have only a leaky block of B-cell differentiation at the pro-B-cell stage.
In general, mutations of λ5 or BLNK causes a profound block of B-cell differentiation in humans.
Bruton Agammaglobulinemia (OMIM No. 300300)
In 1952, Bruton described a male child with hypogammaglobulinemia and early onset of bacterial infections (224), later found to be inherited in an X-linked pattern that became known as X-linked agammaglobulinemia (225). The constellation of findings consists of very low serum Ig levels (i.e., for IgG, approximately 10% of the normal control), no antibody production after immunization, markedly decreased B cells (0.3% of normal levels) (226), and no germinal centers, the hallmark of antibody production, in the lymph nodes. The genetic defect is located in the midportion of the long arm of the X chromosome (i.e., Xq22) (227). There are several variants of the disease in relation to immunologic function and clinical heterogeneity (228). The product of this gene was identified as a Src protein tyrosine kinase (PTK) that was called Bruton tyrosine kinase or Btk (229,230). More than 400 mutations have been characterized to date (231).
Btk is a member of the Btk/Tec family of PTKs, which includes Btk, Tec, Itk, Rlk, and Bmx (232). In the C-terminal end, Btk contains the catalytic (SH1) domain, and next to it lies the SH2 domain, which associates with phosphorylated tyrosines in other signaling molecules. The SH2is followed by one SH3 domain, which binds proline-rich sequences in protein–protein interactions, and next to it is a Tec homology (TH) domain, which contains the Zn2+-binding Btk motif and a proline-rich stretch. At the N terminus is the pleckstrin homology (PH) domain, which binds with high-affinity phosphatidylinositol-3,4,5-triphosphate (PIP3) and is responsible for the translocation of Btk to the membrane. Btk and Tec kinases lack the unique N-terminal myristylated SH4 region, which is carried by other Src kinases. Btk occupies a central position in BCR signaling and regulation of lineage development, and is linked to multiple downstream signaling pathways through BLNK (also known as SH2 domain–containing leukocyte protein [SLP]-65). BLNK functions as a scaffolding protein and binds to the SH2 domain of Btk (233). It connects Syk to Btk and links Btk to downstream signaling molecules, like PLCγ2, a linkage essential for Ca2+ signals (234). Btk also associates with Wiskott-Aldrich syndrome protein via the SH3 domain; this is the reason that in XLA patients with defective Btk, the collagen-induced tyrosine phosphorylation of Wiskott-Aldrich syndrome protein in platelets is reduced (235). Different regions of Btk are critical for Btk activation and signal transduction, that is, the PH domain binds to Ca2+-dependent (α, βI, βII) and Ca2+-independent isoforms of protein kinase C (PKC) (∊ and ζ), to IP3 (236), and to heterotrimeric G proteins (237). Sequential phosphorylations of regulatory tyrosines by the BRC-associated Src kinases activate Btk, such as the phosphorylation of Tyr551 in the kinase domain or Tyr 223 in the SH3 domain (238,239).
In Src kinases, negative regulation is mediated by the Tyr527 in their C-terminal region, but Btk has no equivalent residue, and it is likely that such a function is mediated by the recently identified inhibitor of Btk (IBtk). IBtk binds to the PH domain and down- regulates Btk function, such as Ca2+ mobilization and NF-κ B activation (240).
It appears that Btk is a bidirectional regulator with the capacity to deliver survival or apoptotic signals depending on the expression of surface receptors and the stage of the cell differentiation (241,242). The molecular mechanism of Btk function in XLA is still not quite clear. Mutations of Btk gene, i.e., deletions, insertions, or substitutions (243), produce variable degrees of immunodeficiency, whether in the kinase domain (230) or in other domains.
Evidence from the bone marrow of XLA patients shows that there is an expansion of pro-B cells, whereas the numbers of more mature B cells are negligible (244). Based on the expression of VpreB, the pre-BI cells are SLC-positive, and a number of them are large in size and cycling, whereas the pre-BII cells are -negative. It appears that Btk blocks normal B-cell maturation at a point just before that stage (i.e., it interferes with the transition of pro-B cells to pre-BI stage) (244). Some patients express an inactive form of Btk and have a more severe form of XLA than those expressing no Btk molecule (245).
Phenotypic Changes in the Early Stages of B-Cell Development
The study of phenotypes of B-cell precursors in normal adult bone marrow is hampered by the relative paucity of these cells. Therefore, some investigators have resorted to the study of leukemias under the assumption that they accurately reflect normal B-cell development. Because aberrant phenotypes in acute leukemias have been identified, suggesting asynchronous antigen expression, the validity of the results from such an approach has been challenged (1,246). Normal adult bone marrow (247) or fetal bone marrow and fetal liver (248,249) have been used as a source of B-cell precursors in cultures in vitro or after injection in SCID mice. B lymphocytes derive from cells expressing high levels of CD34 (250,251).
Phenotypically, B-cell development can be separated into three stages: (a) precursor B cells (which includes pro-B cells and pre-B cells), (b) immature B cells, and (c) mature B cells (Fig. 15.1). Markers defining the first stage are CD19, and, occasionally, some other “promiscuous” markers, such as CD2 or CD7, are detected, but probably the best marker is the detection of the SLC by a monoclonal antibody, a hallmark of a B cell, progressing through the progenitor stages. Entrance into the immature B-cell stage is defined by the surface expression of IgM, with antisera not only for μ chains but also for light chain κ or λ. This indicates that the μ chain is associated with a light chain and not with the SLC, suggesting that the Ig gene rearrangements are complete. The immature B-cell stage is characterized by expression of only IgM, without IgD. Functionally, this is important because the former phenotype defines a cell that is vulnerable to tolerogenic stimuli, indicative of its “immaturity.” At this stage, the cell expresses other markers, such as CD10 and CD24, at high density. The mature B-cell stage is simply identified by coexpression of IgD as well as other markers such as CD20, CD21, and CD22.
Another useful phenotypic marker for precursor B cells is the nuclear enzyme TdT (252). TdT is a DNA polymerase catalyzing the elongation of polynucleotide chains without template (253) and adds N-nucleotides at the DNA cleavage site during V(D)J recombination. CD24 is expressed at a very early stage, and it is present at a much higher density as compared with the mature sIg+ B lymphocyte (254). CD2 expression has been detected on biphenotypic ALL cells and also on their normal counterparts in human fetal hematopoietic tissues (255,256). Whether they represent common T- and B-lineage progenitors remains to be seen. Pre-B cells have been detected during fetal life first in the liver (by 8 weeks) and later in the bone marrow (by 12 weeks) (257,258). In the liver, they are mixed among other myeloid cells next to liver parenchyma in extrasinusoidal areas (259).
The small pre-B cell is the immediate precursor of the immature B cells and starts to express sIgM by the end of the first trimester. In adults, pre-B cells are present in the bone marrow, where they constitute 0 to 7% of the nucleated cells and only rarely are noted in the peripheral lymphoid organs.
During the last phase of pre-B cell, CD34 and TdT disappear, CD10 is down-regulated, and the cell expresses first CD22 and later CD20. Before its appearance on the cell surface, CD22 is detected in the cytoplasm very early during development. It is identical to the surface CD22 and is B-lineage–restricted (260). The receptors for Fc are not present on pre-B cells, but receptors for C3b have been detected in some large pre-B cells and in approximately 40% of the small pre-B cells (261).
Mature B Lymphocytes
The hallmark of maturing B lymphocytes is the appearance of sIg, and the first one expressed is IgM. During fetal life, the IgM+ cells (which are mostly CD5+) populate the lymph nodes and settle in the primary lymphoid follicles after 16 to 20 weeks of gestation. By 13 weeks, most of the B cells are IgM+IgD+, and later IgG or IgA is added. This triple phenotype of fetal B cells persists until birth but is converted to the adult single phenotype during the first few months of life. In adult life, cells expressing IgG or IgA are mostly IgM- and IgD-. By 10 weeks of age, practically all B cells in the spleen are IgM+IgD+, whereas in the bone marrow only 30 to 40% coexpress the two isotypes. Maturation of B lymphocytes is associated with a change in the density of the two isotypes from IgMhighIgD- to IgMhighIgDlow and finally to IGMlowIgDhigh. New markers appear on B lymphocytes with the expression of IgM (Fig. 15.1). CD21 is present in more than 90% of resting B lymphocytes and is the same as the C3d/EBV receptor. With activation, CD21 and CD22 are lost, and, concomitantly, CD23 is expressed. CD23 is up-regulated by IL-4 (262) and the EBV nuclear antigen-2 (EBNA-2) (263) and is down-regulated by interferon-γ. CD24 expression decreases with B-cell maturation, and, in combination with the expression of CD45, it can be used to determine stages of B-cell maturation relevant to B-cell neoplasias (264). CD45 expression also varies with B-cell maturation. Its expression is low on the most immature precursors in the bone marrow, is up-regulated as normal B-cell differentiation progresses, and then declines at the terminal stages of differentiation as plasma cells become negative for CD45 (265).
Probably the most important application of the phenotypic changes occurring during B-cell differentiation is in B-cell malignancies, because it is widely accepted that B-cell leukemias/ lymphomas correspond to distinct stages of differentiation. An attempt to incorporate immunophenotypes (and genetic techniques) into the traditional morphologic features has resulted in a revised classification with well-defined disease entities (266) and more recently in the revised World Health Organization classification (267).
Surface Immunoglobulin
sIg serves as the receptor for antigen on B lymphocytes. It is identified most commonly with the use of fluorochrome-conjugated anti-Ig antisera. The first Ig to appear on the membrane of B lymphocytes is IgM. In contrast to the secreted pentameric form, the membrane IgM (mIgM) is composed of only one subunit (8S) containing two H and two L chains. The second isotype to appear is IgD, which is present either as a complete four-chain molecule or as a half-molecule. IgD is present in the serum in very low concentrations, and it is not known to have a role in humoral defect mechanisms. It has been suggested that B-cell surface IgD may modulate humoral responses by finely tuning the strength of BCR upon encounter with antigen (268). The ratio of secretory to mIg increases with maturation from resting B cells to plasma cells. The transmembrane and secreted forms are encoded by the same gene. There are two transcription termination of polyadenylation sites: One is 3′ to the last constant chain exon, and the second is 3′ to the second transmembrane exon (Fig. 15.7). Termination at the first site results in loss of transmembrane exons and production of the secreted form, whereas polyadenylation at the second site produces the membrane form.
Between 50,000 and 100,000 molecules of Ig are found on each B lymphocyte. This number is an average, because the density of sIg varies among individual B cells (269). The mIg is distributed throughout the membrane in small clusters, including the microvilli (270,271). In human B lymphocytes, the clusters are separated from each other by a few thousand angstroms of bare membrane, indicating restriction in the free distribution of mIg. In the mouse, the clusters are interconnected by strands composed of a few molecules, forming a lacy, continuous network. When examined at 4°C in the fluorescent microscope with fluorescein-conjugated anti-Ig antibodies, the mIg appears distributed as a ring, but an increase in the temperature to 37°C prompts the formation of clusters (patching) that move rapidly toward one pole of the cell, forming a cap (272) (Figs. 15.8 and 15.9). Shortly after the formation of the cap, changes in cell shape occur, accompanied by cell movement. The cell pushes out a projection located opposite to the cap that corresponds to membrane ruffles noted on scanning electron microscopic analysis (273) (Fig. 15.10). After the formation of the cap, a constriction under the cap encircles the cell, and the cell takes the shape of a hand mirror, with the cap occupying the area of the uropod with numerous microvilli. A dense band of microfilaments is noted under the constriction that separates the cell into the uropod containing the cap and the area opposite the cap containing the cell organelles and the nucleus. The uropod is well formed after the cap is completed (274).
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Figure 15.7. Expression of membrane versus secreted immunoglobulin (Ig) depends on alternative processing of the primary RNA transcript. There are two polyadenylation (poly A) sites, one for secreted (Se) and one for the membrane (Me) forms. Cμ1–Cμ4 are the exons of IgM. Cy, cytoplasmic region; mRNA, messenger RNA; TM, transmembrane region. |
Capping requires energy provided by the respiratory chain and glycolysis. In most human B lymphocytes, capping does not take place because the Ig clusters on the surface of the cells are widely separated from each other. As a result, cross-linking is not possible because cell motility can be blocked without any effect on Ig capping (274), and capping in many B cells is already complete before any amoeboid movement is noticed.
Colchicine, which disrupts microtubules, has no effect on capping, and only a slight effect is noted with cytochalasin B, which disrupts the microfilaments. The combination of these drugs, however, inhibits mIg capping profoundly. Calcium, which plays an important role in contractile systems, is also critically involved in mIg capping (274,275).
The fate of the complexes in the cap has been studied morphologically using autoradiography, electron microscopy, and measurements of radioactivity. The bulk of the complexes is internalized (endocytosed) in the presence of adequate antibody. Endocytosed material is catabolized within the lysosomes, and small fragments are released into the culture medium. With the completion of endocytosis, the B lymphocyte remains free of mIg until it is resynthesized to the original level by 24 hours.
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Figure 15.8. Demonstration of surface immunoglobulin (Ig) by immunofluorescence on B lymphocytes. Viable normal B lymphocytes treated with anti-IgM antiserum at 4°C present a staining pattern of a uniform ring around the periphery of the cell (A) or discrete patches uniformly dispersed over the cell surface (B). (From Zucker-Franklin D, Greaves MF, Grossi CF. Atlas of blood cells, 2nd ed. Philadelphia: Lea & Febiger, 1988, with permission.) |
B-Cell Antigen Receptor Complex: Structure and Signaling
The sIg of B lymphocytes forms a complex with several components assembled into two structurally and functionally distinct modules: an antigen-recognition module (sIg), and a signal-transducer module (a heterodimer of two polypeptide chains, Igα/β) (Fig. 15.11) (276,277,278). sIg provides the specificity for antigen recognition through its antigen-binding sites. However, the cytoplasmic tails of the two heavy chains of mIgM consist of three amino acids and therefore are not suitable in signal transduction. Two other polypeptide chains, known as Igα, containing a 61-residue cytoplasmic tail, and Igβ, with a 48-residue tail, are associated with mIgM. The genes that encode for these proteins are also known as mb-1 and B-29, respectively. The transmembrane region of Cμ is required for binding to Igα/Igβ heterodimer, and is one of the highly conserved portions of the Ig molecule between species and isotypes. The conserved amino acids are not all hydrophobic but contain several hydrophilic residues. This amphipathic nature indicates again that mIg forms complexes with other proteins. In their cytoplasmic tails, these proteins carry a sequence motif of six conserved and precisely distributed amino acids (tyrosines and negatively charged amino acids) over a sequence of 26 residues. This motif has been termed immunoreceptor tyrosine-based activation motif or ITAM (279). ITAMs have been found in γ, δ, ∊, ζ, and η chains of CD3 and Fc receptors. Following the binding of the ligand on BCR, the ITAMs transmit activation signals initiating proliferation and differentiation of the B cell. However, ITAMs also transmit what have been called “tonic signals,” which have a role in B cell development and survival (280). The molecular mechanism for generation of tonic signals is not known. Association of BCR with lipid rafts (see later) or the balance of PTK (enhancing signaling) and PTP (attenuating signaling) may play a role.
The genes mb-1 and B-29 are active only in B lineage and are expressed even before the assembly of the V gene. In myeloma cells, however, only the B-29 is expressed, not the mb-1, and that prevents expression of Ig in plasma cells. For surface expression of Ig, the complete assembly of the Ig with Igα/Igβ heterodimers is required. This explains the lack of detection of mIg on plasma cells (281). Transfection of plasma cells with the mb-1 gene results in expression of sIg (282). A patch of polar amino acids within the transmembrane region of Cμ signals retention of IgM within the ER until the Igα/Igβ heterodimer associates with IgM for transportation to the cell surface.
In the B-cell receptor complex, the IgM component is in the center, with one Igα/Igβ heterodimer on each side (283). Based, however, on new evidence, this model has been challenged. It is believed that each IgM molecule is associated with only one Igα/Igβ heterodimer (Fig. 15.11) (284). If this model is correct, the question remains whether both chains are linked to IgM, or only Igα binds to both heavy chains, and Igα-BCR forms oligomers with additional molecules of the same isotype.
B-Cell Receptor and Lipid Rafts
The oligomers continuously deliver signals at a low level needed to maintain the B cell alive. Ligand binding changes the orientation of the BCR molecules so that their ITAMs become more accessible to phosphorylation that causes signal spreading restricted to BCRs of the same isotype (284). Signal spreading indicates that the oligomeric receptors form arrays in which the receptors are in close proximity (285). These early events of BCR reorganization provide a mechanism for detecting low concentrations of antigens, thus increasing the threshold of activation.
Cross-linking of BCR by multivalent antigen leads to a series of morphologic and molecular events that are interrelated (i.e., BCR aggregation and loading to “lipid rafts,” signaling, internalization of BCR, and antigen presentation).
In the resting state, BCRs “float” on the cell membrane as monomers, but at the time of “signal spreading” they form oligomers and gather in specialized membrane microdomain referred to as lipid rafts within seconds. Lipid rafts are estimated to represent 30 to 40% of the cell surface in lymphocytes (286). Their size varies from submicroscopic dimensions that may contain only a few hundred phospholipid molecules and four to ten protein molecules. Cross-linking with antigen enhances the transfer of BCRs to lipid rafts (285,286,287,288,289), but the pre-BCRs are constitutively located on lipid rafts (290). Lipid rafts are areas of the membrane enriched in cholesterol and glycosphingolipids with saturated fatty acid side chains consisting of GM1 gangliosides. They are resistant to solubilization in nonionic detergents at low temperatures. Most of the proteins are excluded from the lipid rafts, except proteins modified by saturated fatty acids. Such proteins include acylated Src kinases and the α subunits of trimeric G proteins. Lipid rafts facilitate BCR signaling by colocalizing signaling molecules, i.e., Lyn, Fyn, and Blk, members of the Src family of kinases, but also by excluding molecules that inhibit BCR signaling, such as CD22. Furthermore, in anergic or tolerized B cells, the BCR is unable to enter lipid rafts, indicating that inclusion of BCR within these microdomains is an absolute requirement for initiation of signaling (291). Similarly, localization of BCR within the rafts occurs only in mature, but not immature, B cells (292). The translocation of the BCR into lipid rafts is independent of any signaling initiated by the receptor and does not require actin cytoskeleton polymerization or the Igα/Igβ complex (293). Lipid rafts facilitate not only activation but also apoptosis, because cross-linking of CD24, a glycosylphosphatidylinositol-anchored protein that is down-regulated during B-cell differentiation, induces apoptosis via a lipid raft signaling system (294). CD24 cross-linking brings some BCR within the lipid rafts, activating Lyn kinase. Ezrin is a member of the ezrin–radixin–moesin (ERM) family, is dephosphorylated by BCR, and is detached from the actin as well as from lipid rafts, resulting in greater coalescence of lipid rafts and more effective B-cell activation (295).
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Figure 15.9. Redistribution of surface immunoglobulin (Ig). A: Mouse B lymphocytes treated with fluorescein-conjugated antimouse Ig antibody. a: Surface Ig is uniformly distributed around the cell (ring pattern). This pattern is seen only if the cell is kept at 0°C. b: Warming the cell to room temperature causes redistribution of the Ig so that it occupies only one pole (cap pattern). (From Taylor RB, et al. Redistribution and pinocytosis of lymphocyte surface immunoglobulin molecules induced by anti-immunoglobulin antibody. Nature 1971;233:225, with permission. Copyright (c)1971 Macmillan Magazines Limited.) B: Mouse B lymphocytes treated with rabbit antimouse Ig-conjugated with iodine-125 were examined by high-resolution radioautography. a: At the ultrastructural level, Ig is distributed around the entire surface of the cell, if it is maintained at 4°C. b:Surface Ig accumulates to one of the poles of the cell (uropod), forming a cap when it is warmed at 37°C. c and d: The cap is eventually endocytosed. (From Unanue ER, Perkins WD, Karnovsky MJ. Ligand-induced movement of lymphocyte membrane macromolecules. I. Analysis by immunofluorescence and ultrastructural radioautography. J Exp Med 1972;136:885–906. Reproduced by copyright permission of the Rockefeller University Press.) |
B cells are not only antibody-producing cells after BCR-mediated differentiation, but in addition are antigen-processing and presenting cells. With this function, they can attract antigen-specific T cells so they receive T-cell help (296). On the lipid rafts, B cells divert antigen captured by them to the endocytic pathway for further processing (297). The CD21/CD19 complex coreceptor, which markedly enhances BCR activation, significantly stabilizes the residence of BCR within rafts and thus prolongs B-cell activation (298). The FcγRII, which acts as an inhibitor of B-cell activation, on the other hand, destabilizes BCR–raft association (299).
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Figure 15.10. Ultrastructure of a cap. A: Anti-immunoglobulin (Ig) labeled with hemocyanin in mouse spleen B lymphocyte is heavily concentrated in the uropod around short microvilli. Some of the capped Ig has been endocytosed (E). The label in the cap is densely packed, whereas the adjacent areas are clear of label (arrows). (From Karnovsky MJ, Unanue ER, Leventhal M. Ligand-induced movement of lympho-cyte membrane macromolecules. II. Mapping of surface moieties. J Exp Med 1972;136:907–930. Reproduced by copyright permission of the Rockefeller University Press.) B: A cell with a tightly packed cap with smooth surface in the remaining body. C: The cap is separated by a constriction from the rest of the body, which shows intense membrane ruffling. G, Golgi apparatus. (B and C from Karnovsky MJ, Unanue E. Cell surface changes in capping studied by correlated fluorescence and scanning electron microscopy. Lab Invest 1978;39:554–564, with permission.) |
After translocation of BCR (or other immune receptors) onto lipid rafts, the rafts appear to form clusters [i.e., clustered rafts (285)] increasing the range of their diameter from hundreds of nanometers to micrometers. Clustering results from cross-linking of receptors located on separate rafts and eventually from the linking of rafts with linker proteins. In addition, cytosolic proteins that have been associated with cytoplasmic domains of receptors residing on a raft may also contribute to the bridging of individual lipid rafts to form larger conglomerates. Cytoskeletal components are also linked to activated receptors, and their reorganization bridges clustered rafts.
Continuation of raft clustering eventually leads to the formation of the “synapse,” a highly ordered membrane structure in which immune receptors, signaling molecules, and cell adhesion molecules are clustered (300). Synapse formation is a highly organized structure with a cluster of immune receptors in the center ringed by adhesion molecules and several signaling molecules and cytoskeletal components on the cytoplasmic side. The formation of the synapse was first identified on T cells, but it has now been identified also on B cells (301). The B-cell synapse is associated with antigen capturing for processing, and when antigen is captured by other antigen-presenting cells, such as dendritic cells, they trigger the formation of the synapse on B cells. During this close encounter between the dendritic cell with synapse formation, the B cells sample and gather antigen for internalization and processing.
The mechanisms of internalization have been discussed earlier with the Ig redistribution after anti-Ig binding (see “Surface Immunoglobin”). Lipid rafts are the middle point between signaling and internalization, and these two events are interrelated (302) because signaling regulates the BCR internalization (303). Cells with deficiency of expression of Src kinases, which normally initiate B-cell signaling, fail to internalize the receptor (304), and the targeting of antigen to the major histocompatibility complex class II peptide-loading compartment is also disrupted (305). Internalization of BCR occurs when clathrin is associated with rafts and is tyrosine phosphorylated after BCR cross-linking (299). When lipid rafts are disrupted either by expression of LMP2A protein of EBV or by reagents that sequester cholesterol, internalization does not occur (304,306).
B-Cell Receptor Signaling
The most important structural elements of signaling molecules for the initiation of B-cell activation and differentiation are the ITAM motifs of the Igα/Igβ heterodimer and the PTKs associated with the complex (Fig. 15.12). The ITAM is characterized by a sequence of 26 amino acids, D/E-X7-D/E-X2-YXXL/I-X6(7)-YXXL/I, with six of them being conserved (X = any amino acid). Critical for signaling are the two tyrosines (Y), and one or both of them are phosphorylated when the BCR is engaged by antigen. Some evidence suggests that activation of B cells may be triggered not only by the tyrosine-based activation motif (ITAM), but by a critical non-ITAM tyrosine 204 residue that is conserved in evolution (307).
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Figure 15.11. The B-cell receptor (BCR). The surface immunoglobulin (Ig) (two heavy and two light chains) constitutes the antigen-specific component of BCR, which is associated noncovalently with a heterodimer consisting of two chains, Igα and Igβ. This heterodimer constitutes the signal-transduction component. The Igαchain associates with the H chains through ionic interactions. ITAM, immunoreceptor tyrosine-based activation motif; mIgM, membrane immunoglobulin M. |
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Figure 15.12. B-cell activation. Signal transduction initiated by the engagement of the B-cell receptor (BCR) is a highly complex process. For better understanding, it is divided arbitrarily into three main signaling pathways: (a) initiation: with the assembly by adapter proteins such as BLNK (B-cell linker protein), Cbl, and others of signaling protein tyrosine kinase (PTK) complexes such as Src (Lyn, Fyn), Syk, and Btk; (b) inositol phosphatides generation by the activation of PI3K and PLC-γ; (c) the ras/Erk pathway assembled by adapter proteins Grb, Shc, and Sos. These pathways carry the signals downstream to other signaling molecules Akt, calcineurin, and Erk/1,2 and to activation of transcription factors nuclear factor-AT (NF-AT), NF-κB, Creb, c-fos, etc. |
BCR signaling is a highly complex process (308,309,310), but for the sake of understanding, the interactions are divided somewhat arbitrarily into three major pathways: (a) initial interactions, (b) phosphoinositide pathways, and (c) the ras pathway.
Initiation Pathway
BCR has no intrinsic protein tyrosine kinase (PTK) activity but uses several distinct families of cytoplasmic PTKs. Three distinct types of PTKs are activated on BCR engagement: (a) the Src-PTKs (Lyn, Blk, and Fyn), (b) Syk, and (c) Btk (308). Activation of Lyn is one of the earliest events in BCR-induced signaling, which is constitutively acylated and as a result localizes in the cell membrane. It is responsible for the initial phosphorylation of the ITAMs of Igα and Igβ. The kinase activity of Lyn is regulated by phosphorylation of a carboxy-terminal regulatory tyrosine by the kinase Csk (311) and dephosphorylation by the phosphatase CD45 (312). Lyn has one SH3 domain on its N terminus, followed by one SH2domain and the kinase domain in the C terminus. SH2 domains bind phos-phorylated tyrosines, whereas the SH3 domains bind proline-rich sequences. BCR engagement triggers dephosphorylation of Tyr508 by the CD45 phosphatase, whereas Ty394 within the catalytic domain is rapidly phosphorylated. Syk kinase is recruited to the phosphorylated ITAMs of Igα and Igβ, and is phosphorylated by Lyn or by an autophosphorylation mechanism (313).
Activation of Syk is a critical event in BCR signaling and for recruitment of BLNK, or SLP-65, a linker protein with a major scaffolding function connecting several downstream signaling molecules (314). BLNK assembles macromolecular complexes that include PLCγ, Vav, and Btk and additional linker proteins Grb2 and Nck (315,316). Phosphorylation of five tyrosine residues of BLNK is required for coordination of the assembly of multimolecular complexes.
Btk belongs to the TEC family of kinases and differs in several aspects from the Src kinases (i.e., it is not myristoylated, does not contain a negative regulatory phosphorylation site in the C terminus, and has a pleckstrin homology domain [PH] in the N terminus). Btk function is regulated by phosphorylation of the Tyr551, which is essential for Btk participation in signal transduction (317). Btk also interacts with phosphatidylinositol-3,4,5-tripho-phosphate (PIP3), an interaction required for recruitment of Btk to the cell membrane (318). Because PIP3 is generated from activated PI3 kinase, Btk is targeted to the membrane after PI3 kinase activation, where it could be phosphorylated by Lyn or Syk.
Generation of Phosphoinositides
Hydrolysis of inositol-containing phospholipids is mediated by the lipid metabolizing enzymes PLC-γ and PI3K. There are several pathways leading to activation of PLC-γ in B cells (319). BLNK associated with PLC-γ2 (which is the main isoform in B cells) brings it to an appropriate position for activation by Syk kinase. On the other hand, Btk also contributes to PLC-γ2 activation because B cells from XLA patients show a profound reduction of IP3 production on BCR engagement (320). PIP3, the product of PI3 kinase activation, binds to the PH domain of PLC-γ2, thus providing another pathway for PLC-γ2 activation. PLC-γ2 activation leads to hydrolysis of phospholipids, generating IP3 and DAG (diacylglycerol). IP3 binds to appropriate receptors on the ER, leading to Ca2+ release from internal stores.
PI3 kinase is activated by at least two pathways. A prominent substrate for tyrosine phosphorylation is the product of the oncogene c-cbl. BCR engagement phosphorylates p12-cbl, which is then associated with the 85-kDa component of PI3 kinase (321). A second pathway is through the CD19/CD21 coreceptor complex (322,323). BCR stimulation phosphorylates the two YXXM motifs of CD19, which then bind the SH2 domain of the p85 regulatory subunit of PI3 kinase. Enhanced phosphorylation occurs when the CD19/CD21 complex binds C3d fragments. Furthermore, the proline-rich region of the p85 subunit binds to SH3 domains of Lyn and Fyn, further enhancing PI3K activation.
Ca2+ binds to calmodulin and promotes calmodulin-dependent protein kinase activation and calcineurin, a serine-threonine–specific protein phosphatase. Calcineurin directly dephosphorylates the NFAT family of transcription factors (324,325). DAG, one of the two second messengers produced by PLC-γ2 activation, activates PKC. One target of PKC on B cells is MARCKS, a protein that regulates actin reorganization. Ca2+elevation also activates a number of other transcription factors, such as NF-κB and ATF-2. Phosphoinositides generated by PI3K bind to Akt/PKB kinase, an evolutionarily conserved kinase across species (326). Akt is the product of an oncogene transduced by the acute transforming retrovirus (Akt-8). The viral and cellular oncogenes encode a serine-threonine protein kinase consisting of a C-terminal kinase domain and an N-terminal PH domain. Mutations of the PH domain blocks Akt activation by growth factors or phosphoinositides. Akt activation is also mediated by the serine/threonine kinase PDK1, which is stimulated by PIP3 (not PIP2) and phosphorylates Akt on Thr308. Akt inhibits glycogen synthase kinase 3 (GSK3), which destabilizes Myc and cyclin D, both required for cell-cycle progression. A combination of various Akt activation effects plays a role for its transforming and oncogenic potential. Some of its multiple effects include transcriptional regulation of gene expression, inhibition of apoptosis, cell-cycle regulation, insulin-induced metabolic signals, endocytosis, etc.
The Ras Pathway
BCR cross-linking leads to an increase of the guanosine triphosphate (GTP)–bound ras and its accumulation in the membrane under BCR. Ras is a guanine nucleotide–binding protein, which cycles between a guanosine diphosphate (GDP)–bound (inactive) and a GTP-bound (active) state. The proteins of the Ras family are proto-oncogenes, which, on mutation, accumulate in the GTP-bound state in human tumors. On activation of B lymphocytes, Ras is rapidly converted to the GTP-bound state. The cycle between GDP and GTP binding is controlled by guanine nucleotide exchange factors (GEFs) that promote the transition from a GDP- to a GTP-bound state. This is reversed by guanosine triphosphatase (GTPase)– activating proteins, which stimulate GTPase activity of Ras and result in hydrolysis of GTP to GDP. The balance between GEFs and GTPase-activating proteins regulates Ras activity (327). The most likely pathway of ras activation is through the adapter protein Shc, which is phosphorylated after BCR engagement. Shc binds to a second adapter protein Grb-2, which in turn binds to Sos, a nucleotide exchange factor. This multimolecular complex is associated with the membrane (328,329). A guanine nucleotide exchanger protein, Vav, which is a GEF in the Rho family of GTPases, is recruited to the phosphorylated Tyr341 and Tyr345 of Syk through its SH2 domain and is subsequently phosphorylated (330). These Ras-like proteins are molecular switches that are active in the GTP-bound state and can promote site-specific actin polymerization to create alterations in plasma membrane structures such as filopodia and lamellipodia. Vav has a Dbl homology domain, which has the GEF activity, a PH domain (binds phosphoinositides), an SH2 domain, and two SH3 domains. The PH domain of Vav uses PIP3 for recruitment to the cell membrane. Vav may also be recruited to the membrane by binding to Tyr391 of CD19. A negative regulator of the ras pathway is Cbl, which competes for the binding to Sos. Cbl (casitas B-lineage lymphoma) is the cellular homolog of v-Cbl, part of the transforming gene of the Cas-NS1 retrovirus, a murine virus, capable of causing pre-B-cell lymphomas. It contains a proline-rich region (residues 481 to 688), a leucine zipper motif (residues 855 to the C terminus), and multiple potential SH2-binding motifs. It binds to PTKs such as Fyn, ZAP-70, and Btk, to the adapter molecule Grb2, and to PI3K (331). In B-cell signaling, Cbl binds to BLNK through its SH2 domain and inhibits association of PLC-γ2phosphorylation (332). Binding of Sos and Cbl to Grb-2 is mutually exclusive, because the proline-rich domains of these proteins compete for the same SH3 domain of Grb2. A group of signal-transduction pathways is characterized by successive phosphorylations of serine/threonine kinases. This group consists of a mitogen-activated protein kinase (MAPK), a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK). Ras-GTP phosphorylates Raf-1, which is the MAPKKK of this cascade. Raf-1 phosphorylates and activates MEK1 and MEK2, which in turn phos-phorylates ERK1 and ERK2. Phosphorylated ERKs form dimers and are translocated to the nucleus, where they phosphorylate transcription factors c-fos and Jun members of the Ets family.
Adapter Molecules: Signaling Plasticity and Diversity
Adapter proteins possess domains that mediate protein–protein or protein–lipid interactions, but they have no enzymatic activity. Two groups of adapter proteins can be identified: transmembrane adapter proteins and cytosolic adapter proteins (CAPs). In general, adapter proteins assemble multimolecular signaling complexes and direct their formation to specific cellular locations (333,334,335,336). At the initial stages of B-cell activation, Lyn, Syk, and Btk kinases are activated.
A transmembrane adapter protein, PAG (phosphoprotein associated with glycosphingolipid-enriched microdomains), links Lyn to the kinase that phosphorylates the C-terminal tyrosine for Lyn activation. The CAP adapter protein BLNK (or SLP-65, SH2 containing Linker Protein of 65 kDa) connects Btk to Syk and brings PLC-γ2 on the lipid rafts. There are inhibitors for adapter proteins, such as IBtk, which binds to the pleckstrin domain of Btk and negatively regulates its activation. BLNK which connects several important molecules to BCR for the initial stage of signaling, is indispensable for LMP2A (latent membrane protein), a constitutively activated EBV protein in infected B cells. LMP2A signaling can substitute for the signaling of BCR and maintains survival of EBV-infected B cells, leading to infectious mononucleosis or Burkitt lymphoma. The CAP BLNK is clearly required for the translocation of PLCγ2 from the cytosol to the plasma membrane and its subsequent activation. BLNK (SLP-65) is essential in pre-B-cell development and, with BtK, induces cell proliferation.
B-Cell Signaling Through Accessory Structures
Many other cell-surface molecules participate in B-cell signaling. The coreceptor complex CD19/CD21 activates the CD19-associated PTKs, which induce phosphorylation of tyrosine residues on CD19 motifs (337). These then become potential SH2-binding sites for PI3 kinase, which generates phosphoinositides. Recruitment of PI3K to CD19 requires phosphorylation of Tyr484 and Tyr515. Activation of PI3K generates PIP3, which functions to localize Btk to the plasma membrane, where it is phosphorylated and activated by Src and Syk kinases. BCR activation of Btk is dependent on CD19 expression, whereas activation of Lyn and Syk is not (338). PLC activation generates DAG, which activates PKC, and 1,4,5-triphosphate, which increases cellular Ca2+ (353). CD22 is associated with BCR, which on engagement phosphorylates some of the six tyrosines of CD22. This in turn leads to recruitment of SHP-1 via its SH2 domain to CD22 (339). CD22 contains ITAMs and four immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic region. PI3K and PLC-γ1 associate with CD22 through the YXXM motif recognized by the N-terminal SH2 domain of the p85 subunit of PI3K, whereas the protein tyrosine phosphatase SHP1 binds to ITIMs. As a result of multiple ITIMs, CD22 is probably a negative regulator of B-cell activation. (For more details, see CD22 in Appendix A.)
Interleukin-4
Although several cytokines act on B cells, the action of IL-4 was first demonstrated on B lymphocytes, and on these cells IL-4 evokes the strongest reactions. IL-4 binds to a transmembrane receptor and results in cross-linking with another protein termed common gamma chain (γc), which is shared by other cytokines (340,341,342). Mutations in γc chain results in X-linked SCID, an inherited disease with profound suppression of cell-mediated and humoral immunity (343,344). IL-4 binding induces phosphorylation of a substrate of 170 kDa designated as 4PS, which is unique because no other cytokine except IL-13 binding phosphorylates a similar substrate. However, insulin and insulin growth factor-1 result also in 4PS phosphorylation. 4PS is structurally similar to insulin receptor substrate-1 (IRS-1). The gene for 4PS has been cloned and is called IRS-2. IRS-1/2 contains more than 20 potential tyrosine phosphorylation sites and 30 potential threonine/ serine phosphorylation sites. The tyrosine phosphorylation sites bind with high affinity to cellular proteins possessing SH2 domains and thus act as docking sites for several signal- transducing proteins such as PI3 kinase and growth-factor receptor-bound protein 2 (Grb2). Mutational analysis has mapped a region of the IL-4R between amino acids 437 and 557, which is important for IRS-1 phosphorylation and therefore signal transduction (345). This region contains a single tyrosine, is shared by the IL-4 receptor (IL-4R) and the insulin receptor, and is known as the I4R motif.
Although the insulin receptor and IL-4R have many similarities, they have an important difference. The insulin receptor is a receptor tyrosine kinase, whereas the IL-4R and γc are not, but are associated with nonreceptor PTKs. Association of IL-4R and γc activates kinases associated with IL-4R, which then phosphorylates the tyrosine in the I4R motif. This allows 4PS to bind to the IL-4R and to be phosphorylated by the kinases associated with the IL-4R. The phosphorylated motif I4R interacts with the PTB domain of IRS1/2. PTB domains are found in adapter proteins, such as Shc, and bind phosphopeptides. IRS1/2 becomes phosphorylated as a result of interaction with IL-4Rα receptor and binds to the p85 subunit of PI3K, which as an adapter links IRS1/2 to the catalytic subunit p110. PI3K is activated as a result of conformational changes, acts on phosphatidylinositol of the cell membrane, and transfers phosphate groups from adenosine triphosphate to the D3 position of inositol-generating PIP3 and phosphatidylinositol 3,4-biphosphate. The phosphoinositides act on downstream kinases (i.e., PKC and Akt) that make important contributions to cell survival. Activation of the IRS1/2 signaling proteins is associated with activation of the ras/MAPK pathway (see “The Ras Pathway”).
IL-4R is also associated with the Janus kinases (or JAKs) (346,347). The α chain binds to JAK1, whereas the γc chain binds to JAK3. The receptors bind to these kinases through their membrane-proximal domains, which are known as box 1 and box 2 motifs and have some similarity among cytokine receptors.
The sequence between residues 557 and 657 of the α chain (known as the gene-regulation domain) is critical for expression of IL-4–responsive genes. It contains three conserved Tyr residues (Y575, Y603, and Y631), which can potentially be phosphorylated and thus be able to associate with SH2 domains. IL-4 utilizes the STAT-6 member of the transcription factor family known as STATs (signal transducers and activators of transcription). IL-4–responsive genes include class II HLA, CD23, germline Ig∊ and γ1 chains, and IL-4Rα chain.
Engagement of IL-4R results in the activation of JAK1 and JAK3, which phosphorylate tyrosines of the cytoplasmic region of the receptor. STAT-6 binds through its SH2 domains to the phosphorylated tyrosines and becomes itself phosphorylated at its C terminus by the activated JAK kinases. The phosphorylated STAT-6 dimerizes and binds to promoters of the IL-4–responsive genes (348). (See Chapter 17 for more on IL-4.)
Interleukin-5
The α chain of IL-5R is a type I membrane protein of 415 amino acids, its extracellular region comprising three sets of fibronectin type III domains, whereas the intracellular domain does not contain sequences of tyrosine kinase but shows homology with a part of the actin-binding domain of β-spectrin (349). It also has a region rich in prolines conserved among receptors of other cytokines (e.g., IL-3, granulocyte-macrophage colony-stimulating factor receptor or CD116; see Appendix A).
A second β chain is important for signal transduction, but it does not contribute to IL-5 binding. A membrane-proximal region contains a conserved box 1/box 2 motif that is responsible for the interaction with JAK2, and the distal domain is responsible for the activation of the ras-related pathways. IL-5 signaling increases Btk activity.
Interleukin-6
IL-6 is a pleiotropic cytokine that, among many other functions, is involved in terminal differentiation of B cells (350,351). The extracellular region of the IL-6R consists of one constant-region domain of the Ig superfamily and two fibronectin type III domains, which have four conserved cysteine residues and a motif containing two tryptophans and two serines. This motif is located in a groove between the two fibronectin domains. The intracellular domain is short and is not involved in signal transduction. Associated with the IL-6R is a protein known as gp130, which dimerizes when IL-6 binds to the IL-6R. The gp130 protein is shared by other cytokine receptors and initiates signal transduction. Homo-dimerization of gp130 induces activation of JAK kinases, which are associated with the membrane-proximal region of gp130, also known as box 1. This leads into phosphorylation of a tyrosine in the distal part of gp130 (box 3), resulting in binding through an SH2 domain of the transcription factor STAT-3, previously known as acute-phase response factor or APRF. JAK kinases activate STAT-3 by phosphorylation.
Another nuclear target for gp130 signaling is the transcription factor NF-IL-6 with a leucine zipper motif. This factor is inducible in hepatocytes and monocytes by IL-6 and other cytokines and mediates the expression of IL-6–inducible genes. NF-IL-6 has a consensus sequence for MAP kinase, suggesting that it is activated through this pathway, which is ras-dependent. (For further details, see Chapter 17.)
CD40
CD40 is a member of the tumor necrosis factor receptor family, which interacts with its ligand, CD154, expressed on T cells. [See “CD40 and CD40 Ligand (CD154),” Chapter 17.] Signal transduction by CD40 is mediated by certain proteins that bind to its cytoplasmic region and are known as TRAFs (tumor necrosis factor receptor activation factor). (See Appendix A: CDs.) Engagement of CD40 by its ligand activates the Src kinases Lyn and Fyn and Btk. Signaling follows the Ras pathway via the nucleotide exchanging factor SOS, leading to JNK and ERK activation. The functional outcome of CD40 depends on the state of activation of B cells and the intensity of stimulation. On naïve B cells it induces proliferation and Ig production, but on memory cells it induces apoptosis. CD40 activation induces homotypic adhesion of B cells mediated by CD54 (intercellular adhesion molecule-1)–CD11/CD18 (LFA-1), or CD23–CD21. The functional outcomes depend on the type of TRAFs that are associated with CD40. For example, trimers of TRAF-2 mediate apoptosis, whereas trimers of TRAF-6 or TRAF-5 mediate proliferation (352). Germinal-center formation and Ig class switch are hallmarks of T-cell–dependent responses, and TRAF-6 plays a role in class switch (see Chapter 17).
Polyclonal Activation
Certain substances can activate B lymphocytes independent of their antigenic specificity. The response to these substances involves all B-cell clones, and, for this reason, these substances became known as polyclonal B-cell activators (PBAs).
PBAs are primarily microbial cell constituents such as lipopolysaccharide (LPS), purified protein derivative, staphylococcal protein A, streptolysin O, pneumococcal polysaccharide III, a water-soluble antigen from Nocardia, EBV dextrans, etc.
PBAs can be categorized on the basis of their effect on B lymphocytes; some promote only B-cell proliferation, whereas others, in addition, stimulate Ig secretion.
LPS has been used experimentally as PBA for several years, but only recently was its receptor shown to be the CD14 molecule (353). LPS binding to CD14 is enhanced in the presence of a plasma protein, LPS-binding protein (354). CD14 is expressed primarily on monocytes and granulocytes. Its structure, function, and role in human disease have been reviewed (355).
Morphologic Changes associated with B-Cell Differentiation
Plasma Cell
Elegant studies of morphologic differentiation in immunized animals at the ultrastructural level were performed by Harris et al., who isolated individual cells involved in antibody synthesis (356) (Fig. 15.13). These authors showed that antibody production is detected while the cell still retains a “lymphocytic” morphology and contains no endoplasmic reticulum. These cells can be differentiated from inactive lymphocytes by the abundance of free polyribosomes and a large nucleolus. A spectrum of cells that actively secrete antibody can be ranked according to the size and development of the ER (357,358,359,360,361). During early differentiation, the ER is scarce and unorganized. Later, the lamellae increase in length and become parallel, until they fill the entire cytoplasm and give rise to its onion-skin appearance. The Golgi apparatus increases concomitantly. No dividing line exists that distinguishes the cells that make IgM from those that make IgG antibody, although a preponderance of IgM producers with lymphocytic morphology and of IgG producers with plasmacytic morphology is noted (357).
The term plasma cell was first used by Waldeyer in 1875 (362). His description, however, included several types of cells, and in 1881, Unna (363) redefined the cells as he observed them in a case of lupus, emphasizing the characteristic basophilia of the cytoplasm (“granuloplasm”). In a subsequent report (364) containing pictures of methyl green– and pyronin-stained cells, several cells are identified easily as characteristic plasma cells. In 1895, Marschalko took issue with Unna’s description and emphasized that the appearance of the nucleus with its characteristic arrangement of angular chromatin blocks and its eccentric position within the cell are to be used as stringent criteria for the identification of plasma cells (365). The characteristic nuclear morphology was given the name radkern by Pappenheim. Early analytic reviews of plasma cells were provided by Downey (366) and later by Michels (367). In those early years, whether the plasma cell was a normal constituent of tissues was the subject of considerable debate. Its origin was disputed, but several prominent investigators believed that it originated from lymphocytes.
The modern period of plasma cell study was introduced in 1937 by the clinical observations of Bing and Plum, who noted the close association of hyperglobulinemia and the presence of plasma cells (368). Subsequent studies in hyperimmunized rabbits were carried out by Bjornboe and Gormsen (369), who demonstrated that antibody production correlated with massive plasma cell proliferation in the spleen. In his doctoral thesis, Fagraeus (370) left little doubt about the importance of plasma cells in antibody formation. Differences among animals in their capacity to produce antibody could be related to differences in the number of plasma cells, particularly immature plasma cells. Fagraeus thought that mature plasma cells had “passed the stage of their greatest functional intensity.”
Indisputable evidence in favor of antibody production by plasma cells was provided by Coons, who introduced the powerful technique of immunofluorescence to immunology (371,372). Plasma cells containing antibody were detected in the red pulp of the spleen, the medullary cords of the lymph nodes, and focal granulomata of immunized animals. Excellent detailed descriptions of the morphology and ultrastructure of plasma cells have been published (373,374).
The plasma cell is round or oval, with an eccentrically located nucleus and chromatin arranged in pyramidal blocks against the nuclear membrane, giving the characteristic “cartwheel” appearance (Fig. 15.14). The cytoplasm is intensely basophilic because of the high content of ribonucleoprotein. Certain plasma cells stain red to violaceous rather than blue and are known as flaming plasma cells, a name coined by Undritz. This coloration is attributed to the accumulation within the ER cisternae of Ig with a high carbohydrate content. Electron microscopic studies revealed that the nucleus is surrounded by a double membrane. The outer membrane is covered with ribonucleoprotein particles and is continuous with the cytoplasmic ER. The Golgi is well developed and consists of vesicles and tubules. The centrosome lies next to the nucleus, surrounded by the Golgi apparatus. Several microtubules radiate from the centriole. Many prominent mitochondria are scattered between the ER lamellae. A striking ultrastructural feature of the plasma cell is the rich and well-organized ER. It consists of membranes studded on one side by ribosomal particles and arranged in parallel arrays. In the mature plasma cell, the ER fills the entire cytoplasm. The cisternae are sometimes distended with granular or homogeneous material, giving rise to cytoplasmic inclusions known as Russell bodies (375). The Russell bodies are composed of Ig (375,376), although sometimes Ig cannot be demonstrated (377). One suggestion is that these inclusions are not made from Ig aggregates. Alternatively, the Ig is condensed to such a degree that it cannot be penetrated by the dyes. Russell bodies sometimes are detected within the nucleus (intranuclear inclusions). Under certain circumstances, the plasma cell contains large quantities of a homogeneous material that distends the cell and stains gray or sometimes red as in the flaming cells. These cells, called thesaurocytes (378), reveal, under electron microscopic analysis, dilation of the ER cisternae (479) (Fig. 15.15). The flaming cell likely represents an early stage of the thesaurocyte in terms of storage of synthesized Ig (380,381). Two lines of evidence suggest that these forms are the result of disturbances in the secretion of Ig. In nonsecretory myelomas, the cells often are similar to thesaurocytes (382) or flaming plasma cells (383). Mott cells, which are considered plasma cells with multiple Russell bodies, also can result from a complete or partial block in the secretion of Ig, causing localized distention of ER cisternae (384).
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Figure 15.13. Morphology of antibody production. Terminal B-cell differentiation (antigen-dependent) is characterized morphologically by the gradual increase of the amount of cytoplasm and, concomitantly, the appearance of strands and eventually well-organized endoplasmic reticulum. A: An antibody-producing lymphoid cell with sparse endoplasmic reticulum (ER). (From Harris TN, Hummeler K, Harris S. Electron microscopic observations on antibody-producing lymph node cells. J Exp Med 1966;123:161–172, with permission.) B: Endoplasmic reticulum (ERVS) is slightly distended, containing amorphous material. (From Hummeler K, Harris TN, Tomassini N, et al. Electron microscopic observations on antibody-producing cells in lymph and blood. J Exp Med 1966;124:255–262, with permission.) C: Mature plasma cell displaying well-organized ER and distinct Golgi apparatus (G). (From Gudat FG, Harris TN, Harris S, Hummeler K. Studies on antibody-producing cells. I. Ultrastructure of 19S and 7S antibody-producing cells. J Exp Med 1970;132:448–474, with permission.) M, mitochondrion; N, nucleus; NOS, nucleolus; NU, nucleus. (Figures A, B, and C, reproduced from J Exp Med, as cited, by copyright permission of the Rockefeller University Press.) |
The most immature plasma cell is the plasmablast. It has a nucleus with evenly dispersed chromatin and a large nucleolus. The ER is sparse, and the cytoplasm is filled with clusters of polyribosomes. As the cell matures, the chromatin forms clumps, and the ER becomes more abundant and well organized. As typical lymphocytes mature into plasma cells, they pass through intermediate stages. These cells are evident in the blood of patients who have plasma cell dyscrasias or immunologic diseases characterized by hypergammaglobulinemia. Similar cells sometimes are encountered in the blood of patients with viral infections (Turk cells), including infectious mononucleosis. The intermediate forms have blue cytoplasm with abundant ER, but not as much as that seen in mature plasma cells. Some intermediate forms resemble the less mature transformed lymphocytes with simple ER and many ribosomes and polyribosomes.
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Figure 15.14. Plasma cells. A: Normal. B, C: Plasmacytes with vacuoles from the bone marrow of a patient with infection and arthritis. |
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Figure 15.15. Unusual forms of plasma cells. Under certain circumstances related to immunoglobulin (Ig) secretion, the cisternae of endoplasmic reticulum are distended as a result of the accumulation of Ig. Plasma cells with accumulation of a homogeneous material that sometimes stains pink or red on Giemsa preparations (thesaurocytes). A, B:Giemsa stain of bone marrow from two patients with IgA myeloma. Cell in A is bright red; that in B stains bluish-gray. C, D: Ultrastructure of thesaurocytes with different degrees of distention of the cisternae of the endoplasmic reticulum. (From Bessis M, et al. Étude comparée du plasmacytome et du syndrome de Waldenstrom. Nouv Rev Fr Hematol 1963;3:159, with permission.) |
Plasma cells down-regulate certain cell-surface molecules, such as BCR, major histocompatibility complex class II, CD19, and CD20, but express others such as Syndecan-1 (CD138) and CD38. Differentiation of plasma cells requires the transcription factor Blimp-1, which is a transcriptional repressor (385). Blimp-1 represses c-myc transcription, which explains cessation of cell cycle in plasma cells, as well as represses Pax5, which is required for lineage commitment of B-cell development and for isotype switching in germinal centers. Down-regulation of Pax5 is necessary for development of antibody-producing cells, because it represses XBP-1, J-chain, and Ig heavy-chain gene transcription (386). Blimp-1 promotes plasmacytic differentiation by extinguishing expression of genes that are important for BCR signaling, germinal-center function, and proliferation, but allows expression of XBP-1. XBP-1 is the only transcription factor required specifically for terminal differentiation of B lymphocytes to plasma cells (387). XBP-1 (X-box–binding protein) is a basic-region leucine zipper protein essential for the growth of hepatocytes and has been implicated in the proliferation of malignant plasma cells (388).
A small subpopulation of germinal-center cells in the light zone express Blimp-1, which functions upstream from XBP-1 (389). These cells have survived the selection and are probably destined to become plasma cells. Blimp-1 represses BCL-6, a germinal-center–restricted transcriptional repressor required for germinal-center function. A negative feedback loop operates in the germinal centers between Blimp-1 and BCL-6. When BCL-6 is expressed, Blimp-1 expression and plasma cell differentiation are blocked. However, Blimp-1 activation represses BCL-6, and plasmacytic differentiation is irreversible. (See also Chapter 17.)
Molecular and Ultrastructural Aspects of Immunoglobulin Biosynthesis
Heavy and light chains are synthesized separately, the H chain on large 270S to 300S polyribosomes composed of 16 to 20 subunits, the L chains on smaller 190S to 200S polyribosomes composed of 7 to 8 subunits. The size of these polysomes is such as to suggest synthesis of each chain as a single unit. Under normal conditions, L chains may be synthesized in slight excess.
After separate synthesis of H and L chains, intramolecular folding and assembly of the individual chains begins on nascent proteins, and continues into the cisternae of the ER. Depending on the Ig class, the assembly begins with the formation of the H-L half-molecules, two of which then combine to form a complete Ig monomer. Alternatively, two H chains combine to form H2, followed by H2L, suggesting that the final H2L2 structure may be reached by several pathways (390). Assembly of H chains is restricted between chains of the same class so that dimers between different H-chain isotypes are not formed in cells expressing both. In most instances, polymeric Ig, such as IgM and IgA (9S,11S,13S), are assembled intracellularly from their constituent subunits.
The attachment of core oligosaccharides to the N-glycosylation acceptor site begins on the ribosome, but glycosylation is completed in the Golgi apparatus, where the polypeptides are transported from the ER (391). The Golgi apparatus is also the site of final processing of the carbohydrate (392) and where the molecule is attached to membrane and then packaged into vesicles for secretion or incorporation into the plasma membrane (392). This mechanism of secretion is a form of reverse pinocytosis. Disruption of the traffic of the vesicles containing Ig inhibits Ig secretion.
The carbohydrate moiety may facilitate the secretion of Ig by the cell, although this effect depends on the Ig class and the amount of Ig synthesized. For some classes, such as IgM, Ig secretion is blocked when glycosylation is inhibited. H chains that have not assembled with L chains form complexes with an H-chain–binding protein (BiP) (393,394). BiP prevents transportation of H chains to the Golgi complex until they become associated with the L chains that displace BiP. The presence of such complexes provides an explanation for the lack of secretion of μ chains in pre-B cells when L chains are not available. In contrast to the normal situation, free H chains are secreted in association with certain lymphomas such as H-chain disease (395,396). In these disorders, the H chains have a large deletion involving the CH1 domain (397). This deletion explains how these mutant chains can be secreted, i.e., complexes with BiP can be formed only through the CH1 domain (398,399).
Although the synthesis of L chains and H chains takes only 30 and 60 seconds, respectively, the addition of carbohydrates and the process of secretion take at least 30 minutes. An adult synthesizes approximately 2.3 g of Ig daily (400).
Most Ig-synthesizing cells contain one type of H chain and one type of L chain (401), but a few cells (usually <1%) contain more than one type of H chain, usually μ and γ.
Antibody first appears in the perinuclear cisternae (402) and eventually is detected in the rest of the ER (403,404) (Fig. 15.16). Activation of the ER cisternae is gradual, because not all of them within the same cell contain antibody at any one moment. Antibody is found in association with the ribosomes of ER and is not detected outside the ER cisternae. In most immature blasts with a sparse or nonexistent ER, antibody is present in the cytoplasm, synthesized on polyribosomal clusters. Not only the intracellular distribution but also the rate of synthesis increases with time, and Ig, in some cases, distends the cisternae, forming large spheric masses reminiscent of Russell bodies. Not all plasma cells contain antibody during the primary response. In the first 2 to 3 days after immunization, most of the plasma cells contain Ig that has no antibody activity (405). Antibody-containing plasma cells appear later in the response. Throughout plasmacytic differentiation, the Golgi apparatus always contains antibody.
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Figure 15.16. Ultrastructural localization of antibody. A: An immature plasma cell demonstrates antibody primarily in the perinuclear area. Antibody is also detected in the few strands of endoplasmic reticulum (ER, arrows) and the Golgi (GA) apparatus. B: Plasma cell with well-developed endoplasmic reticulum filled with antibody. Both cells were isolated from rabbits immunized with peroxidase. For the intracellular localization of the antiperoxidase antibody, the cells were treated with peroxidase before incubation with diaminobenzidine. (From Leduc EH, Avrameas S, Bouteille M. Ultrastructural localization of antibody in differentiating plasma cells. J Exp Med 1968;127:109–118. Reproduced by copyright permission of the Rockefeller University Press.) |
Global Gene View of B-Cell Activation and Differentiation
The Ig gene is assembled from multiple gene segments, and every B cell is characterized by its own unique rearrangement [see “V(D)J Recombination”] maintained throughout its life and transferred to all the members of its progeny when it forms a clone of antibody-producing cells, with idiotypes characteristic of the parent B cell.
Malignancies arise from one cell, therefore are monoclonal, and inherit this unique Ig rearrangement (107). The new technology of “gene profiling” enables identification of the genes that are activated and, very likely, characteristic of each stage in the life of the B cell. This technology has already been applied on normal B cells to characterize their genetic profile at various functional states.
Gene profiling inspects activation and gene transcription of hundreds or thousands of genes, providing a global “genomic” view (406). In contrast to gene sequencing (“structural genomics”), gene profiling is considered a new field of “functional genomics.”
Serial analysis of gene expression (SAGE) uses gene-specific 14-bp sequence tags for enumeration of genes expressed in a cell (407). In the new methods, DNA fragments from individual genes are placed on a solid support in an ordered array. Total cellular mRNA isolated from cells is used to generate cDNA probes by reverse transcription, which are tagged by a radioactive or fluorescent probe. The cDNA probes are then applied to the arrays for hybridization with the DNA on the array. The hybridization results are quantitated by phosphorimagers for radioactive probes or scanning confocal microscopy for fluorescent probes. In one commonly used microarray system, oligonucleotides are produced by in situ synthesis in a technique called photolithography and are hybridized by fluorescent cDNA probes (408,409). In another technique, PCR products from cDNA clones are spotted onto coated glass slides (410). In this technique, two cDNA probes from different samples, labeled with different fluorochromes (usually Cy3 or Cy5), are applied on the microarray simultaneously for hybridization. The expression for each gene is evaluated by the ratio of the fluorescence, and interpretation of the data uses algorithms. Various analytical tools are available, and no single tool is better than others. Their use may well depend on the experimental design and the questions asked (411,412).
One approach uses hierarchical clustering (413). This algorithm begins by clustering pairs of genes with most similar pattern of expression, eventually building larger and larger clusters. Clustering may be unsupervised (i.e., the arrangement of gene expression follows predefined parameters built into the algorithm). In supervised clustering, the investigator introduces other parameters, such as clinical data. Several algorithms have been designed for data analysis, and more than one may be necessary for the analysis.
Three B-cell functional states were evaluated in an experimental model: naïve B cells, B cells activated in vitro by a foreign antigen (hen egg lysozyme, HEL), and B cells tolerant against self. In this case, B cells were obtained from transgenic mice (i.e., expressing HEL from birth); therefore, the B cells have developed tolerance against HEL as for any other natural self-antigen (414,415).
The results show that the naïve or resting state is maintained by several inhibitory genes, whereas activation is associated with loss of expression of some of the inhibitory genes rather than induction of genes that regulate entry into the cell cycle. B cells tolerant to “self” are characterized by increasing expression of more inhibitory genes at the same time that they maintain the expression of the basal inhibitory gene profile.
Some other data reveal that the gene profile of in vitro activated B cells differs from that of germinal-center B cells, where they are activated by T cells (406). Germinal-center B cells actually differ from naïve B cells in the expression of hundreds of genes (416). The function of signaling molecules has been studied by inactivation of the appropriate gene, but repercussions from changes in the expression of other genes cannot be heard. An attempt was made to provide an answer to this question from the effect of reduction of the function of PI3K or Btk to other genes. Reduced function of PI3K or Btk significantly affected 5% of BCR-dependent gene expression (417). The data also suggested that PI3K acts through Btk in regulating genes that are critical for determining entry into the cell cycle.
By hierarchical clustering, it has been possible to identify groups of genes that are characteristic of a lineage or a specific stage of differentiation or proliferation (i.e., germinal-center B cell). Each group of genes defines a “signature” of a cell type or of a function, etc.
Plasma cells, which represent the end stage of B-cell differentiation, were found to have 1,476 known genes, which were differently expressed compared to B cells (418). A number of factors characteristic of terminal differentiation (i.e., related to RNA polymerase I) were down-regulated, while a number of transcription factors were maintained (e.g., AP-1, NFAT, and NF-κB). Two genes for factors associated with neuronal cell positioning, reeling and neuropilin-1, were unexpectedly expressed, and their role in plasma cell life remains to be determined.
A large body of data has already been collected in the short period since this technology was developed for large-scale application. The early findings in B-cell malignancies hold a number of unexpected results (419). Diffuse large B-cell lymphomas (DLBCLs) have been shown to have two different gene profiles (419). In one group of patients, the cells had a germinal-center gene profile, whereas in the second group, the lymphoma cells showed a profile of mitogenically activated B cell. These two groups also differ in several other genes. DLBCL with the germinal-center B-cell profile has retained the hypermutation machinery, but the second group, resembling mitogenically activated B cells, has not (420). Germinal-center B-like DLBCL also closely resembles follicular lymphoma (421). On the basis of sharing some gene expressions with a small population of B cells within the germinal center as well as with plasma cells, it has been suggested that the activated B-like DLBCL may arise from a cell in its transition to becoming a plasma cell.
Another important finding is the possibility that B-CLL, which, phenotypically, has been separated into two groups on the basis of CD38 expression, may actually be one “genomic” disease with two variants (422). A small number of <30 genes may, however, be able to distinguish cases with IgV-mutated versus -unmutated profiles (423). Furthermore, the gene profiles have indicated that in the majority of cases, the cells are related to memory B cells rather than naïve B cells or B cells of any other category. This may not be unexpected, because the somatic hypermutation machinery is active in CLL and could play a role in intraclonal diversification development (424). Microarray gene profiling has been used to identify B-CLL cases with cells resistant to apoptosis after DNA damage. Thirteen of 16 genes were found that were specific for resistant B-CLL cells (425). A study of mantle cell lymphoma also detected resistance to apoptosis gene profiling. The FADD gene, a key gene associated with Fas-mediated apoptosis, was down-regulated 10-fold in this lymphoma (426).
In multiple myeloma, the profiling suggests a classification into four subgroups (427,428). Group 1 patients (MM1) have a gene profile similar to MGUs, whereas the patients in MM4 group have poor prognosis, with a gene profile similar to myeloma cell lines. Numerous unaccounted genes with “unknown” function have been detected, so that only 10% of all these genes have matched entries in the database Expressed Sequence Tags (429).
The origin of Hodgkin and Reed-Sternberg (HRS) cells have remained elusive, and, recently, strong evidence has been presented that in most cases with this disease, the cell is of B-lineage origin and only rarely originates from a T cell (430). Identification of gene profiling demonstrated that the HRS cell expresses a distinct gene profile regardless of its B- or T-cell derivation. The gene profile is similar to that of EBV-transformed B cells or cell lines from large cell lymphomas with features of in vitro–activated B cells (431). Among the genes specifically identified in HRS are a cluster of genes for transcription factors (i.e., GATA-3, ABF-1, EAR3, and Nrf3). Down-regulation of several genes that are active in B cells were identified. Several of these genes are positively regulated by the transcription factor Pax5 [see “Stem Cell to Progenitor-B (Pro-B) Cell”]. However, the Pax5 gene is still expressed in HRS cells, and therefore the loss of B-cell–specific gene expression remains unknown (432).
Btk mutations influence the expression of other genes in EBV-transformed cell lines, and, in the absence of functional Btk, 11 genes were identified that were induced more than 1.9-fold (433).
Microarray gene profiling has been applied to develop a molecular predictor of survival after chemotherapy for DLBCL. The study used 17 genes to construct a predictor of overall survival (434). Four gene groups were identified and clustered within individual signatures. The proliferation signature was the best predictor for adverse outcome. Signatures identifying good prognosis were the lymph node signature, encoding extracellular matrix, and the connective tissue growth factor, which promotes fibrosis and synthesis of extracellular matrix. Some of these genes are linked to histologic or other lymph node reactions, known already for their favorable prognosis. For example, the lymph node signature is associated with expression of genes that are also expressed in macrophages and NK cells, presumed to indicate a cellular antitumor response. The other favorable signature is the major histocompatibility complex class II gene expression.
B-Cell Subpopulations
A subpopulation of B cells can be detected on the basis of expression of the CD5 antigen ordinarily present on T cells. The CD5 antigen (formerly T1 or Leu1) is a 67,000-dalton gp detected on all normal T cells (435,436), on a small subpopulation of normal B cells (437), and on all cells from patients with CLL (438,439,440). The CD5 B cells possess unique properties. They are phenotypically identical to CD5- B cells (441), but they are larger; in mice, they have 10 times more mIgM with λ chains, which is expressed only rarely in mice. The CD5+ B cells are present in high numbers in fetal and neonatal life (50% of all IgM+ B cells), but they progressively diminish in number after birth and are present in small numbers in secondary lymphoid organs in adult life (442,443,444). At birth, most of the B cells in cord blood are CD5+ (443), but they constitute less than 10 to 30% of B cells in adult spleen, lymph nodes, and peripheral blood. Their proliferative capacity is high, and as a result, they give rise spontaneously to cell lines that demonstrate c-myc amplification (445). The CD5 binds to CD72.
Presently, there are two views regarding the origin of the CD5+ subpopulation. According to one of them, the CD5+ cells (also known as B1a) belong to a lineage of B cells distinct from the conventional B lymphocytes (CD5-) (446,447,448). The CD5+ (B-1) cells develop early in ontogeny and, in the adult, predominate in peritoneal and pleural cavities and are self-replenished—that is, they do not arise from undifferentiated progenitors. Progenitors of CD5+ cells are present in fetal liver and omentum but not adult bone marrow, whereas conventional B-cell progenitors are present in fetal liver and adult bone marrow. According to the second hypothesis, the B-1 cells derive from conventional B cells (449,450), based on in vitro evidence of stimulation of conventional B cells with anti-IgM antibodies and IL-6, which generated the B-1 phenotype (451). B-1 cells may have different antibody repertoires (e.g., have few N-region insertions in their rearranged V genes), and the VHrepertoire is biased toward V-gene families proximal to JH, whereas in adult B cells it is more randomized.
Perhaps the most controversial aspect is the production of autoantibodies. In NZB mice, a strain well known for autoimmune phenomena, the CD5+ B cells are increased in number and spontaneously secrete IgM autoantibodies (452). Increased numbers of CD5+ B cells are found in patients with rheumatoid arthritis (453,454), Sjögren syndrome (455), and progressive systemic sclerosis (456), but not in patients with systemic lupus erythematosus (456). Numbers of CD5+ B cells are also increased after bone marrow transplantation (457). In 95% of patients with B-cell CLL, the leukemic cells express the CD5 antigen (438,439,458), which is also detected on cells from other B-cell lymphomas. The CD5+ B cells from normal subjects (459) or from patients with CLL (460) produce autoantibodies, such as cold agglutinins, antibodies against cytoskeletal elements, and rheumatoid factor, but according to another view, these Ig are not autoantibodies but are polyreactive or natural antibodies with specificities against some self-antigens, such as the Fc fragment of IgG, DNA, and thyroglobulin (459). The CD5+ B-cell subpopulation may therefore play an important role in the development of B-cell repertoire related to natural immunity, which develops in the absence of an encounter with exogenous antigens. Certain V genes are selectively expressed in CD5+ B cells, and those are not changed by somatic hypermutations normally observed in CD5- B cells responding to exogenous antigens (461).
The marginal-zone B lymphocytes share many phenotypic characteristics with B-1 cells and, like them, develop in response to T-independent type 2 antigens. The B-2 repertoire is selected by self-antigen and therefore tends to be autoreactive. Selection into B-1a population is favored during fetal life because TdT is not expressed during this period; therefore, the repertoire is limited in its specificity range. This dangerous repertoire is kept under control by the CD5-mediated negative signaling, thus preventing inappropriate activation. On the other hand, this repertoire is useful because the B-1 cell specificities are directed against several pathogens and are important in mucosal immunity (462).
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