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Cell surface immunoglobulin receptors in B cell development
seminars in IMMUNOLOGY, Vol. 14, 2002: pp. 351–358 doi:10.1016/S1044–5323(02)00068-4, available online at http://www.idealibrary.com on
Cell surface immunoglobulin receptors in B cell development Kelly A. Pike a,b and Michael J.H. Ratcliffe a,b,∗ ity of cells entering the B lineage developmental pathway fail to express a functional surface immunoglobulin receptor (BcR). However, the end product of B lineage development is the generation of a population of B lymphocytes, all of which express a functional BcR. Consequently, B cell lymphopoiesis is regulated to eliminate those precursors that fail to express a BcR complex, while supporting the further development of those precursors that express a functional receptor. Pathways of B cell lymphopoiesis have diverged during evolution. Thus while B cell development in some species, such as rodents and primates, occurs primarily in the bone marrow, other species use a variety of gut associated lymphoid tissues as their primary B lymphoid organ. Nonetheless, selection for cells expressing a functional BcR has been maintained throughout evolution. In this review we will draw on insights derived from different models of B cell development to address the role of the BcR complex in supporting B cell development.
Expression of surface immunoglobulin (sIg) related receptors has been conserved in phylogenetically distinct species as a critical checkpoint in B cell development. The sIg receptor comprises extracellular IgM heavy and light chains, with the potential for ligand binding, complexed to the Igα/Igβ heterodimer that is responsible for signal transduction through sIg. Experimental systems, from both avian and murine models of B cell development, have been designed to identify the function of individual receptor components in B cell development. In this review, we assess the regulatory functions of different components of the sIg receptor complex during early development in experimental systems from evolutionarily distinct species. Key words: B lymphocytes / surface immunoglobulin / bursa of fabricius / Igα / Igβ © 2002 Elsevier Science Ltd. All rights reserved.
Introduction
The BcR complex
A diverse repertoire of antibodies is required for protection against the heterogeneous array of pathogens in the environment. The molecular processes governing the generation of diverse Ig repertoires vary between species but in virtually all cases require modification of germ line sequences. These modifications include rearrangement of V, D and J gene segments that in some species subsequently undergo further diversification by gene conversion or hypermutation. Each of these processes is error prone, and the major-
IgM is expressed at the surface of B cells in association with the Igα/Igβ heterodimer. The products of V(D)J recombination of both immunoglobulin heavy and light loci covalently associate, forming a ligand recognition domain. The short three amino acid cytoplasmic tail of the µ chain has no intrinsic signaling capacity.1 Rather surface IgM (sIgM) is non-covalently associated with the disulphide bonded Igα/β heterodimer. The Igα/Igβ heterodimer has been conserved during evolution and each chain contains an extracellular Ig domain, a transmembrane region and a cytoplasmic domain that contains conserved signaling motifs (reviewed in Reference 2). The cytoplasmic domains of both Igα and Igβ contain immunoreceptor tyrosine-based activation motifs (ITAMs)3 as well as other residues important for signaling through the BcR (reviewed in Reference 2).
From the a Department of Immunology, University of Toronto, 1 King’s College Circle, Toronto, Ont., Canada M5S 1A8 and b Sunnybrook and Women’s College Health Sciences Center, 2075 Bayview Avenue, Toronto, Ont., Canada M4N 3M5. * Corresponding author. E-mail: [email protected] © 2002 Elsevier Science Ltd. All rights reserved. 1044–5323 / 02 / $– see front matter
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In contrast to murine B cell development, the rearrangement of chicken Ig VH and VL genes occurs stochastically rather than sequentially.17 Consequently, there is no apparent role for expression of a pre-BcR in chicken B cell development and indeed, to this point, chicken homologues to the VpreB and λ5 genes have not been identified. However, in contrast to the mouse, V(D)J rearrangement in the chicken generates minimal antibody diversity.18 At the light chain locus there is only one functional VL segment, which rearranges to the unique JL segment.19 Similarly, heavy chain rearrangement utilizes one functional VH segment, a cluster of highly conserved DH segments and a unique JH segment.20 As such, rearrangement of chicken Ig genes generates BcRs of limited diversity that have been referred to as the ‘pre-diversified BcR’. BcR diversity is subsequently generated through somatic gene conversion events that occur within bursal follicles.21 The initial checkpoint in chicken B cell development is therefore expression of the pre-diversified BcR, a complex that contains µ and light chains of restricted diversity in association with the Igα/Igβ heterodimer. Expression of this receptor requires productive rearrangement at both Ig heavy and light chain loci and is required for productive colonization of bursal follicles and subsequent Ig diversification by gene conversion.
Checkpoints in murine B cell development After birth, murine B cell development occurs within the bone marrow. Prior to the onset of the VH gene rearrangement pro-B cells express the Igα/β heterodimer at the cell surface as part of a complex that includes the chaperone calnexin which can be defined as the pro-BcR.4, 5 Following productive Ig heavy chain rearrangement, the µ heavy chain is expressed at the cell surface with the surrogate light chain molecules VpreB and λ5 and in association with the Igα/Igβ heterodimer as a complex defined as the pre-BcR. Cells that fail to productively rearrange the heavy chain locus and generate a pre-BcR undergo apoptosis.6 Pre-BcR expression results in the termination of further rearrangement at the heavy chain locus and the induction of a burst of pre-B cell proliferation.7, 8 Pre-B cells downregulate transcription of VpreB and λ5 and surface pre-BcR levels are diluted during this round of proliferation. As a consequence, pre-B cells stop proliferating and undergo rearrangement of the light chain locus.8 Those cells that have productively rearranged the light chain locus, and express a BcR (µ and κ or λ, associated with the Igα/Igβ complex), subsequently migrate out of the bone marrow into the periphery. In contrast, cells unable to generate a functional BcR are eliminated (reviewed in Reference 9). The expression of pre-BcR and BcR therefore represent checkpoints that define the productivity of rearrangement at the heavy and light chain loci, respectively.
The Igα/β heterodimer is required for completion of VDJH rearrangement (the pro-B cell to pre-B cell transition) As described above, the Igα/β heterodimer is expressed on the surface of pro-B cells as part of the pro-BcR complex prior to the completion of VDJH rearrangement. The functional importance of pro-BcR expression is evidenced in mice deficient for Igβ. In such mice B lineage cells undergo normal levels of DJH rearrangement but the levels of V–DJ rearrangement are drastically reduced.22 Therefore surface pro-BcR expression appears to play a critical role in regulating induction of the murine heavy chain V gene rearrangements required for the pro-B to pre-B transition, prior to the pre-BcR checkpoint. Surface expression of murine sIgM, and by extension the pre-BcR requires co-expression of both Igα and Igβ.23–27 In consequence, complete deletion of either Igα or Igβ cannot address the question as to whether surface expression of both Igα and Igβ (as opposed to either Igα or Igβ) are required to support B
Checkpoints in avian B cell development In the avian system, the bursa of Fabricius is the primary lymphoid organ for B cell lymphopoiesis. Nevertheless, the bursal microenvironment is not required for B lineage commitment or the induction of Ig gene rearrangement.10–13 Starting at day 8 of embryogenesis the bursal mesenchyme is colonized by a single wave of B cell precursors14 some of which migrate across the bursal epithelial basement membrane and undergo proliferation within the epithelial buds that eventually develop into bursal follicles.11, 15 Only those B cell precursors that have undergone productive Ig gene rearrangement and express a sIg receptor are selected for clonal expansion within the bursa.16 Therefore, as in the murine system, surface expression of a BcR complex represents a critical checkpoint in early B cell development. 352
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lineage development. Therefore, as an alternative to deletion of the complete Igα or Igβ molecule, truncation of the cytoplasmic domains of Igα or Igβ has been used to disrupt their signaling ability while maintaining their capacity to form a sIg related receptor complex. In mice containing truncations of both Igα and Igβ, reduced numbers of cells expressing cytoplasmic µ chains were observed. It was therefore concluded that truncations of both Igα and Igβ results in a reduction of V–DJ rearrangement, similar to that observed in Igβ −/− mice.28 Conversely, in mice containing either a truncated Igα or a truncated Igβ, the B lineage cells proceed through this checkpoint, undergo VH to DJH rearrangement and establish a pre-B cell population.28, 29 Consequently, the pro-B to pre-B transition is supported by the cytoplasmic domain of either Igα or Igβ. At this point it remains unclear how pro-BcR expression may regulate the induction of VH rearrangement. Nonetheless, IL-7 receptor signaling modifies the accessibility of the VH locus for rearrangement.30, 31 Moreover, as observed in Igβ −/− mice, IL-7Rα −/− mice undergo normal levels of D–J rearrangement while displaying a marked reduction of V–DJ rearrangement. In particular a decreased usage of 5 VH J558 segments was observed in the IL-7Rα −/− mice.32 Given the demonstrated interplay between IL-7 receptor signaling and signaling through the Igα/β heterodimer in pre-B cells,33 pro-BcR expression and consequent signaling through Igα and/or Igβ may increase the sensitivity of pro-B cells to IL-7 modulated VH locus accessibility for rearrangement.
receptor could not. Moreover, when introduced into recombinase competent mice, the YS:VV mµ receptor was not able to support allelic exclusion of V–DJ rearrangement.36 Consequently the association between µ and the Igα/β heterodimer as part of the pre-BcR is required for the expansion of a functional pre-B cell population.
Igα/β in pre-B cell expansion While the transgene encoding the YS:VV mutant µ chain failed to support pre-B cell expansion, the same mutant µ chain when fused directly to the cytoplasmic domain of either Igα or Igβ (YS:VV–IgM:Igα or YS:VV–IgM:Igβ, respectively) supported the progression of B cell development through the pro-B to pre-B transition in RAG1−/− mice.36, 37 This transition was characterized not only on the basis of B lineage phenotype but also by the establishment of allelic exclusion at the heavy chain locus. Whereas mice containing the Igβ truncation contained normal numbers of pre-B cells, indicative of substantial pre-B expansion, mice containing the Igα truncation contain reduced numbers of pre-B cells. Thus signals downstream of Igα appear more efficient at supporting the pro-B to pre-B transition or expanding pre-B cells.28, 29 However, in contradiction to this conclusion, the relative efficiencies of the YS:VV–IgM:Igα and YS:VV–IgM:Igβ chimeric receptors in supporting the pro-B to pre-B transition were indistinguishable.38, 39 This may be a reflection of the organization of the cytoplasmic domains of Igα and Igβ in the plasma membrane. In BcR related complexes containing the truncation mutants of Igα or Igβ, each Igα/β heterodimer contains one functioning cytoplasmic domain per heterodimer. In contrast, the YS:VV–IgM:Igα and YS:VV–IgM:Igβ chimeric proteins would each be expressed as surface homodimers, as a consequence of µ chain dimerization, containing two functional cytoplasmic Igα or Igβ domains, respectively.
Pre-B cell expansion requires association between pre-BcR and Igα/β Following VH rearrangement, the expression of a functional pre-BcR, µ/VpreB/λ5, is required for pre-B cell expansion and the downregulation of further heavy chain rearrangement. The association between sIgM heavy chains and the Igα/β heterodimer depends on polar amino acids in the transmembrane region of µ chain. Reducing the polarity of the µ transmembrane region resulted in surface expression of the mutant IgM (YS:VV mµ) in the absence of Igα/β.1 B cell development in RAG1−/− or RAG2−/− mice is blocked at the pro-B cell stage of development.34, 35 While a wild-type µ chain, that can associate with Igα/β, supported the generation and expansion of a pre-B cell population in RAG2−/− mice, the YS:VV mµ mutant
Igα/β in the pre-B to B cell transition Pre-B cells undergo rearrangement at the light chain locus and those that express a functional BcR make the transition to immature B cells. In mice containing the Igβ truncation and therefore expressing a pre-BcR containing only functional Igα, normal levels of immature B cells were generated.28 In contrast, mice 353
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containing a truncated Igα, and therefore expressing a pre-BcR containing only Igβ, reduced levels of pre-B cells were matched by an equivalent reduction in immature B cell numbers.29 This suggests the possibility that the transition from pre-B to immature B cell is supported by either Igα or Igβ with equal efficiency. Following productive light chain rearrangement resulting in immature B cells subsequent maturation generated a pool of mature recirculating B cells. The transition from immature to mature B cells is strikingly reduced in mice containing either the Igα or Igβ truncation, with most immature B cells undergoing apoptotic cell death.29 Consequently, while expression of the cytoplasmic domains of either Igα or Igβ is sufficient to support B lineage development to the immature B cell stage, albeit with differing efficiencies, the cytoplasmic domains of both Igα and Igβ are required for the efficient establishment of a mature B cell pool. It is important to consider, however, that in both experimental models, it is impossible to assess the efficiency with which the chimeric YS:VV–IgM:Igα and YS:VV–IgM:Igβ receptors or the Igα/Igβ truncations support B cell development relative to normal B cell development. Thus the YS:VV–IgM:Igα and YS:VV–IgM:Igβ chimeric receptors were introduced into RAG1−/− mice in which normal B lineage development is compromised. Similarly in mice containing truncations of Igα or Igβ, there is no development of the B cell lineage supported by the intact Igα/β heterodimer.
development at the pre-B cell stage as well as a failure to establish allelic exclusion.41 Similarly, B cell development in λ5−/− ,42 and VpreB1−/− /VpreB2−/− ,43 mice is largely blocked at the pre-B cell stage. Importantly, B cell development is not completely abrogated, in either λ5−/− or VpreB1−/− /VpreB2−/− mice. This is most likely a consequence of low level light chain gene rearrangement, allowing a small proportion of developing B lineage cells to express a µκ or µλ BcR at the pre-B developmental stage. More dramatically, transgenic expression of light chains in pre-B cells can overcome the developmental block in λ5−/− ,44 mice arguing that while expression of a sIg related receptor is critical, recognition of ligand by the VpreB/λ5 determinants of the pre-BcR is not required for B cell development.
Truncated sIgM receptors support B cell development Further support for the contention that ligand recognition by the pre-BcR is not required to support B cell development came from mice transgenic for the expression of a truncated IgM (Tµ) that lacked VH and a part of the Cµ1 domain. This Tµ failed to associate with λ5 or VpreB and, as a consequence of failing to associate with BiP, was expressed at the cell surface in the absence of surrogate light chain proteins. This Tµ receptor supported the generation of pre-B cells, and induced the downregulation of λ5 transcription in Rag1−/− mice. Moreover, the introduction of the Tµ into µMT mice rescued allelic exclusion and induced rearrangement at the light chain locus.45 Again, the efficiency with which the Tµ construct supported B cell development is difficult to assess as these observations were made in RAG1−/− mice in which normal B cell development was abrogated. In an alternative approach, retroviral gene transfer was used to introduce a Tµ receptor into chicken B cell precursor cells in vivo. The Tµ construct in these experiments contained a deletion of the entire VH and Cµ1 domains and was expressed at the B cell surface in the absence of light chain. Expression of this receptor was sufficient to support the early stages of avian B cell development: colonization of bursal follicles, clonal expansion of B cells within bursal follicles and the induction of gene conversion. Crucially, however, since the Tµ encoding virus was introduced into normal B cell precursors, B cell development supported by the Tµ receptor occurred in competition with B cell development supported by expression of
Igα/β expression versus receptor ligation While the BcR is clonally diverse, this is not the case for either the avian pre-diversified BcR or mammalian pre-BcR. In the case of the pre-BcR, the VpreB/λ5 surrogate light chain complex is invariant and the pre-diversified BcR also has limited diversity. This has suggested the possibility that these receptors may bind ligand(s), as distinct from specific antigen, in their environment and that this recognition is required to support subsequent B lineage development. Indeed, the observation that a soluble pre-BcR construct binds to stromal cells has been taken to support this contention.40 Failure to generate a sIg receptor related complex at the pre-B stage of murine B cell development results in developmental arrest. Thus genetic disruption of the exon encoding the transmembrane domain of murine IgM membrane (µMT) resulted in an arrest of B cell 354
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the endogenous pre-diversified BcR. Under these circumstances, the Tµ receptor supported development with an efficiency indistinguishable from the endogenous receptor.46 Both Tµ models demonstrate that a specific interaction between ligand and the V(D)J (or VpreB/λ5) encoded determinants of either the preBcR or pre-diversified BcR is not required for the early stages of B cell development. However, these experiments did not exclude the possibility that development required an interaction between a ligand and the residual extracellular domains of either the µ chain or the Igα/β heterodimer.
sIg demonstrated that expression of the chimeric receptor supported the early stages of B cell development with an efficiency equivalent to that observed following expression of the pre-diversified BcR (Pike et al., manuscript in preparation). This provides further support for the argument that ligation of either the pre-diversified BcR or pre-BcR is not required for the efficient progression of B cell development.
Basal signaling through Igα/β in the absence of receptor ligation Taken together the experimental systems outlined above strongly suggest that membrane proximal expression of the cytoplasmic domains of Igα and/or Igβ is sufficient to support the generation of immature B cells. Under these circumstances the role of µ/VpreB/λ5 in the pre-BcR or µ/L in the BcR or avian pre-diversified BcR would be to function as a chaperone to facilitate the expression of the Igα/β heterodimer at the pre-B or B cell surface. The cytoplasmic domains of Igα and Igβ contain ITAM motifs.3 Disrupting these ITAM motifs disrupted the ability of the Igα/β cytoplasmic domains to support B cell development.36, 48 Similarly, disrupting the Igα ITAM of the CD8α:Igα chimeric receptor disrupted its ability to support bursal colonization, even in the presence of a CD8β:Igβ chain containing a functional Igβ ITAM. Following receptor expression in the absence of overt ligation, the ITAM motifs of Igα and Igβ are constantly being phosphorylated and dephosphorylated. This has best been demonstrated by the incubation of cells with pervanadate, which blocks phosphotyrosine phosphatase activity, and rapidly induces high levels of Igα and Igβ tyrosine phosphorylation despite the lack of deliberate activation of tyrosine kinases.49 We, and others, would support the conclusion that the steady state levels of Igα and Igβ phosphorylation in unstimulated cells is sufficient to allow basal signal transduction downstream of Igα and/or Igβ and that this basal signaling is required to support the progression of B cell development. The ability of the cytoplasmic domains of Igα and Igβ to independently support the generation of immature murine B cells suggests that at least some aspects of downstream signaling are shared between the two chains. In contrast, the requirement for the cytoplasmic domains of both Igα and Igβ in the generation of mature B cells suggests that there must be unique functions associated with each domain or a
BcR expression restricted to the cytoplasmic domains of Igα/β The ability of the chimeric YS:VV–IgM:Igα and YS:VV– IgM:Igβ chains to support B lineage development in the absence of endogenous receptor expression argued that ligand recognition by the extracellular domains of Igα or Igβ is not an obligatory requirement in B lineage progression. However, two experimental systems have been developed to address this issue more specifically. In one model, a Lck myristoylation/palmitoylation signal was used to target a fusion protein containing the cytoplasmic domains of Igα and Igβ to the cell surface in the absence of their extracellular domains. Expression of lipid targeted Igα/β cytoplasmic domains in B lineage precursors supported their development into immature B cells, under circumstances where the endogenous pre-BcR/BcR was not synthesized.47 While these studies demonstrate that there is no obligate requirement for extracellular ligation of the pre-BcR in B cell development, again the efficiency with which the lipid targeted Igα and Igβ cytoplasmic domains supported B cell development relative to endogenous intact receptors is difficult to judge, as these experiments were performed in µMT B cell precursors. In an alternative model, a chimeric receptor, in which the extracellular and transmembrane domains of mouse CD8α and CD8β were fused to the cytoplasmic domains of chicken Igα and Igβ, respectively, was introduced into developing chicken B cell precursors by retroviral gene transfer. These receptors were expressed in normal embryos fully competent to express an endogenous pre-diversified BcR. The presence of roughly equivalent numbers of developing B cells expressing either endogenous sIg or the chimeric CD8:Igα/β receptor in the absence of endogenous 355
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combination of the Igα and Igβ cytoplasmic domains that are required for the later transition. Further support for unique biological properties associated with the cytoplasmic domains of Igα and Igβ is evidenced in avian B cell development where bursal colonization is supported by the chimeric CD8α:Igα alone, but not by a CD8α:Igβ chimeric receptor alone (Pike et al., manuscript in preparation).
indicates that there are unique downstream consequences of signaling through each of these chains that are required for B cell development. The majority of in vitro assays have demonstrated that Igα and Igβ induce differing levels of Ca2+ mobilization and differing tyrosine phosphorylation patterns.1, 55–59 However, at this point these unique signaling outcomes are unclear, although recent evidence is emerging to support an involvement of SLP-65 (BLNK/BASH). SLP-65 associates with the cytoplasmic domain of Igα through a third tyrosine residue that is conserved between chicken and mouse Igα.60 SLP-65 is a cytoplasmic adaptor protein which is phosphorylated only through Igα mediated signaling59 and when phosphotylated, associates with the downstream effectors Btk and PLCγ 2. As such Igα mediated signaling may be required for recruitment of either the SLP-65/Btk/PLCγ 2 protein complex, or an as yet unidentified SLP-65 based complex, that is of specific importance during development. The observation that SLP-65 deficient mice have a defect in normal B cell development61 would be consistent with such a postulate. Specifically, a fivefold reduction of pre-B cells was observed in SLP-65 deficient mice. Moreover in such mice immature and re-circulating mature cell populations were also reduced significantly.62 Therefore SLP-65 activation through Igα may be required to efficiently support the earlier stages of B cell development. Other Igα specific sequences may be involved in these later stages of development. In particular the exchange of a QTAT sequences present in the murine Igβ cytoplasmic tail with a DCSM sequence in the Igα tail was sufficient to generate an Igα signaling phenotype from the mutated Igβ cytoplasmic tail. As such it has been suggested that these sequences may be involved in regulating the cytoplasmic tail conformation and, as a result, regulating interactions between Igα and/or Igβ with downstream signaling effectors.55 Nonetheless, these sequences are not fully conserved (GIAT and QCSM, respectively in chickens) arguing that the regulatory sequences may be more narrowly constrained than previously suggested.
Common function of Igα and Igβ The requirement for signaling downstream of Igα and/or Igβ in B cell development is further supported by the block in B lineage development in mice deficient in the Syk protein tyrosine kinase. While V–DJH chain rearrangement does occur in such mice, there is a lack of clonal expansion and selection for productive rearrangement.50 Therefore Syk is critical in cell proliferation at the pre-B cell stage of development. Syk can associate with the phosphorylated ITAMs of both Igα and Igβ,51 consistent with the evidence that either chain can support these earlier stages of development. Nonetheless, the induction of VH rearrangement in Syk−/− mice suggests that signals distinct from Syk mediate B lineage progression supported by the pro-BcR. In addition to the tyrosine residues of the ITAM motifs, the cytoplasmic tails of Igα and Igβ contain serine and threonine residues that can become phosphorylated. Phosphorylation of ITAM proximal serine/threonine residues results in negative regulation of ITAM-mediated signaling.52 Following BcR crosslinking, Igβ is much more weakly phosphorylated on tyrosine residues than Igα.53, 54 This may be a consequence of both ITAM tyrosines within Igβ being surrounded by serine/threonine residues resulting in heavy negative regulation. As further evidence of the regulatory function of ITAM-surrounding sequences, the isolated Igβ ITAM is capable of inducing a full calcium influx whereas the full length Igβ is not.55 Consequently quantitative differences in the ability of Igα and Igβ to independently support early stages of B cell development may be a reflection of differences in the basal levels of Igα and Igβ ITAM tyrosine phosphorylation.
Conclusion Evidence accumulating from a number of experimental systems supports the notion that expression rather than ligation of the BcR, and its related complexes (pre-BcR, pre-diversified BcR) is sufficient to support B cell development. BcR expression must result in
Unique functions of Igα and Igβ B cell development to the mature B cell stage requires the cytoplasmic domains of both Igα and Igβ. This 356
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a basal level of signaling through the BcR although the nature of such basal signaling remains undefined. However, we would anticipate that surface expression of the Igα/β heterodimer would organize signaling and/or effector proteins into signaling complexes resulting in a constant dynamic equilibrium between phosphorylated and de-phosphorylated forms of the receptor. This might result, at any given point in time in a certain proportion of BcR complexes being associated with downstream signaling molecules. This proportion would be subject to levels of kinase and phosphatase activity within the unstimulated cell. Thus a developing B lineage cell could distinguish too little signal (as a consequence of non-productive V(D)J rearrangement), too much signal (as a consequence of receptor ligation by self antigen) and an intermediate level of signal (as a consequence of receptor expression in the absence of ligation). The regulation of B lineage development by receptor complex signaling intensity bears a striking similarity to the selection of T cells in the thymus, where only those cells receiving an intermediate level of signaling are selected into the mature T cell repertoire. It will clearly be of interest to determine whether homologous signaling pathways regulate development of both B and T cell lineages.
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serine/threonine-kinase recruitment. Proc Natl Acad Sci USA 91:474–478 Reichlin A, Hu Y, Meffre E, Nagaoka H, Gong S, Kraus M, Rajewsky K, Nussenzweig MC (2001) B cell development is arrested at the immature B cell stage in mice carrying a mutation in the cytoplasmic domain of immunoglobulin β. J Exp Med 193:13–23 Torres RM, Flaswinkel H, Reth M, Rajewsky K (1996) Aberrant B cell development and immune response in mice with a compromised BCR complex. Science 272:1804–1808 Yancopoulos GD, Alt FW (1985) Developmentally controlled and tissue-specific expression of unrearranged VH gene segments. Cell 40:271–281 Yancopoulos GD, Alt FW (1986) Regulation of the assembly and expression of variable-region genes. Annu Rev Immunol 4:339– 368 Corcoran AE, Riddell A, Krooshoop D, Venkitaraman AR (1998) Impaired immunoglobulin gene rearrangement in mice lacking the IL-7 receptor. Nature 391:904–907 Marshall AJ, Fleming HE, Wu GE, Paige CJ (1998) Modulation of the IL-7 dose-response threshold during pro-B cell differentiation is dependent on pre-B cell receptor expression. J Immunol 161:6038–6045 Shinkai Y, Rathbun G, Lam KP, Oltz EM, Stewart V, Mendelsohn M, Charron J, Datta M, Young F, Stall AM (1992) RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68:855–867 Mombaerts P, Iacomini J, Johnson RS, Herrup K, Tonegawa S, Papaioannou VE (1992) RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68:869–877 Papavasiliou F, Misulovin Z, Suh H, Nussenzweig MC (1995) The role of Igβ in precursor B cell transition and allelic exclusion. Science 268:408–411 Papavasiliou F, Jankovic M, Suh H, Nussenzweig MC (1995) The cytoplasmic domains of immunoglobulin (Ig)α and Igβ can independently induce the precursor B cell transition and allelic exclusion. J Exp Med 182:1389–1394 Paramithiotis E, Jacobsen KA, Ratcliffe MJ (1995) Loss of surface immunoglobulin expression precedes B cell death by apoptosis in the bursa of Fabricius. J Exp Med 181:105–113 Paramithiotis E, Ratcliffe MJ (1993) Bursa-dependent subpopulations of peripheral B lymphocytes in chicken blood. Eur J Immunol 23:96–102 Bradl H, Jack HM (2001) Surrogate light chain-mediated interaction of a soluble pre-B cell receptor with adherent cell lines. J Immunol 167:6403–6411 Kitamura D, Roes J, Kuhn R, Rajewsky K (1991) A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin µ chain gene. Nature 350:423–426 Kitamura D, Kudo A, Schaal S, Muller W, Melchers F, Rajewsky K (1992) A critical role of λ5 protein in B cell development. Cell 69:823–831 Mundt C, Licence S, Shimizu T, Melchers F, Martensson IL (2001) Loss of precursor B cell expansion but not allelic exclusion in VpreB1/VpreB2 double-deficient mice. J Exp Med 193:435–445 Pelanda R, Schaal S, Torres RM, Rajewsky K (1996) A prematurely expressed Igκ transgene, but not VκJκ gene segment targeted into the Igκ locus, can rescue B cell development in λ5-deficient mice. Immunity 5:229–239 Shaffer AL, Schlissel MS (1997) A truncated heavy chain protein relieves the requirement for surrogate light chains in early B cell development. J Immunol 159:1265–1275
46. Sayegh CE, Demaries SL, Iacampo S, Ratcliffe MJ (1999) Development of B cells expressing surface immunoglobulin molecules that lack V(D)J-encoded determinants in the avian embryo bursa of Fabricius. Proc Natl Acad Sci USA 96:10806–10811 47. Bannish G, Fuentes-Panana EM, Cambier JC, Pear WS, Monroe JG (2001) Ligand-independent signaling functions for the B lymphocyte antigen receptor and their role in positive selection during B lymphopoiesis. J Exp Med 194:1583–1596 48. Kraus M, Pao LI, Reichlin A, Hu Y, Canono B, Cambier JC, Nussenzweig MC, Rajewsky K (2001) Interference with immunoglobulin (Ig)α immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation modulates or blocks B cell development, depending on the availability of an Igβ cytoplasmic tail. J Exp Med 194:455–469 49. Wienands J, Larbolette O, Reth M (1996) Evidence for a preformed transducer complex organized by the B cell antigen receptor. Proc Natl Acad Sci USA 93:7865–7870 50. Cheng AM, Rowley B, Pao W, Hayday A, Bolen JB, Pawson T (1995) Syk tyrosine kinase required for mouse viability and B-cell development. Nature 378:303–306 51. Rowley RB, Burkhardt AL, Chao HG, Matsueda GR, Bolen JB (1995) Syk protein-tyrosine kinase is regulated by tyrosinephosphorylated Igα/Igβ immunoreceptor tyrosine activation motif binding and autophosphorylation. J Biol Chem 270:11590–11594 52. Muller R, Wienands J, Reth M (2000) The serine and threonine residues in the Igα cytoplasmic tail negatively regulate immunoreceptor tyrosine-based activation motif-mediated signal transduction. Proc Natl Acad Sci USA 97:8451–8454 53. Clark MR, Campbell KS, Kazlauskas A, Johnson SA, Hertz M, Potter TA, Pleiman C, Cambier JC (1992) The B cell antigen receptor complex: association of Igα and Igβ with distinct cytoplasmic effectors. Science 258:123–126 54. Flaswinkel H, Reth M (1994) Dual role of the tyrosine activation motif of the Igα protein during signal transduction via the B cell antigen receptor. EMBO J 13:83–89 55. Cassard S, Choquet D, Fridman WH, Bonnerot C (1996) Regulation of ITAM signaling by specific sequences in Igβ B cell antigen receptor subunit. J Biol Chem 271:23786–23791 56. Baumann G, Maier D, Freuler F, Tschopp C, Baudisch K, Wienands J (1994) In vitro characterization of major ligands for Src homology 2 domains derived from protein tyrosine kinases, from the adaptor protein SHC and from GTPase-activating protein in Ramos B cells. Eur J Immunol 24:1799–1807 57. Lin J, Justement LB (1992) The MB-1/B29 heterodimer couples the B cell antigen receptor to multiple src family protein tyrosine kinases. J Immunol 149:1548–1555 58. Taddie JA, Hurley TR, Hardwick BS, Sefton BM (1994) Activation of B- and T-cells by the cytoplasmic domains of the B-cell antigen receptor proteins Igα and Igβ. J Biol Chem 269:13529–13535 59. Kim KM, Alber G, Weiser P, Reth M (1993) Differential signaling through the Igα and Igβ components of the B cell antigen receptor. Eur J Immunol 23:911–916 60. Engels N, Wollscheid B, Wienands J (2001) Association of SLP-65/BLNK with the B cell antigen receptor through a non-ITAM tyrosine of Igα. Eur J Immunol 31:2126–2134 61. Jumaa H, Mitterer M, Reth M, Nielsen PJ (2001) The absence of SLP65 and Btk blocks B cell development at the preB cell receptor-positive stage. Eur J Immunol 31:2164–2169 62. Xu S, Tan JE, Wong EP, Manickam A, Ponniah S, Lam KP (2000) B cell development and activation defects resulting in xid-like immunodeficiency in BLNK/SLP-65-deficient mice. Int Immunol 12:397–404
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Cell surface immunoglobulin receptors in B cell development Kelly A. Pike a,b and Michael J.H. Ratcliffe a,b,∗ ity of cells entering the B lineage developmental pathway fail to express a functional surface immunoglobulin receptor (BcR). However, the end product of B lineage development is the generation of a population of B lymphocytes, all of which express a functional BcR. Consequently, B cell lymphopoiesis is regulated to eliminate those precursors that fail to express a BcR complex, while supporting the further development of those precursors that express a functional receptor. Pathways of B cell lymphopoiesis have diverged during evolution. Thus while B cell development in some species, such as rodents and primates, occurs primarily in the bone marrow, other species use a variety of gut associated lymphoid tissues as their primary B lymphoid organ. Nonetheless, selection for cells expressing a functional BcR has been maintained throughout evolution. In this review we will draw on insights derived from different models of B cell development to address the role of the BcR complex in supporting B cell development.
Expression of surface immunoglobulin (sIg) related receptors has been conserved in phylogenetically distinct species as a critical checkpoint in B cell development. The sIg receptor comprises extracellular IgM heavy and light chains, with the potential for ligand binding, complexed to the Igα/Igβ heterodimer that is responsible for signal transduction through sIg. Experimental systems, from both avian and murine models of B cell development, have been designed to identify the function of individual receptor components in B cell development. In this review, we assess the regulatory functions of different components of the sIg receptor complex during early development in experimental systems from evolutionarily distinct species. Key words: B lymphocytes / surface immunoglobulin / bursa of fabricius / Igα / Igβ © 2002 Elsevier Science Ltd. All rights reserved.
Introduction
The BcR complex
A diverse repertoire of antibodies is required for protection against the heterogeneous array of pathogens in the environment. The molecular processes governing the generation of diverse Ig repertoires vary between species but in virtually all cases require modification of germ line sequences. These modifications include rearrangement of V, D and J gene segments that in some species subsequently undergo further diversification by gene conversion or hypermutation. Each of these processes is error prone, and the major-
IgM is expressed at the surface of B cells in association with the Igα/Igβ heterodimer. The products of V(D)J recombination of both immunoglobulin heavy and light loci covalently associate, forming a ligand recognition domain. The short three amino acid cytoplasmic tail of the µ chain has no intrinsic signaling capacity.1 Rather surface IgM (sIgM) is non-covalently associated with the disulphide bonded Igα/β heterodimer. The Igα/Igβ heterodimer has been conserved during evolution and each chain contains an extracellular Ig domain, a transmembrane region and a cytoplasmic domain that contains conserved signaling motifs (reviewed in Reference 2). The cytoplasmic domains of both Igα and Igβ contain immunoreceptor tyrosine-based activation motifs (ITAMs)3 as well as other residues important for signaling through the BcR (reviewed in Reference 2).
From the a Department of Immunology, University of Toronto, 1 King’s College Circle, Toronto, Ont., Canada M5S 1A8 and b Sunnybrook and Women’s College Health Sciences Center, 2075 Bayview Avenue, Toronto, Ont., Canada M4N 3M5. * Corresponding author. E-mail: [email protected] © 2002 Elsevier Science Ltd. All rights reserved. 1044–5323 / 02 / $– see front matter
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In contrast to murine B cell development, the rearrangement of chicken Ig VH and VL genes occurs stochastically rather than sequentially.17 Consequently, there is no apparent role for expression of a pre-BcR in chicken B cell development and indeed, to this point, chicken homologues to the VpreB and λ5 genes have not been identified. However, in contrast to the mouse, V(D)J rearrangement in the chicken generates minimal antibody diversity.18 At the light chain locus there is only one functional VL segment, which rearranges to the unique JL segment.19 Similarly, heavy chain rearrangement utilizes one functional VH segment, a cluster of highly conserved DH segments and a unique JH segment.20 As such, rearrangement of chicken Ig genes generates BcRs of limited diversity that have been referred to as the ‘pre-diversified BcR’. BcR diversity is subsequently generated through somatic gene conversion events that occur within bursal follicles.21 The initial checkpoint in chicken B cell development is therefore expression of the pre-diversified BcR, a complex that contains µ and light chains of restricted diversity in association with the Igα/Igβ heterodimer. Expression of this receptor requires productive rearrangement at both Ig heavy and light chain loci and is required for productive colonization of bursal follicles and subsequent Ig diversification by gene conversion.
Checkpoints in murine B cell development After birth, murine B cell development occurs within the bone marrow. Prior to the onset of the VH gene rearrangement pro-B cells express the Igα/β heterodimer at the cell surface as part of a complex that includes the chaperone calnexin which can be defined as the pro-BcR.4, 5 Following productive Ig heavy chain rearrangement, the µ heavy chain is expressed at the cell surface with the surrogate light chain molecules VpreB and λ5 and in association with the Igα/Igβ heterodimer as a complex defined as the pre-BcR. Cells that fail to productively rearrange the heavy chain locus and generate a pre-BcR undergo apoptosis.6 Pre-BcR expression results in the termination of further rearrangement at the heavy chain locus and the induction of a burst of pre-B cell proliferation.7, 8 Pre-B cells downregulate transcription of VpreB and λ5 and surface pre-BcR levels are diluted during this round of proliferation. As a consequence, pre-B cells stop proliferating and undergo rearrangement of the light chain locus.8 Those cells that have productively rearranged the light chain locus, and express a BcR (µ and κ or λ, associated with the Igα/Igβ complex), subsequently migrate out of the bone marrow into the periphery. In contrast, cells unable to generate a functional BcR are eliminated (reviewed in Reference 9). The expression of pre-BcR and BcR therefore represent checkpoints that define the productivity of rearrangement at the heavy and light chain loci, respectively.
The Igα/β heterodimer is required for completion of VDJH rearrangement (the pro-B cell to pre-B cell transition) As described above, the Igα/β heterodimer is expressed on the surface of pro-B cells as part of the pro-BcR complex prior to the completion of VDJH rearrangement. The functional importance of pro-BcR expression is evidenced in mice deficient for Igβ. In such mice B lineage cells undergo normal levels of DJH rearrangement but the levels of V–DJ rearrangement are drastically reduced.22 Therefore surface pro-BcR expression appears to play a critical role in regulating induction of the murine heavy chain V gene rearrangements required for the pro-B to pre-B transition, prior to the pre-BcR checkpoint. Surface expression of murine sIgM, and by extension the pre-BcR requires co-expression of both Igα and Igβ.23–27 In consequence, complete deletion of either Igα or Igβ cannot address the question as to whether surface expression of both Igα and Igβ (as opposed to either Igα or Igβ) are required to support B
Checkpoints in avian B cell development In the avian system, the bursa of Fabricius is the primary lymphoid organ for B cell lymphopoiesis. Nevertheless, the bursal microenvironment is not required for B lineage commitment or the induction of Ig gene rearrangement.10–13 Starting at day 8 of embryogenesis the bursal mesenchyme is colonized by a single wave of B cell precursors14 some of which migrate across the bursal epithelial basement membrane and undergo proliferation within the epithelial buds that eventually develop into bursal follicles.11, 15 Only those B cell precursors that have undergone productive Ig gene rearrangement and express a sIg receptor are selected for clonal expansion within the bursa.16 Therefore, as in the murine system, surface expression of a BcR complex represents a critical checkpoint in early B cell development. 352
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lineage development. Therefore, as an alternative to deletion of the complete Igα or Igβ molecule, truncation of the cytoplasmic domains of Igα or Igβ has been used to disrupt their signaling ability while maintaining their capacity to form a sIg related receptor complex. In mice containing truncations of both Igα and Igβ, reduced numbers of cells expressing cytoplasmic µ chains were observed. It was therefore concluded that truncations of both Igα and Igβ results in a reduction of V–DJ rearrangement, similar to that observed in Igβ −/− mice.28 Conversely, in mice containing either a truncated Igα or a truncated Igβ, the B lineage cells proceed through this checkpoint, undergo VH to DJH rearrangement and establish a pre-B cell population.28, 29 Consequently, the pro-B to pre-B transition is supported by the cytoplasmic domain of either Igα or Igβ. At this point it remains unclear how pro-BcR expression may regulate the induction of VH rearrangement. Nonetheless, IL-7 receptor signaling modifies the accessibility of the VH locus for rearrangement.30, 31 Moreover, as observed in Igβ −/− mice, IL-7Rα −/− mice undergo normal levels of D–J rearrangement while displaying a marked reduction of V–DJ rearrangement. In particular a decreased usage of 5 VH J558 segments was observed in the IL-7Rα −/− mice.32 Given the demonstrated interplay between IL-7 receptor signaling and signaling through the Igα/β heterodimer in pre-B cells,33 pro-BcR expression and consequent signaling through Igα and/or Igβ may increase the sensitivity of pro-B cells to IL-7 modulated VH locus accessibility for rearrangement.
receptor could not. Moreover, when introduced into recombinase competent mice, the YS:VV mµ receptor was not able to support allelic exclusion of V–DJ rearrangement.36 Consequently the association between µ and the Igα/β heterodimer as part of the pre-BcR is required for the expansion of a functional pre-B cell population.
Igα/β in pre-B cell expansion While the transgene encoding the YS:VV mutant µ chain failed to support pre-B cell expansion, the same mutant µ chain when fused directly to the cytoplasmic domain of either Igα or Igβ (YS:VV–IgM:Igα or YS:VV–IgM:Igβ, respectively) supported the progression of B cell development through the pro-B to pre-B transition in RAG1−/− mice.36, 37 This transition was characterized not only on the basis of B lineage phenotype but also by the establishment of allelic exclusion at the heavy chain locus. Whereas mice containing the Igβ truncation contained normal numbers of pre-B cells, indicative of substantial pre-B expansion, mice containing the Igα truncation contain reduced numbers of pre-B cells. Thus signals downstream of Igα appear more efficient at supporting the pro-B to pre-B transition or expanding pre-B cells.28, 29 However, in contradiction to this conclusion, the relative efficiencies of the YS:VV–IgM:Igα and YS:VV–IgM:Igβ chimeric receptors in supporting the pro-B to pre-B transition were indistinguishable.38, 39 This may be a reflection of the organization of the cytoplasmic domains of Igα and Igβ in the plasma membrane. In BcR related complexes containing the truncation mutants of Igα or Igβ, each Igα/β heterodimer contains one functioning cytoplasmic domain per heterodimer. In contrast, the YS:VV–IgM:Igα and YS:VV–IgM:Igβ chimeric proteins would each be expressed as surface homodimers, as a consequence of µ chain dimerization, containing two functional cytoplasmic Igα or Igβ domains, respectively.
Pre-B cell expansion requires association between pre-BcR and Igα/β Following VH rearrangement, the expression of a functional pre-BcR, µ/VpreB/λ5, is required for pre-B cell expansion and the downregulation of further heavy chain rearrangement. The association between sIgM heavy chains and the Igα/β heterodimer depends on polar amino acids in the transmembrane region of µ chain. Reducing the polarity of the µ transmembrane region resulted in surface expression of the mutant IgM (YS:VV mµ) in the absence of Igα/β.1 B cell development in RAG1−/− or RAG2−/− mice is blocked at the pro-B cell stage of development.34, 35 While a wild-type µ chain, that can associate with Igα/β, supported the generation and expansion of a pre-B cell population in RAG2−/− mice, the YS:VV mµ mutant
Igα/β in the pre-B to B cell transition Pre-B cells undergo rearrangement at the light chain locus and those that express a functional BcR make the transition to immature B cells. In mice containing the Igβ truncation and therefore expressing a pre-BcR containing only functional Igα, normal levels of immature B cells were generated.28 In contrast, mice 353
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containing a truncated Igα, and therefore expressing a pre-BcR containing only Igβ, reduced levels of pre-B cells were matched by an equivalent reduction in immature B cell numbers.29 This suggests the possibility that the transition from pre-B to immature B cell is supported by either Igα or Igβ with equal efficiency. Following productive light chain rearrangement resulting in immature B cells subsequent maturation generated a pool of mature recirculating B cells. The transition from immature to mature B cells is strikingly reduced in mice containing either the Igα or Igβ truncation, with most immature B cells undergoing apoptotic cell death.29 Consequently, while expression of the cytoplasmic domains of either Igα or Igβ is sufficient to support B lineage development to the immature B cell stage, albeit with differing efficiencies, the cytoplasmic domains of both Igα and Igβ are required for the efficient establishment of a mature B cell pool. It is important to consider, however, that in both experimental models, it is impossible to assess the efficiency with which the chimeric YS:VV–IgM:Igα and YS:VV–IgM:Igβ receptors or the Igα/Igβ truncations support B cell development relative to normal B cell development. Thus the YS:VV–IgM:Igα and YS:VV–IgM:Igβ chimeric receptors were introduced into RAG1−/− mice in which normal B lineage development is compromised. Similarly in mice containing truncations of Igα or Igβ, there is no development of the B cell lineage supported by the intact Igα/β heterodimer.
development at the pre-B cell stage as well as a failure to establish allelic exclusion.41 Similarly, B cell development in λ5−/− ,42 and VpreB1−/− /VpreB2−/− ,43 mice is largely blocked at the pre-B cell stage. Importantly, B cell development is not completely abrogated, in either λ5−/− or VpreB1−/− /VpreB2−/− mice. This is most likely a consequence of low level light chain gene rearrangement, allowing a small proportion of developing B lineage cells to express a µκ or µλ BcR at the pre-B developmental stage. More dramatically, transgenic expression of light chains in pre-B cells can overcome the developmental block in λ5−/− ,44 mice arguing that while expression of a sIg related receptor is critical, recognition of ligand by the VpreB/λ5 determinants of the pre-BcR is not required for B cell development.
Truncated sIgM receptors support B cell development Further support for the contention that ligand recognition by the pre-BcR is not required to support B cell development came from mice transgenic for the expression of a truncated IgM (Tµ) that lacked VH and a part of the Cµ1 domain. This Tµ failed to associate with λ5 or VpreB and, as a consequence of failing to associate with BiP, was expressed at the cell surface in the absence of surrogate light chain proteins. This Tµ receptor supported the generation of pre-B cells, and induced the downregulation of λ5 transcription in Rag1−/− mice. Moreover, the introduction of the Tµ into µMT mice rescued allelic exclusion and induced rearrangement at the light chain locus.45 Again, the efficiency with which the Tµ construct supported B cell development is difficult to assess as these observations were made in RAG1−/− mice in which normal B cell development was abrogated. In an alternative approach, retroviral gene transfer was used to introduce a Tµ receptor into chicken B cell precursor cells in vivo. The Tµ construct in these experiments contained a deletion of the entire VH and Cµ1 domains and was expressed at the B cell surface in the absence of light chain. Expression of this receptor was sufficient to support the early stages of avian B cell development: colonization of bursal follicles, clonal expansion of B cells within bursal follicles and the induction of gene conversion. Crucially, however, since the Tµ encoding virus was introduced into normal B cell precursors, B cell development supported by the Tµ receptor occurred in competition with B cell development supported by expression of
Igα/β expression versus receptor ligation While the BcR is clonally diverse, this is not the case for either the avian pre-diversified BcR or mammalian pre-BcR. In the case of the pre-BcR, the VpreB/λ5 surrogate light chain complex is invariant and the pre-diversified BcR also has limited diversity. This has suggested the possibility that these receptors may bind ligand(s), as distinct from specific antigen, in their environment and that this recognition is required to support subsequent B lineage development. Indeed, the observation that a soluble pre-BcR construct binds to stromal cells has been taken to support this contention.40 Failure to generate a sIg receptor related complex at the pre-B stage of murine B cell development results in developmental arrest. Thus genetic disruption of the exon encoding the transmembrane domain of murine IgM membrane (µMT) resulted in an arrest of B cell 354
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the endogenous pre-diversified BcR. Under these circumstances, the Tµ receptor supported development with an efficiency indistinguishable from the endogenous receptor.46 Both Tµ models demonstrate that a specific interaction between ligand and the V(D)J (or VpreB/λ5) encoded determinants of either the preBcR or pre-diversified BcR is not required for the early stages of B cell development. However, these experiments did not exclude the possibility that development required an interaction between a ligand and the residual extracellular domains of either the µ chain or the Igα/β heterodimer.
sIg demonstrated that expression of the chimeric receptor supported the early stages of B cell development with an efficiency equivalent to that observed following expression of the pre-diversified BcR (Pike et al., manuscript in preparation). This provides further support for the argument that ligation of either the pre-diversified BcR or pre-BcR is not required for the efficient progression of B cell development.
Basal signaling through Igα/β in the absence of receptor ligation Taken together the experimental systems outlined above strongly suggest that membrane proximal expression of the cytoplasmic domains of Igα and/or Igβ is sufficient to support the generation of immature B cells. Under these circumstances the role of µ/VpreB/λ5 in the pre-BcR or µ/L in the BcR or avian pre-diversified BcR would be to function as a chaperone to facilitate the expression of the Igα/β heterodimer at the pre-B or B cell surface. The cytoplasmic domains of Igα and Igβ contain ITAM motifs.3 Disrupting these ITAM motifs disrupted the ability of the Igα/β cytoplasmic domains to support B cell development.36, 48 Similarly, disrupting the Igα ITAM of the CD8α:Igα chimeric receptor disrupted its ability to support bursal colonization, even in the presence of a CD8β:Igβ chain containing a functional Igβ ITAM. Following receptor expression in the absence of overt ligation, the ITAM motifs of Igα and Igβ are constantly being phosphorylated and dephosphorylated. This has best been demonstrated by the incubation of cells with pervanadate, which blocks phosphotyrosine phosphatase activity, and rapidly induces high levels of Igα and Igβ tyrosine phosphorylation despite the lack of deliberate activation of tyrosine kinases.49 We, and others, would support the conclusion that the steady state levels of Igα and Igβ phosphorylation in unstimulated cells is sufficient to allow basal signal transduction downstream of Igα and/or Igβ and that this basal signaling is required to support the progression of B cell development. The ability of the cytoplasmic domains of Igα and Igβ to independently support the generation of immature murine B cells suggests that at least some aspects of downstream signaling are shared between the two chains. In contrast, the requirement for the cytoplasmic domains of both Igα and Igβ in the generation of mature B cells suggests that there must be unique functions associated with each domain or a
BcR expression restricted to the cytoplasmic domains of Igα/β The ability of the chimeric YS:VV–IgM:Igα and YS:VV– IgM:Igβ chains to support B lineage development in the absence of endogenous receptor expression argued that ligand recognition by the extracellular domains of Igα or Igβ is not an obligatory requirement in B lineage progression. However, two experimental systems have been developed to address this issue more specifically. In one model, a Lck myristoylation/palmitoylation signal was used to target a fusion protein containing the cytoplasmic domains of Igα and Igβ to the cell surface in the absence of their extracellular domains. Expression of lipid targeted Igα/β cytoplasmic domains in B lineage precursors supported their development into immature B cells, under circumstances where the endogenous pre-BcR/BcR was not synthesized.47 While these studies demonstrate that there is no obligate requirement for extracellular ligation of the pre-BcR in B cell development, again the efficiency with which the lipid targeted Igα and Igβ cytoplasmic domains supported B cell development relative to endogenous intact receptors is difficult to judge, as these experiments were performed in µMT B cell precursors. In an alternative model, a chimeric receptor, in which the extracellular and transmembrane domains of mouse CD8α and CD8β were fused to the cytoplasmic domains of chicken Igα and Igβ, respectively, was introduced into developing chicken B cell precursors by retroviral gene transfer. These receptors were expressed in normal embryos fully competent to express an endogenous pre-diversified BcR. The presence of roughly equivalent numbers of developing B cells expressing either endogenous sIg or the chimeric CD8:Igα/β receptor in the absence of endogenous 355
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combination of the Igα and Igβ cytoplasmic domains that are required for the later transition. Further support for unique biological properties associated with the cytoplasmic domains of Igα and Igβ is evidenced in avian B cell development where bursal colonization is supported by the chimeric CD8α:Igα alone, but not by a CD8α:Igβ chimeric receptor alone (Pike et al., manuscript in preparation).
indicates that there are unique downstream consequences of signaling through each of these chains that are required for B cell development. The majority of in vitro assays have demonstrated that Igα and Igβ induce differing levels of Ca2+ mobilization and differing tyrosine phosphorylation patterns.1, 55–59 However, at this point these unique signaling outcomes are unclear, although recent evidence is emerging to support an involvement of SLP-65 (BLNK/BASH). SLP-65 associates with the cytoplasmic domain of Igα through a third tyrosine residue that is conserved between chicken and mouse Igα.60 SLP-65 is a cytoplasmic adaptor protein which is phosphorylated only through Igα mediated signaling59 and when phosphotylated, associates with the downstream effectors Btk and PLCγ 2. As such Igα mediated signaling may be required for recruitment of either the SLP-65/Btk/PLCγ 2 protein complex, or an as yet unidentified SLP-65 based complex, that is of specific importance during development. The observation that SLP-65 deficient mice have a defect in normal B cell development61 would be consistent with such a postulate. Specifically, a fivefold reduction of pre-B cells was observed in SLP-65 deficient mice. Moreover in such mice immature and re-circulating mature cell populations were also reduced significantly.62 Therefore SLP-65 activation through Igα may be required to efficiently support the earlier stages of B cell development. Other Igα specific sequences may be involved in these later stages of development. In particular the exchange of a QTAT sequences present in the murine Igβ cytoplasmic tail with a DCSM sequence in the Igα tail was sufficient to generate an Igα signaling phenotype from the mutated Igβ cytoplasmic tail. As such it has been suggested that these sequences may be involved in regulating the cytoplasmic tail conformation and, as a result, regulating interactions between Igα and/or Igβ with downstream signaling effectors.55 Nonetheless, these sequences are not fully conserved (GIAT and QCSM, respectively in chickens) arguing that the regulatory sequences may be more narrowly constrained than previously suggested.
Common function of Igα and Igβ The requirement for signaling downstream of Igα and/or Igβ in B cell development is further supported by the block in B lineage development in mice deficient in the Syk protein tyrosine kinase. While V–DJH chain rearrangement does occur in such mice, there is a lack of clonal expansion and selection for productive rearrangement.50 Therefore Syk is critical in cell proliferation at the pre-B cell stage of development. Syk can associate with the phosphorylated ITAMs of both Igα and Igβ,51 consistent with the evidence that either chain can support these earlier stages of development. Nonetheless, the induction of VH rearrangement in Syk−/− mice suggests that signals distinct from Syk mediate B lineage progression supported by the pro-BcR. In addition to the tyrosine residues of the ITAM motifs, the cytoplasmic tails of Igα and Igβ contain serine and threonine residues that can become phosphorylated. Phosphorylation of ITAM proximal serine/threonine residues results in negative regulation of ITAM-mediated signaling.52 Following BcR crosslinking, Igβ is much more weakly phosphorylated on tyrosine residues than Igα.53, 54 This may be a consequence of both ITAM tyrosines within Igβ being surrounded by serine/threonine residues resulting in heavy negative regulation. As further evidence of the regulatory function of ITAM-surrounding sequences, the isolated Igβ ITAM is capable of inducing a full calcium influx whereas the full length Igβ is not.55 Consequently quantitative differences in the ability of Igα and Igβ to independently support early stages of B cell development may be a reflection of differences in the basal levels of Igα and Igβ ITAM tyrosine phosphorylation.
Conclusion Evidence accumulating from a number of experimental systems supports the notion that expression rather than ligation of the BcR, and its related complexes (pre-BcR, pre-diversified BcR) is sufficient to support B cell development. BcR expression must result in
Unique functions of Igα and Igβ B cell development to the mature B cell stage requires the cytoplasmic domains of both Igα and Igβ. This 356
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a basal level of signaling through the BcR although the nature of such basal signaling remains undefined. However, we would anticipate that surface expression of the Igα/β heterodimer would organize signaling and/or effector proteins into signaling complexes resulting in a constant dynamic equilibrium between phosphorylated and de-phosphorylated forms of the receptor. This might result, at any given point in time in a certain proportion of BcR complexes being associated with downstream signaling molecules. This proportion would be subject to levels of kinase and phosphatase activity within the unstimulated cell. Thus a developing B lineage cell could distinguish too little signal (as a consequence of non-productive V(D)J rearrangement), too much signal (as a consequence of receptor ligation by self antigen) and an intermediate level of signal (as a consequence of receptor expression in the absence of ligation). The regulation of B lineage development by receptor complex signaling intensity bears a striking similarity to the selection of T cells in the thymus, where only those cells receiving an intermediate level of signaling are selected into the mature T cell repertoire. It will clearly be of interest to determine whether homologous signaling pathways regulate development of both B and T cell lineages.
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