- Email: [email protected]
Selection events operating at various stages in B cell development
202
Selection events operating at various stages in B cell development Antonius G Rolink*, Christoph Schaniel, Jan Andersson and Fritz Melchers B cells have to progress through various checkpoints during their process of development. The three transcription factors E2A, EBF (early B cell factor) and Pax5 play essential roles in B cell commitment checkpoints. The various forms of the BCR and their downstream signaling molecules, which are expressed at different stages of B cell development, act as critical checkpoint guards allowing (positive selection) or preventing (negative selection) developmental progression. The recent advances on the molecular mechanisms operating at these various checkpoints are here summarized and discussed. Addresses Basel Institute for Immunology, Grenzacherstrasse 487, CH-4005 Basel, Switzerland *e-mail: [email protected] Current Opinion in Immunology 2001, 13:202–207 0952-7915/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations btk Bruton’s tyrosine kinase CDR3 complementarity-determining region 3 EBF early B cell factor FLOC fetal liver organ culture IgH immunoglobulin heavy chain IgL immunoglobulin light chain sIgM surface IgM SL surrogate light chain
Introduction The development of B cells in bone marrow has been dissected into several discrete stages by the discovery and use of a series of surface-bound and cytoplasmically expressed markers (for a review, see [1]). These developmental stages have subsequently been ordered according to the rearrangement status of the immunoglobulin heavy (IgH) and light (IgL) chain gene loci at the single-cell level (for a review, see [1]). Over the past decade, both the introduction of numerous mutations into the germline and the identification of natural mutants have shown that B cells must pass several checkpoints in order to progress through these distinct developmental stages. Here we review recent progress in research in three of these checkpoints. In addition, we will discuss the evidence for positive selection of B cells.
encoding components of the rearrangement machinery nor the pre-BCR is a sign of commitment. These conclusions are based on recent findings made with mice deficient in a third transcription factor, Pax5. Pax5–/– mice have an absolute block in B cell development at the pre-B type I cell stage [5,6]. The pre-B I cells found in these mice have DJ-rearranged IgH loci and express genes encoding E2A, EBF, λ5, VpreB, Igα and Igβ [5,6]. Moreover, like wild-type pre-B I cells, Pax5–/– pre-B I cells can be propagated in vitro in the presence of stromal cells and IL-7 [5,6]. Unlike wild-type cells, however, Pax5–/– preB I cells possess stem-cell-like activity; that is, under appropriate in vitro and in vivo conditions they can give rise to cells of multiple hematopoietic lineages and they possess self-renewal capacity [7••,8••]. Thus, Pax5 appears to be absolutely essential in the first checkpoint of B cell development, namely commitment. But whether Pax5 expression results in irreversible or reversible commitment — that is, whether normal pre-B I cells that loose Pax5 expression regain multilineage potential — remains unknown.
Selection at the pre-BCR checkpoint VH to DHJH rearrangement is initiated in the transition from pre-B I to large pre-B II cells (for a review, see [1]). The µH chain protein that is expressed when the VHDHJH rearrangement is in-frame can pair with the surrogate light chain (SL) components λ5 and VpreB, and, in association with the signal-transducing Igα and Igβ subunits, can form the pre-BCR complex at the cell surface. About 10% of pre-B I cells in mouse bone marrow express µH chain in the cytoplasm. Not all of these µH chains can form a preBCR, however, as about half of them seem unable to do so [9]. Although not yet proven, this is the probable consequence of special CDR3 (complementarity-determining region 3), structures that do not allow pairing with the SL.
B cell commitment
Non-pairing µH chains, which contain VH gene segments of various VH gene families, have been found. Moreover, two µH chains are expressed in 2–4% of pre-B II and B cells. Of these two µH chains, only one can form a preBCR [10]. Thus, at the transition from pre-B I to pre-B II, those cells expressing a µH chain capable of forming a preBCR are positively selected, because they enter into cellular division and expansion (see below).
To date, three transcription factors have been identified as essential for the commitment of hematopoietic precursor cells to the B lymphoid lineage. E2A [2,3] and early B cell factor (EBF; [4]), both basic helix–loop–helix proteins, are required for initiation of B lymphopoiesis [2,3]; in the absence of either of these proteins, not even the earliest B cell progenitors develop. However, E2A and EBF, although required, do not determine B cell commitment. Moreover, neither the expression of genes
About 20% of pre-B I cells that express a µH chain use the most 3′ VH gene element, called VH81x. In marked contrast, peripheral B cells very rarely use VH81x. This negative selection of B cells expressing VH81x µH chains appears to be caused by the inability of these µH chains to form a pre-BCR. In fact, all tested VH81x µH chains derived from bone marrow pre-B I cells are unable to pair with the SL [9]. Moreover, the finding that a mouse strain
Selection events in B cell development Rolink et al.
carrying a VH81x µH transgene, that cannot form a preBCR, does not express this transgene in the periphery supports this hypothesis [11]. However, about 30% of VH81x µH chains expressed in fetal liver pre-B I cells do form a pre-BCR (XC Kong, F Melchers, AG Rolink, unpublished data). Moreover, such a fetal-liver-derived VH81x µH chain transgene is also expressed by peripheral B cells [12••]. Thus, the CDR3 region, which is less diverse in fetal-liver-derived µH chains owing to the absence of the enzyme TdT, appears to determine whether a VH81x µH chain can form a pre-BCR and thereby be selected into the peripheral B cell repertoire. Because the frequency of in-frame VH81x rearrangements in bone marrow pre-B I cells is high, it is somewhat surprising that, in the 2–4% of B cells in which two in-frame VHDHJH rearrangements are found, an in-frame VH81x–DHJH rearrangement has not yet been detected [10]. This counter-selection of VH81x µH chains might be explained by toxicity of these chains. Alternatively, and more interestingly, a secondary VH rearrangement (replacement) — as observed in complete immunoglobulin knockin mice — might also account for this counter-selection. Because no counter-selection for out-of-frame VH81x–DHJH rearrangements is observed, the protein coded by the inframe rearrangement must have a role in selection. For future studies, the production of an in-frame non-pre-BCRforming VH81x–DHJH knockin mouse might shed light on the mechanism controlling this negative selection. The deposition of the pre-BCR, presumably in the surface membrane, induces the pre-B II cells to enter cell cycle and divide. Pre-B cells of λ5–/– [13], µMT–/– [14] or Igβ–/– [15] pre-BCR-defective mice or of syk–/– pre-BCR-signaling-defective mice (syk is a protein tyrosine kinase) [16,17] do not enter this expansion phase. As yet, whether the preBCR needs a ligand to signal this proliferative expansion remains unambiguous; however, several observations argue in favour of ligand-independent pre-BCR signaling. First, a number of light chains expressed as transgenes prematurely in λ5–/– mice repair the proliferative defect [18,19]. Hence, a variety of endogenously produced µH chains form IgMs with these transgenic light chains to function in a pre-BCR-like fashion in large pre-B II cells. This makes it unlikely that a limited number of ligands can function via recognition of the CDRs of the VH and the CDR-like structures of the SL; and it does not rule out the possibility of ligand recognition by VpreB, which could still be associated with the pre-BCR-like IgM on the surface of the λ5–/–, light-chain-transgenic pre-B cells. Second, monoclonal antibodies specific for the preBCR — that is, specific for VpreB, λ5 and µH chains — do not perturb B cell development either when injected in vivo or when added to fetal liver organ culture (FLOC) [20]. By contrast, either in vivo or in FLOC, monoclonal antibodies specific for µH chains are perfectly capable of
203
inhibiting the development at a later stage — that is, at the stage of the development of immature, surface-IgM+ (sIgM+) B cells [20]. Hence, it appears that engagement of the pre-BCR by specific antibodies does not influence B cell development whereas engagement of the BCR on immature cells does. Third, ex vivo isolated c-Kit+CD19+ pre-B I cells proliferate and differentiate in vitro in a pre-BCR-dependent manner [21••]. Because this pre-BCR-dependent proliferation and differentiation does not require stromal cells or any exogenous cytokines, it is most likely to be ligand independent. Moreover, as occurs in vivo and in FLOC, addition of µH-chain-specific monoclonal antibody inhibits the differentiation of the cells into immature, sIgM+ B cells only (AG Rolink, unpublished data). Interestingly, single ex vivo isolated pre-B cells proliferate to a different clone size in vitro [21••]. This may be taken as an indication that different µH chains form pre-BCRs of different quality and thus allow a different number of divisions. The molecular mechanism by which the pre-BCR signals a pre-B I cell to divide still remains to be investigated in detail. However, the partial blocks in B cell development at the transition from pre-B to pre-B I in syk- [16,17] and BLNK-deficient mice (BLNK is an adapter protein) [22•–24•] indicate that these molecules have a role in this signaling event. Moreover, reconstitution of RAG-deficient mice with constitutive active forms of Ras [25•] or Raf [26•] results in restoration of pre-B cell development, which suggests that these proteins are also involved in signaling. Although the pre-BCR has a critical role in B cell development, λ5-deficient mice still make B cells — albeit in strongly reduced numbers. In a low percentage of pre-B cells, IgL chains are rearranged before IgH chain rearrangements are completed [27]. Moreover, B cell development in λ5–/– mice can be rescued by premature expression of IgL chains. On the basis of these two findings, it has been argued that B cells found in λ5–/– mice are derived from preB cells that have undergone IgL chain rearrangements before completing the IgH chains. A prediction based on this hypothesis is that IgL chain rearrangements in λ5–/– mice have taken place at a stage of development in which the enzyme TdT is expressed and thus would result in IgL chains containing nontemplate-encoded nucleotides (N-regions) in their VL–JL junctions [28]. However, sequence analysis of IgH and IgL of peripheral B cells derived from λ5–/– mice show no evidence for N-nucleotides in the IgL chains whereas the IgH chains have N-sequences like those of the IgH chains of wild-type B cells [19]. Hence, the order of IgH and IgL chain gene rearrangements in developing λ5–/– B cells appears to be identical to that observed in most wild-type pre-B cells — that is, the IgH chain rearranges before the IgL chain. Another issue is whether the pre-BCR is involved in mediating allelic exclusion. Expression of the pre-BCR results
204
Lymphocyte development
Figure 1
Immature B cells Selection
sIgM
Immature B cells Selection
Mature B cells Selection
sIgM
Production 2 x 107 / day
CD45–/– OBF–/– Igα∆c/∆c
Selection steps and proteins involved in late B cell development. The various selection steps (boxed) and important proteins (shown at the bottom of the figure) that are involved in B cell development in the bone marrow and spleen are indicated.
Spleen
Bone marrow
2 x 106 / day
Btk–/– Ii–/– Aα–/– C57Bl/6
in a very rapid downregulation of the genes and proteins involved in the rearrangement process, which could argue in favor of such a role [29]. However, allelic exclusion is not perturbed in λ5–/– mice, which indicates, at least, that a complete pre-BCR is not required [10].
Selection of sIgM+ immature B cells in the bone marrow Large pre-B II cells, which have lost expression of the preBCR, re-express the RAG-1 and RAG-2 genes at the mRNA but not the protein level [29]. Moreover, sterile transcripts from κL chain gene loci become detectable, although the IgL chain loci are still in germline configuration. When the large, cycling cells become resting and small, IgL chain gene rearrangement is induced [30]. In the mouse, peripheral B cells express κL and λL chains in a 10:1 ratio [30,31•,32•]. Single-cell light chain rearrangement analysis has revealed that this ratio is already established at the small pre-B II stage. Experiments by Engel et al. [33•] suggest that the κ locus opens before the λ locus and indicate that the κ locus has earlier access to the rearrangement machinery. This might therefore be the explanation for the high ratio of κ : λL chains found in mouse B cells. In a wild-type mouse, about half of the sIg+ B cells have rearranged only one allele at the κL chain locus. Most of these cells show only one single rearrangement, preferentially to Jκ1, the most V-proximal of the functional J segments. In marked contrast, small pre-B II cells show a dramatically increased frequency of multiple κL chain rearrangements [31•,32•]. Moreover, about 20% of small pre-B II cells express a κL chain protein in the cytoplasm but not on the cell surface although half of these cells have productively rearranged VκJκ segments. As over 95% of all pre-B II cells express µH chains in their cytoplasm, there must be reasons why these µH–κL chain combinations are not expressed on the surface.
sIgM sIgD
2 x 106 / day
Current Opinion in Immunology
We can envisage two possible reasons for the lack of surface expression of the µH–κL chain pairs. First, certain µH–κL chain combinations may not be able to form an IgM molecule. Although the peripheral, sIgM+ B cell pool should contain only pairing combinations, the small preB II cell pool may be enriched for those that do not fit together. This possibility is being tested currently by experiments in which genes encoding the µH and κL chains expressed in single small pre-B II and mature B cells are cloned and their paring capacities subsequently examined. Second, a given µH–κL chain combination may have, in fact, paired properly and been deposited on the surface. However, if this IgM were autoreactive, it would have found its autoantigen in the bone marrow and been downregulated from the surface. Nemazee and colleagues [34], and Weigert and co-workers [35,36] have shown that recognition of autoantigen by immature B cells induces secondary rearrangement at the light chain loci and thus can change the cells’ receptor specificity from self to non-self. In humans, a similar type of BCR editing has been observed [37]. The contribution of B cells that have undergone receptor editing to the total peripheral B cell repertoire is not yet clear. Moreover, it should be noted that in Cκ-deficient mice — that is, in mice that cannot make a κL chain protein — the κL chain gene rearrangement patterns in small pre-B II cells are indistinguishable from those found in their wild-type counterparts. Thus, several κL chain gene rearrangements in these pre-B cells are at least in part not immunoglobulin-receptor-induced [31•,32•].
Selection at the transition from immature to mature B cells Osmond and colleagues (reviewed in [38]) have determined that mice produce roughly 2 × 107 immature B cells per day in the bone marrow. These immature B cells then
Selection events in B cell development Rolink et al.
migrate through the terminal branches of central arterioles into the spleen. There, these immature B cells, which have half-lifetimes of about 4 days, differentiate into mature B cells with lifespans of around 15 weeks [39]. The first selection event operating at these late stages of B cell development is the transition from the bone marrow to the spleen (Figure 1). At this stage, about 90% of the immature B cells produced in the bone marrow are lost. A large part of this loss can probably be explained by the deletion of autoreactive B cells in the bone marrow. Moreover, signaling through the BCR in general appears to play an important role at this checkpoint. Thus, both syk-deficient mice and mice expressing an Igα lacking the cytoplasmic tail (Igα∆c/∆c) show a much more marked loss of B cells at this transition [40,41]. Interestingly, immature B cells in Igα∆c/∆c mice have increased levels of tyrosine phosphorylation and expression of activation antigen [42•], suggesting that the intensity of signaling may be very critical for B cells passing this checkpoint. Similar findings were made in Igα∆c/∆c mice carrying a soluble hen egg lysozyme (HEL) and an anti-HEL-immunoglobulin transgene [43•]. The finding that in CD45–/– mice more immature B cells enter the spleen, therefore, might indicate that the phosphatase CD45 is critically involved in this signaling intensity [44]. Mice deficient for the transcriptional coactivator OBF (also called OCA-B or Bob-1) show a severe reduction of immature B cells in the spleen [45]. This reduction is even more dramatic in OBF/Oct-2 [46] and OBF/btk (Bruton’s tyrosine kinase) [47•] double-mutant mice. The molecular mechanisms underlying these defects are still unclear. However, a bcl-2 transgene expressed in the B lineage rescues, to a large extent, this defect in OBF/btk double-mutant mice, suggesting that lifespans of immature B cells may play a role in this transition (AG Rolink, unpublished data). On the basis of the expression of CD21 and CD23, immature B cells in the spleen can be subdivided into two populations, namely CD21–CD23– and CD21+CD23+ [48]. Crosslinking the BCR on these two immature B cell populations causes induction of apoptosis ([39]; AG Rolink, unpublished data), indicating that they are still sensitive for undergoing negative selection. Nevertheless, in wild-type mice most immature splenic B cells enter the pool of long-lived mature B cells ([39]; Figure 1). The finding that apoptosis induced by BCR crosslinking can be blocked by antiCD40, IL-4 and BAFF ([39]; AG Rolink, unpublished data) may indicate that these molecules are involved in the transition from immature to mature B cells. btk-defective mice have normal numbers of immature splenic B cells; however, their number of mature B cells is reduced about five-fold. As the lifespan of mature B cells in btk–/– mice is indistinguishable from that found for wild-type B cells, the defect most probably reflects a reduced efficiency of btk–/– immature splenic B cells to enter the mature compartment [44]. The finding that a bcl-2 transgene expressed in B cells can rescue this defect suggests that the Btk molecule might be involved in induction of survival.
205
MHC class II has also been implicated in the transition from immature to mature B cells in the spleen. Thus, both mice deficient in the invariant chain (Ii–/–; [49]) and MHC classIIα–/– C57Bl/6 mice [44] have a severely reduced mature B cell compartment. In fact, the mature B cell pool is completely absent in btk/Aα double-mutant mice. The defect in Aα–/– mice appears to be intrinsic to the B cells and to be due to a 4–5-fold reduced lifespan of mature B cells [44]. The molecular mechanisms underlying these defects are still unclear. In particular, the finding that CTIIA–/– [50] and Aβ–/– [51,52] mice, as well as mice lacking all conventional MHC class II genes [53], do not show such a defect makes the role of MHC class II in this transition very puzzling.
Are mature peripheral B cells positively selected? It is well established that B cells, like T cells, undergo negative selection. But whether B cells undergo positive selection, as do T cells, remains to be elucidated. The finding that VH gene repertoire expressed in pre-B cells is different from that found in mature B cells could indicate positive selection [54]. However, negative selection could also account for this difference. A few years ago, Lam et al. [55] showed that ablation of sIg on mature B cells results in rapid cell death. For T cells, the interaction between TCR and MHC is required for long-term survival [56]. In analogy, B cells might require the interaction between their BCR and a specific ligand to survive and, thus, might be positively selected. Recently, Hayakawa et al. [57••] showed that autoreactive B cells can be positively selected into the B-1 cell compartment. The findings that numerous mutants with directly or indirectly perturbed BCR signaling capacity all have some more severe defects in the B-1 than in the B-2 compartment might also argue in favor of positive selection for B-1 cells. The experiments by Martin and Kearney [12••] strongly suggest that also marginal zone B cells are positively selected. Moreover, Kouskoff and colleagues [58] recently showed that application of T-cell-independent antigen results at least in a partial rescue of B cells from peripheral immune tolerance. Similar findings were previously reported by Andersson and colleagues [59]. Thus, the evidence for positive selection of peripheral mature B cells is increasing.
Conclusions It is by now well established that B cells, during their development, have to pass through several critical checkpoints at which transcription factors, as well as signaling molecules, downstream of the different forms of the BCR play critical roles. However, the precise molecular mechanisms operating at these various checkpoints still need to be elucidated.
Acknowledgements We thank Raul Torres for critical reading of this manuscript. The Basel Institute for Immunology was founded and is supported by F Hoffmann-La Roche Ltd., Basel, Switzerland.
206
Lymphocyte development
References and recommended reading
17.
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Melchers F, Rolink AG: B-lymphocyte development and biology. In Fundamental Immunology. Edited by Paul WE. Philadelphia: Lippincott-Raven Publishers; 1998:183-224.
2.
Bain G, Maandag EC, Izon DJ, Amsen D, Kruisbeek AM, Weintraub BC, Krop I, Schlissel MS, Feeney AJ, van Roon M et al.: E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements. Cell 1994, 79:885-892.
3.
Zhuang Y, Soriano P, Weintraub H: The helix-loop-helix gene E2A is required for B cell formation. Cell 1994, 79:875-884.
4.
Lin H, Grosschedl R: Failure of B-cell differentiation in mice lacking the transcription factor EBF. Nature 1995, 376:263-267.
5.
Nutt SL, Urbanek P, Rolink A, Busslinger M: Essential functions of Pax5 (BSAP) in pro-B cell development: difference between fetal and adult B lymphopoiesis and reduced V-to-DJ recombination at the IgH locus. Genes Dev 1997, 11:476-491.
6.
Nutt SL, Morrison AM, Dorfler P, Rolink A, Busslinger M: Identification of BSAP (Pax-5) target genes in early B-cell development by loss- and gain-of-function experiments. EMBO J 1998, 17:2319-2333.
7. ••
Nutt SL, Heavey B, Rolink AG, Busslinger M: Commitment to the B lymphoid lineage depends on the transcription factor Pax5. Nature 1999, 401:556-562. Pax5 is identified as the B lineage commitment factor, which represses lineageinappropriate options in the progenitor cell. First, pre-B cells isolated from bone marrow of Pax5-deficient mice are shown to have multipotent hematopoietic lineage capacity (NK cells, macrophages, osteoclasts, dendritic cells and granulocytes) under appropriate in vitro stimulation. Second, differentiation of these pre-B cells along the B-lineage pathway is observed after retroviral restoration of Pax5 expression. At commitment, Pax5 restricts alternative lineage choices by repressing the transcription of non-B-lineage genes including the myeloid M-CSF-R gene. 8. ••
Rolink AG, Nutt SL, Melchers F, Busslinger M: Long-term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors. Nature 1999, 401:603-606. Pax5-deficient pre-B cells are shown to reconstitute T cell development fully in the thymus and secondary lymphoid organs of injected RAG2-deficient mice. The injected Pax5-deficient pre-B cells home to the bone marrow where they maintain their pre-B cell phenotype, undergo self-renewal and continuously seed the thymus. When transferred into secondary RAG2-deficient recipients, these pre-B cells again give rise to long-term reconstitution of the thymus. 9.
ten Boekel E, Melchers F, Rolink AG: Changes in the V(H) gene repertoire of developing precursor B lymphocytes in mouse bone marrow mediated by the pre-B cell receptor. Immunity 1997, 7:357-368.
10. ten Boekel E, Melchers F, Rolink AG: Precursor B cells showing H chain allelic inclusion display allelic exclusion at the level of pre-B cell receptor surface expression. Immunity 1998, 8:199-207. 11. Kline GH, Hartwell L, Beck-Engeser GB, Keyna U, Zaharevitz S, Klinman NR, Jack HM: Pre-B cell receptor-mediated selection of pre-B cells synthesizing functional mu heavy chains. J Immunol 1998, 161:1608-1618. 12. Martin F, Kearney JF: Positive selection from newly formed to •• marginal zone B cells depends on the rate of clonal production, CD19, and btk. Immunity 2000, 12:39-49. B cells expressing a VH81x µH chain derived from a neonatal mouse as a transgene are found to be positively selected in the marginal zone repertoire.
Turner M, Mee PJ, Costello PS, Williams O, Price AA, Duddy LP, Furlong MT, Geahlen RL, Tybulewicz VL: Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk. Nature 1995, 378:298-302.
18. Pelanda R, Schaal S, Torres RM, Rajewsky K: A prematurely κ transgene, but not Vκ κJκ κ gene segment targeted expressed Igκ κ locus, can rescue B cell development in λ5-deficient into the Igκ mice. Immunity 1996, 5:229-239. 19. Rolink A, Haasner D, Melchers F, Andersson J: The surrogate light chain in mouse B-cell development. Int Rev Immunol 1996, 13:341-356. 20. Ceredig R, Rolink AG, Melchers F, Andersson J: The B cell receptor, but not the pre-B cell receptor, mediates arrest of B cell differentiation. Eur J Immunol 2000, 30:759-767. 21. Rolink AG, Winkler T, Melchers F, Andersson J: Precursor B cell •• receptor-dependent B cell proliferation and differentiation does not require the bone marrow or fetal liver environment. J Exp Med 2000, 191:23-32. This study shows that pre-BCR-dependent proliferation of pre-B cells does not require the bone marrow or fetal liver environment. 22. Jumaa H, Wollscheid B, Mitterer M, Wienands J, Reth M, Nielsen PJ: • Abnormal development and function of B lymphocytes in mice deficient for the signaling adaptor protein SLP-65. Immunity 1999, 11:547-554. These three papers [22•−24•] describe the BLNK-deficient mouse. The reported findings indicate a potential role for BLNK in the transition from preB I to pre-B II cells. 23. Pappu R, Cheng AM, Li B, Gong Q, Chiu C, Griffin N, White M, • Sleckman BP, Chan AC: Requirement for B cell linker protein (BLNK) in B cell development. Science 1999, 286:1949-1954. See annotation to [22•]. 24. Hayashi K, Nittono R, Okamoto N, Tsuji S, Hara Y, Goitsuka R, • Kitamura D: The B cell-restricted adaptor BASH is required for normal development and antigen receptor-mediated activation of B cells. Proc Natl Acad Sci USA 2000, 97:2755-2760. See annotation to [22•]. 25. Shaw AC, Swat W, Ferrini R, Davidson L, Alt FW: Activated Ras • signals developmental progression of recombinase-activating gene (RAG)-deficient pro-B lymphocytes. J Exp Med 1999, 189:123-129. Using an active Ras transgene on a RAG-deficient background, the authors show that not only the pre-B cell stage but also more mature B cell stages are reconstituted by the active Ras. 26. Iritani BM, Alberola-Ila J, Forbush KA, Perimutter RM: Distinct signals • mediate maturation and allelic exclusion in lymphocyte progenitors. Immunity 1999, 10:713-722. Using an active Raf transgene on a RAG-deficient background, the authors show, in contrast to [25•], that active Raf can substitute for only a pre-BCR signal. Although the pre-BCR-dependent differentiation is reconstituted, the VH selection process mediated by the formation of the pre-BCR is impaired in these mice, indicating the requirement for distinct signals in maturation and allelic exclusion. 27.
Novobrantseva TI, Martin VM, Pelanda R, Muller W, Rajewsky K, Ehlich A: Rearrangement and expression of immunoglobulin light chain genes can precede heavy chain expression during normal B cell development in mice. J Exp Med 1999, 189:75-88.
28. Benedict CL, Gilfillan S, Thai TH, Kearney JF: Terminal deoxynucleotide transferase and repertoire development. Immunol Rev 2000, 175:150-157. 29. Grawunder U, Leu TM, Schatz DG, Werner A, Rolink AG, Melchers F, Winkler TH: Down-regulation of RAG1 and RAG2 gene expression in preB cells after functional immunoglobulin heavy chain rearrangement. Immunity 1995, 3:601-608.
13. Kitamura D, Kudo A, Schaal S, Muller W, Melchers F, Rajewsky K: A critical role of λ5 protein in B cell development. Cell 1992, 69:823-831.
30. ten Boekel E, Melchers F, Rolink A: The status of Ig loci rearrangements in single cells from different stages of B cell development. Int Immunol 1995, 7:1013-1019.
14. Kitamura D, Roes J, Kuhn R, Rajewsky K: A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin µ chain gene. Nature 1991, 350:423-426.
31. Yamagami T, ten Boekel E, Schaniel C, Andersson J, Rolink A, κ–/– small pre-BII cells • Melchers F: Four of five RAG-expressing JCκ have no L chain gene rearrangements: detection by highefficiency single cell PCR. Immunity 1999, 11:309-316. These two papers [31•,32•] describe the immunoglobulin light chain (IgL) rearrangements in small pre-B II, immature and mature B cells at the singlecell level. The accumulation of cells having undergone several κL chain rearrangements in the small pre-B II compartment is described. Moreover, it is shown that the high κ : λ ratio observed in mice is already established at the small pre-B II cell stage.
15. Gong S, Nussenzweig MC: Regulation of an early developmental β. Science 1996, checkpoint in the B cell pathway by Igβ 272:411-414. 16. Cheng AM, Rowley B, Pao W, Hayday A, Bolen JB, Pawson T: Syk tyrosine kinase required for mouse viability and B-cell development. Nature 1995, 378:303-306.
Selection events in B cell development Rolink et al.
32. Yamagami T, ten Boekel E, Andersson J, Rolink A, Melchers F: • Frequencies of multiple IgL chain gene rearrangements in single normal or κL chain-deficient B lineage cells. Immunity 1999, 11:317-327. See annotation to [31•]. 33. Engel H, Rolink A, Weiss S: B cells are programmed to activate κ • and λ for rearrangement at consecutive developmental stages. Eur J Immunol 1999, 29:2167-2176. In this study, the accessibility of the κ and λ loci during mouse B cell development is analyzed. The findings clearly indicate that the κ locus opens at an earlier developmental stage than the λ locus and thus offer an explanation for the high κ : λ ratio found in mouse B cells. 34. Tiegs SL, Russell DM, Nemazee D: Receptor editing in self-reactive bone marrow B cells. J Exp Med 1993, 177:1009-1020. 35. Gay D, Saunders T, Camper S, Weigert M: Receptor editing: an approach by autoreactive B cells to escape tolerance. J Exp Med 1993, 177:999-1008. 36. Radic MZ, Erikson J, Litwin S, Weigert M: B lymphocytes may escape tolerance by revising their antigen receptors. J Exp Med 1993, 177:1165-1173. 37.
Dorner T, Foster SJ, Farner NL, Lipsky PE: Immunoglobulin κ chain receptor editing in systemic lupus erythematosus. J Clin Invest 1998, 102:688-694.
38. Osmond DG: Proliferation kinetics and the lifespan of B cells in central and peripheral lymphoid organs. Curr Opin Immunol 1991, 3:179-185. 39. Rolink AG, Andersson J, Melchers F: Characterization of immature B cells by a novel monoclonal antibody, by turnover and by mitogen reactivity. Eur J Immunol 1998, 28:3738-3748. 40. Turner M, Gulbranson-Judge A, Quinn ME, Walters AE, MacLennan IC, Tybulewicz VL: Syk tyrosine kinase is required for the positive selection of immature B cells into the recirculating B cell pool. J Exp Med 1997, 186:2013-2021. 41. Torres RM, Flaswinkel H, Reth M, Rajewsky K: Aberrant B cell development and immune response in mice with a compromised BCR complex. Science 1996, 272:1804-1808. 42. Torres RM, Hafen K: A negative regulatory role for Ig-alpha during • B cell development. Immunity 1999, 11:527-536. In contrast to the prediction that the cytoplasmic region of Igα plays a positive role in BCR signaling, the authors show for the first time that the developmental arrest in Igα∆c/∆c mice may be due to hyperactivation of this mutant BCR complex. α cytoplasmic 43. Kraus M, Saijo K, Torres RM, Rajewsky K: Igα • truncation renders immature B cells more sensitive to antigen contact. Immunity 1999, 11:537-545. Using the hen egg lysozyme (HEL) system, effects of the cytoplasmic domain of Igα on negative selection are reported. In contrast to anergy in wild-type mice harboring double transgenes (BCRs against HEL and soluble HEL), Igα∆c/∆c mice harboring the double transgenes show rearrangements of endogenous κL chain, presumably reflecting receptor editing and/or apoptosis. Furthermore, because BCR-mediated calcium mobilization and tyrosine phosphorylation are augmented in the mutant mice, the authors suggest that this increased signaling strength leads to selection of editing/apoptosis rather than anergy. 44. Rolink AG, Brocker T, Bluethmann H, Kosco-Vilbois MH, Andersson J, Melchers F: Mutations affecting either generation or survival of cells influence the pool size of mature B cells. Immunity 1999, 10:619-628.
207
45. Schubart DB, Rolink A, Kosco-Vilbois MH, Botteri F, Matthias P: B-cell-specific coactivator OBF-1/OCA-B/Bob1 required for immune response and germinal centre formation. Nature 1996, 383:538-542. 46. Schubart K, Massa S, Schubart D, Corcoran LM, Rolink AG, Matthias P: B cell development and immunoglobulin gene transcription in the absence of Oct-2 and OBF-1. Nat Immunol 2001, 2:69-74. 47. •
Schubart DB, Rolink A, Schubart K, Matthias P: Lack of peripheral B cells and severe agammaglobulinemia in mice simultaneously lacking Bruton’s tyrosine kinase and the B cell-specific transcriptional coactivator OBF-1. J Immunol 2000, 164:18-22. The authors analyze OBF/btk double-mutant mice. They find that B cell development in the bone marrow is unaffected but that mature peripheral B cells are absent, which points at the very important role for these two molecules in the transition of immature B cells from the bone marrow to the spleen. 48. Loder F, Mutschler B, Ray RJ, Paige CJ, Sideras P, Torres R, Lamers MC, Carsetti R: B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals. J Exp Med 1999, 190:75-89. 49. Shachar I, Flavell RA: Requirement for invariant chain in B cell maturation and function. Science 1996, 274:106-108. 50. Chang CH, Guerder S, Hong SC, van Ewijk W, Flavell RA: Mice lacking the MHC class II transactivator (CIITA) show tissuespecific impairment of MHC class II expression. Immunity 1996, 4:167-178. 51. Gosgrove D, Gray D, Dierich A, Kaufman J, Lemeur M, Benoist C, Mathis D: Mice lacking MHC class II molecules. Cell 1991, 66:1051-1066. 52. Markowitz JS, Rogers PR, Grusby MJ, Parker DC, Glimcher LH: B lymphocyte development and activation independent of MHC class II expression. J Immunol 1993, 150:1223-1233. 53. Madsen L, Labrecque N, Engberg J, Dierich A, Svejgaard A, Benoist C, Mathis D, Fugger L: Mice lacking all conventional MHC class II genes. Proc Natl Acad Sci USA 1999, 96:10338-10343. 54. Gu H, Tarlinton D, Muller W, Rajewsky K, Forster I: Most peripheral B cells in mice are ligand selected. J Exp Med 1991, 173:1357-1371. 55. Lam KP, Kuhn R, Rajewsky K: In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death. Cell 1997, 90:1073-1083. 56. Takeda S, Rodewald HR, Arakawa H, Bluethmann H, Shimizu T: MHC class II molecules are not required for survival of newly generated CD4+ T cells, but affect their long-term life span. Immunity 1996, 5:217-228. 57. ••
Hayakawa K, Asano M, Shinton SA, Gui M, Allman D, Stewart CL, Silver J, Hardy RR: Positive selection of natural autoreactive B cells. Science 1999, 285:113-116. In this study, the authors elegantly show that natural autoreactive B ells can be positively selected in the B-1 compartment in the presence of the autoantigen. 58. Kouskoff V, Lacaud G, Nemazee D: T cell-independent rescue of B lymphocytes from peripheral immune tolerance. Science 2000, 287:2501-2503. 59. Andersson J, Melchers F, Rolink A: Stimulation by T cell independent antigens can relieve the arrest of differentiation of immature auto-reactive B cells in the bone marrow. Scand J Immunol 1995, 42:21-33.
Selection events operating at various stages in B cell development Antonius G Rolink*, Christoph Schaniel, Jan Andersson and Fritz Melchers B cells have to progress through various checkpoints during their process of development. The three transcription factors E2A, EBF (early B cell factor) and Pax5 play essential roles in B cell commitment checkpoints. The various forms of the BCR and their downstream signaling molecules, which are expressed at different stages of B cell development, act as critical checkpoint guards allowing (positive selection) or preventing (negative selection) developmental progression. The recent advances on the molecular mechanisms operating at these various checkpoints are here summarized and discussed. Addresses Basel Institute for Immunology, Grenzacherstrasse 487, CH-4005 Basel, Switzerland *e-mail: [email protected] Current Opinion in Immunology 2001, 13:202–207 0952-7915/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations btk Bruton’s tyrosine kinase CDR3 complementarity-determining region 3 EBF early B cell factor FLOC fetal liver organ culture IgH immunoglobulin heavy chain IgL immunoglobulin light chain sIgM surface IgM SL surrogate light chain
Introduction The development of B cells in bone marrow has been dissected into several discrete stages by the discovery and use of a series of surface-bound and cytoplasmically expressed markers (for a review, see [1]). These developmental stages have subsequently been ordered according to the rearrangement status of the immunoglobulin heavy (IgH) and light (IgL) chain gene loci at the single-cell level (for a review, see [1]). Over the past decade, both the introduction of numerous mutations into the germline and the identification of natural mutants have shown that B cells must pass several checkpoints in order to progress through these distinct developmental stages. Here we review recent progress in research in three of these checkpoints. In addition, we will discuss the evidence for positive selection of B cells.
encoding components of the rearrangement machinery nor the pre-BCR is a sign of commitment. These conclusions are based on recent findings made with mice deficient in a third transcription factor, Pax5. Pax5–/– mice have an absolute block in B cell development at the pre-B type I cell stage [5,6]. The pre-B I cells found in these mice have DJ-rearranged IgH loci and express genes encoding E2A, EBF, λ5, VpreB, Igα and Igβ [5,6]. Moreover, like wild-type pre-B I cells, Pax5–/– pre-B I cells can be propagated in vitro in the presence of stromal cells and IL-7 [5,6]. Unlike wild-type cells, however, Pax5–/– preB I cells possess stem-cell-like activity; that is, under appropriate in vitro and in vivo conditions they can give rise to cells of multiple hematopoietic lineages and they possess self-renewal capacity [7••,8••]. Thus, Pax5 appears to be absolutely essential in the first checkpoint of B cell development, namely commitment. But whether Pax5 expression results in irreversible or reversible commitment — that is, whether normal pre-B I cells that loose Pax5 expression regain multilineage potential — remains unknown.
Selection at the pre-BCR checkpoint VH to DHJH rearrangement is initiated in the transition from pre-B I to large pre-B II cells (for a review, see [1]). The µH chain protein that is expressed when the VHDHJH rearrangement is in-frame can pair with the surrogate light chain (SL) components λ5 and VpreB, and, in association with the signal-transducing Igα and Igβ subunits, can form the pre-BCR complex at the cell surface. About 10% of pre-B I cells in mouse bone marrow express µH chain in the cytoplasm. Not all of these µH chains can form a preBCR, however, as about half of them seem unable to do so [9]. Although not yet proven, this is the probable consequence of special CDR3 (complementarity-determining region 3), structures that do not allow pairing with the SL.
B cell commitment
Non-pairing µH chains, which contain VH gene segments of various VH gene families, have been found. Moreover, two µH chains are expressed in 2–4% of pre-B II and B cells. Of these two µH chains, only one can form a preBCR [10]. Thus, at the transition from pre-B I to pre-B II, those cells expressing a µH chain capable of forming a preBCR are positively selected, because they enter into cellular division and expansion (see below).
To date, three transcription factors have been identified as essential for the commitment of hematopoietic precursor cells to the B lymphoid lineage. E2A [2,3] and early B cell factor (EBF; [4]), both basic helix–loop–helix proteins, are required for initiation of B lymphopoiesis [2,3]; in the absence of either of these proteins, not even the earliest B cell progenitors develop. However, E2A and EBF, although required, do not determine B cell commitment. Moreover, neither the expression of genes
About 20% of pre-B I cells that express a µH chain use the most 3′ VH gene element, called VH81x. In marked contrast, peripheral B cells very rarely use VH81x. This negative selection of B cells expressing VH81x µH chains appears to be caused by the inability of these µH chains to form a pre-BCR. In fact, all tested VH81x µH chains derived from bone marrow pre-B I cells are unable to pair with the SL [9]. Moreover, the finding that a mouse strain
Selection events in B cell development Rolink et al.
carrying a VH81x µH transgene, that cannot form a preBCR, does not express this transgene in the periphery supports this hypothesis [11]. However, about 30% of VH81x µH chains expressed in fetal liver pre-B I cells do form a pre-BCR (XC Kong, F Melchers, AG Rolink, unpublished data). Moreover, such a fetal-liver-derived VH81x µH chain transgene is also expressed by peripheral B cells [12••]. Thus, the CDR3 region, which is less diverse in fetal-liver-derived µH chains owing to the absence of the enzyme TdT, appears to determine whether a VH81x µH chain can form a pre-BCR and thereby be selected into the peripheral B cell repertoire. Because the frequency of in-frame VH81x rearrangements in bone marrow pre-B I cells is high, it is somewhat surprising that, in the 2–4% of B cells in which two in-frame VHDHJH rearrangements are found, an in-frame VH81x–DHJH rearrangement has not yet been detected [10]. This counter-selection of VH81x µH chains might be explained by toxicity of these chains. Alternatively, and more interestingly, a secondary VH rearrangement (replacement) — as observed in complete immunoglobulin knockin mice — might also account for this counter-selection. Because no counter-selection for out-of-frame VH81x–DHJH rearrangements is observed, the protein coded by the inframe rearrangement must have a role in selection. For future studies, the production of an in-frame non-pre-BCRforming VH81x–DHJH knockin mouse might shed light on the mechanism controlling this negative selection. The deposition of the pre-BCR, presumably in the surface membrane, induces the pre-B II cells to enter cell cycle and divide. Pre-B cells of λ5–/– [13], µMT–/– [14] or Igβ–/– [15] pre-BCR-defective mice or of syk–/– pre-BCR-signaling-defective mice (syk is a protein tyrosine kinase) [16,17] do not enter this expansion phase. As yet, whether the preBCR needs a ligand to signal this proliferative expansion remains unambiguous; however, several observations argue in favour of ligand-independent pre-BCR signaling. First, a number of light chains expressed as transgenes prematurely in λ5–/– mice repair the proliferative defect [18,19]. Hence, a variety of endogenously produced µH chains form IgMs with these transgenic light chains to function in a pre-BCR-like fashion in large pre-B II cells. This makes it unlikely that a limited number of ligands can function via recognition of the CDRs of the VH and the CDR-like structures of the SL; and it does not rule out the possibility of ligand recognition by VpreB, which could still be associated with the pre-BCR-like IgM on the surface of the λ5–/–, light-chain-transgenic pre-B cells. Second, monoclonal antibodies specific for the preBCR — that is, specific for VpreB, λ5 and µH chains — do not perturb B cell development either when injected in vivo or when added to fetal liver organ culture (FLOC) [20]. By contrast, either in vivo or in FLOC, monoclonal antibodies specific for µH chains are perfectly capable of
203
inhibiting the development at a later stage — that is, at the stage of the development of immature, surface-IgM+ (sIgM+) B cells [20]. Hence, it appears that engagement of the pre-BCR by specific antibodies does not influence B cell development whereas engagement of the BCR on immature cells does. Third, ex vivo isolated c-Kit+CD19+ pre-B I cells proliferate and differentiate in vitro in a pre-BCR-dependent manner [21••]. Because this pre-BCR-dependent proliferation and differentiation does not require stromal cells or any exogenous cytokines, it is most likely to be ligand independent. Moreover, as occurs in vivo and in FLOC, addition of µH-chain-specific monoclonal antibody inhibits the differentiation of the cells into immature, sIgM+ B cells only (AG Rolink, unpublished data). Interestingly, single ex vivo isolated pre-B cells proliferate to a different clone size in vitro [21••]. This may be taken as an indication that different µH chains form pre-BCRs of different quality and thus allow a different number of divisions. The molecular mechanism by which the pre-BCR signals a pre-B I cell to divide still remains to be investigated in detail. However, the partial blocks in B cell development at the transition from pre-B to pre-B I in syk- [16,17] and BLNK-deficient mice (BLNK is an adapter protein) [22•–24•] indicate that these molecules have a role in this signaling event. Moreover, reconstitution of RAG-deficient mice with constitutive active forms of Ras [25•] or Raf [26•] results in restoration of pre-B cell development, which suggests that these proteins are also involved in signaling. Although the pre-BCR has a critical role in B cell development, λ5-deficient mice still make B cells — albeit in strongly reduced numbers. In a low percentage of pre-B cells, IgL chains are rearranged before IgH chain rearrangements are completed [27]. Moreover, B cell development in λ5–/– mice can be rescued by premature expression of IgL chains. On the basis of these two findings, it has been argued that B cells found in λ5–/– mice are derived from preB cells that have undergone IgL chain rearrangements before completing the IgH chains. A prediction based on this hypothesis is that IgL chain rearrangements in λ5–/– mice have taken place at a stage of development in which the enzyme TdT is expressed and thus would result in IgL chains containing nontemplate-encoded nucleotides (N-regions) in their VL–JL junctions [28]. However, sequence analysis of IgH and IgL of peripheral B cells derived from λ5–/– mice show no evidence for N-nucleotides in the IgL chains whereas the IgH chains have N-sequences like those of the IgH chains of wild-type B cells [19]. Hence, the order of IgH and IgL chain gene rearrangements in developing λ5–/– B cells appears to be identical to that observed in most wild-type pre-B cells — that is, the IgH chain rearranges before the IgL chain. Another issue is whether the pre-BCR is involved in mediating allelic exclusion. Expression of the pre-BCR results
204
Lymphocyte development
Figure 1
Immature B cells Selection
sIgM
Immature B cells Selection
Mature B cells Selection
sIgM
Production 2 x 107 / day
CD45–/– OBF–/– Igα∆c/∆c
Selection steps and proteins involved in late B cell development. The various selection steps (boxed) and important proteins (shown at the bottom of the figure) that are involved in B cell development in the bone marrow and spleen are indicated.
Spleen
Bone marrow
2 x 106 / day
Btk–/– Ii–/– Aα–/– C57Bl/6
in a very rapid downregulation of the genes and proteins involved in the rearrangement process, which could argue in favor of such a role [29]. However, allelic exclusion is not perturbed in λ5–/– mice, which indicates, at least, that a complete pre-BCR is not required [10].
Selection of sIgM+ immature B cells in the bone marrow Large pre-B II cells, which have lost expression of the preBCR, re-express the RAG-1 and RAG-2 genes at the mRNA but not the protein level [29]. Moreover, sterile transcripts from κL chain gene loci become detectable, although the IgL chain loci are still in germline configuration. When the large, cycling cells become resting and small, IgL chain gene rearrangement is induced [30]. In the mouse, peripheral B cells express κL and λL chains in a 10:1 ratio [30,31•,32•]. Single-cell light chain rearrangement analysis has revealed that this ratio is already established at the small pre-B II stage. Experiments by Engel et al. [33•] suggest that the κ locus opens before the λ locus and indicate that the κ locus has earlier access to the rearrangement machinery. This might therefore be the explanation for the high ratio of κ : λL chains found in mouse B cells. In a wild-type mouse, about half of the sIg+ B cells have rearranged only one allele at the κL chain locus. Most of these cells show only one single rearrangement, preferentially to Jκ1, the most V-proximal of the functional J segments. In marked contrast, small pre-B II cells show a dramatically increased frequency of multiple κL chain rearrangements [31•,32•]. Moreover, about 20% of small pre-B II cells express a κL chain protein in the cytoplasm but not on the cell surface although half of these cells have productively rearranged VκJκ segments. As over 95% of all pre-B II cells express µH chains in their cytoplasm, there must be reasons why these µH–κL chain combinations are not expressed on the surface.
sIgM sIgD
2 x 106 / day
Current Opinion in Immunology
We can envisage two possible reasons for the lack of surface expression of the µH–κL chain pairs. First, certain µH–κL chain combinations may not be able to form an IgM molecule. Although the peripheral, sIgM+ B cell pool should contain only pairing combinations, the small preB II cell pool may be enriched for those that do not fit together. This possibility is being tested currently by experiments in which genes encoding the µH and κL chains expressed in single small pre-B II and mature B cells are cloned and their paring capacities subsequently examined. Second, a given µH–κL chain combination may have, in fact, paired properly and been deposited on the surface. However, if this IgM were autoreactive, it would have found its autoantigen in the bone marrow and been downregulated from the surface. Nemazee and colleagues [34], and Weigert and co-workers [35,36] have shown that recognition of autoantigen by immature B cells induces secondary rearrangement at the light chain loci and thus can change the cells’ receptor specificity from self to non-self. In humans, a similar type of BCR editing has been observed [37]. The contribution of B cells that have undergone receptor editing to the total peripheral B cell repertoire is not yet clear. Moreover, it should be noted that in Cκ-deficient mice — that is, in mice that cannot make a κL chain protein — the κL chain gene rearrangement patterns in small pre-B II cells are indistinguishable from those found in their wild-type counterparts. Thus, several κL chain gene rearrangements in these pre-B cells are at least in part not immunoglobulin-receptor-induced [31•,32•].
Selection at the transition from immature to mature B cells Osmond and colleagues (reviewed in [38]) have determined that mice produce roughly 2 × 107 immature B cells per day in the bone marrow. These immature B cells then
Selection events in B cell development Rolink et al.
migrate through the terminal branches of central arterioles into the spleen. There, these immature B cells, which have half-lifetimes of about 4 days, differentiate into mature B cells with lifespans of around 15 weeks [39]. The first selection event operating at these late stages of B cell development is the transition from the bone marrow to the spleen (Figure 1). At this stage, about 90% of the immature B cells produced in the bone marrow are lost. A large part of this loss can probably be explained by the deletion of autoreactive B cells in the bone marrow. Moreover, signaling through the BCR in general appears to play an important role at this checkpoint. Thus, both syk-deficient mice and mice expressing an Igα lacking the cytoplasmic tail (Igα∆c/∆c) show a much more marked loss of B cells at this transition [40,41]. Interestingly, immature B cells in Igα∆c/∆c mice have increased levels of tyrosine phosphorylation and expression of activation antigen [42•], suggesting that the intensity of signaling may be very critical for B cells passing this checkpoint. Similar findings were made in Igα∆c/∆c mice carrying a soluble hen egg lysozyme (HEL) and an anti-HEL-immunoglobulin transgene [43•]. The finding that in CD45–/– mice more immature B cells enter the spleen, therefore, might indicate that the phosphatase CD45 is critically involved in this signaling intensity [44]. Mice deficient for the transcriptional coactivator OBF (also called OCA-B or Bob-1) show a severe reduction of immature B cells in the spleen [45]. This reduction is even more dramatic in OBF/Oct-2 [46] and OBF/btk (Bruton’s tyrosine kinase) [47•] double-mutant mice. The molecular mechanisms underlying these defects are still unclear. However, a bcl-2 transgene expressed in the B lineage rescues, to a large extent, this defect in OBF/btk double-mutant mice, suggesting that lifespans of immature B cells may play a role in this transition (AG Rolink, unpublished data). On the basis of the expression of CD21 and CD23, immature B cells in the spleen can be subdivided into two populations, namely CD21–CD23– and CD21+CD23+ [48]. Crosslinking the BCR on these two immature B cell populations causes induction of apoptosis ([39]; AG Rolink, unpublished data), indicating that they are still sensitive for undergoing negative selection. Nevertheless, in wild-type mice most immature splenic B cells enter the pool of long-lived mature B cells ([39]; Figure 1). The finding that apoptosis induced by BCR crosslinking can be blocked by antiCD40, IL-4 and BAFF ([39]; AG Rolink, unpublished data) may indicate that these molecules are involved in the transition from immature to mature B cells. btk-defective mice have normal numbers of immature splenic B cells; however, their number of mature B cells is reduced about five-fold. As the lifespan of mature B cells in btk–/– mice is indistinguishable from that found for wild-type B cells, the defect most probably reflects a reduced efficiency of btk–/– immature splenic B cells to enter the mature compartment [44]. The finding that a bcl-2 transgene expressed in B cells can rescue this defect suggests that the Btk molecule might be involved in induction of survival.
205
MHC class II has also been implicated in the transition from immature to mature B cells in the spleen. Thus, both mice deficient in the invariant chain (Ii–/–; [49]) and MHC classIIα–/– C57Bl/6 mice [44] have a severely reduced mature B cell compartment. In fact, the mature B cell pool is completely absent in btk/Aα double-mutant mice. The defect in Aα–/– mice appears to be intrinsic to the B cells and to be due to a 4–5-fold reduced lifespan of mature B cells [44]. The molecular mechanisms underlying these defects are still unclear. In particular, the finding that CTIIA–/– [50] and Aβ–/– [51,52] mice, as well as mice lacking all conventional MHC class II genes [53], do not show such a defect makes the role of MHC class II in this transition very puzzling.
Are mature peripheral B cells positively selected? It is well established that B cells, like T cells, undergo negative selection. But whether B cells undergo positive selection, as do T cells, remains to be elucidated. The finding that VH gene repertoire expressed in pre-B cells is different from that found in mature B cells could indicate positive selection [54]. However, negative selection could also account for this difference. A few years ago, Lam et al. [55] showed that ablation of sIg on mature B cells results in rapid cell death. For T cells, the interaction between TCR and MHC is required for long-term survival [56]. In analogy, B cells might require the interaction between their BCR and a specific ligand to survive and, thus, might be positively selected. Recently, Hayakawa et al. [57••] showed that autoreactive B cells can be positively selected into the B-1 cell compartment. The findings that numerous mutants with directly or indirectly perturbed BCR signaling capacity all have some more severe defects in the B-1 than in the B-2 compartment might also argue in favor of positive selection for B-1 cells. The experiments by Martin and Kearney [12••] strongly suggest that also marginal zone B cells are positively selected. Moreover, Kouskoff and colleagues [58] recently showed that application of T-cell-independent antigen results at least in a partial rescue of B cells from peripheral immune tolerance. Similar findings were previously reported by Andersson and colleagues [59]. Thus, the evidence for positive selection of peripheral mature B cells is increasing.
Conclusions It is by now well established that B cells, during their development, have to pass through several critical checkpoints at which transcription factors, as well as signaling molecules, downstream of the different forms of the BCR play critical roles. However, the precise molecular mechanisms operating at these various checkpoints still need to be elucidated.
Acknowledgements We thank Raul Torres for critical reading of this manuscript. The Basel Institute for Immunology was founded and is supported by F Hoffmann-La Roche Ltd., Basel, Switzerland.
206
Lymphocyte development
References and recommended reading
17.
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Melchers F, Rolink AG: B-lymphocyte development and biology. In Fundamental Immunology. Edited by Paul WE. Philadelphia: Lippincott-Raven Publishers; 1998:183-224.
2.
Bain G, Maandag EC, Izon DJ, Amsen D, Kruisbeek AM, Weintraub BC, Krop I, Schlissel MS, Feeney AJ, van Roon M et al.: E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements. Cell 1994, 79:885-892.
3.
Zhuang Y, Soriano P, Weintraub H: The helix-loop-helix gene E2A is required for B cell formation. Cell 1994, 79:875-884.
4.
Lin H, Grosschedl R: Failure of B-cell differentiation in mice lacking the transcription factor EBF. Nature 1995, 376:263-267.
5.
Nutt SL, Urbanek P, Rolink A, Busslinger M: Essential functions of Pax5 (BSAP) in pro-B cell development: difference between fetal and adult B lymphopoiesis and reduced V-to-DJ recombination at the IgH locus. Genes Dev 1997, 11:476-491.
6.
Nutt SL, Morrison AM, Dorfler P, Rolink A, Busslinger M: Identification of BSAP (Pax-5) target genes in early B-cell development by loss- and gain-of-function experiments. EMBO J 1998, 17:2319-2333.
7. ••
Nutt SL, Heavey B, Rolink AG, Busslinger M: Commitment to the B lymphoid lineage depends on the transcription factor Pax5. Nature 1999, 401:556-562. Pax5 is identified as the B lineage commitment factor, which represses lineageinappropriate options in the progenitor cell. First, pre-B cells isolated from bone marrow of Pax5-deficient mice are shown to have multipotent hematopoietic lineage capacity (NK cells, macrophages, osteoclasts, dendritic cells and granulocytes) under appropriate in vitro stimulation. Second, differentiation of these pre-B cells along the B-lineage pathway is observed after retroviral restoration of Pax5 expression. At commitment, Pax5 restricts alternative lineage choices by repressing the transcription of non-B-lineage genes including the myeloid M-CSF-R gene. 8. ••
Rolink AG, Nutt SL, Melchers F, Busslinger M: Long-term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors. Nature 1999, 401:603-606. Pax5-deficient pre-B cells are shown to reconstitute T cell development fully in the thymus and secondary lymphoid organs of injected RAG2-deficient mice. The injected Pax5-deficient pre-B cells home to the bone marrow where they maintain their pre-B cell phenotype, undergo self-renewal and continuously seed the thymus. When transferred into secondary RAG2-deficient recipients, these pre-B cells again give rise to long-term reconstitution of the thymus. 9.
ten Boekel E, Melchers F, Rolink AG: Changes in the V(H) gene repertoire of developing precursor B lymphocytes in mouse bone marrow mediated by the pre-B cell receptor. Immunity 1997, 7:357-368.
10. ten Boekel E, Melchers F, Rolink AG: Precursor B cells showing H chain allelic inclusion display allelic exclusion at the level of pre-B cell receptor surface expression. Immunity 1998, 8:199-207. 11. Kline GH, Hartwell L, Beck-Engeser GB, Keyna U, Zaharevitz S, Klinman NR, Jack HM: Pre-B cell receptor-mediated selection of pre-B cells synthesizing functional mu heavy chains. J Immunol 1998, 161:1608-1618. 12. Martin F, Kearney JF: Positive selection from newly formed to •• marginal zone B cells depends on the rate of clonal production, CD19, and btk. Immunity 2000, 12:39-49. B cells expressing a VH81x µH chain derived from a neonatal mouse as a transgene are found to be positively selected in the marginal zone repertoire.
Turner M, Mee PJ, Costello PS, Williams O, Price AA, Duddy LP, Furlong MT, Geahlen RL, Tybulewicz VL: Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk. Nature 1995, 378:298-302.
18. Pelanda R, Schaal S, Torres RM, Rajewsky K: A prematurely κ transgene, but not Vκ κJκ κ gene segment targeted expressed Igκ κ locus, can rescue B cell development in λ5-deficient into the Igκ mice. Immunity 1996, 5:229-239. 19. Rolink A, Haasner D, Melchers F, Andersson J: The surrogate light chain in mouse B-cell development. Int Rev Immunol 1996, 13:341-356. 20. Ceredig R, Rolink AG, Melchers F, Andersson J: The B cell receptor, but not the pre-B cell receptor, mediates arrest of B cell differentiation. Eur J Immunol 2000, 30:759-767. 21. Rolink AG, Winkler T, Melchers F, Andersson J: Precursor B cell •• receptor-dependent B cell proliferation and differentiation does not require the bone marrow or fetal liver environment. J Exp Med 2000, 191:23-32. This study shows that pre-BCR-dependent proliferation of pre-B cells does not require the bone marrow or fetal liver environment. 22. Jumaa H, Wollscheid B, Mitterer M, Wienands J, Reth M, Nielsen PJ: • Abnormal development and function of B lymphocytes in mice deficient for the signaling adaptor protein SLP-65. Immunity 1999, 11:547-554. These three papers [22•−24•] describe the BLNK-deficient mouse. The reported findings indicate a potential role for BLNK in the transition from preB I to pre-B II cells. 23. Pappu R, Cheng AM, Li B, Gong Q, Chiu C, Griffin N, White M, • Sleckman BP, Chan AC: Requirement for B cell linker protein (BLNK) in B cell development. Science 1999, 286:1949-1954. See annotation to [22•]. 24. Hayashi K, Nittono R, Okamoto N, Tsuji S, Hara Y, Goitsuka R, • Kitamura D: The B cell-restricted adaptor BASH is required for normal development and antigen receptor-mediated activation of B cells. Proc Natl Acad Sci USA 2000, 97:2755-2760. See annotation to [22•]. 25. Shaw AC, Swat W, Ferrini R, Davidson L, Alt FW: Activated Ras • signals developmental progression of recombinase-activating gene (RAG)-deficient pro-B lymphocytes. J Exp Med 1999, 189:123-129. Using an active Ras transgene on a RAG-deficient background, the authors show that not only the pre-B cell stage but also more mature B cell stages are reconstituted by the active Ras. 26. Iritani BM, Alberola-Ila J, Forbush KA, Perimutter RM: Distinct signals • mediate maturation and allelic exclusion in lymphocyte progenitors. Immunity 1999, 10:713-722. Using an active Raf transgene on a RAG-deficient background, the authors show, in contrast to [25•], that active Raf can substitute for only a pre-BCR signal. Although the pre-BCR-dependent differentiation is reconstituted, the VH selection process mediated by the formation of the pre-BCR is impaired in these mice, indicating the requirement for distinct signals in maturation and allelic exclusion. 27.
Novobrantseva TI, Martin VM, Pelanda R, Muller W, Rajewsky K, Ehlich A: Rearrangement and expression of immunoglobulin light chain genes can precede heavy chain expression during normal B cell development in mice. J Exp Med 1999, 189:75-88.
28. Benedict CL, Gilfillan S, Thai TH, Kearney JF: Terminal deoxynucleotide transferase and repertoire development. Immunol Rev 2000, 175:150-157. 29. Grawunder U, Leu TM, Schatz DG, Werner A, Rolink AG, Melchers F, Winkler TH: Down-regulation of RAG1 and RAG2 gene expression in preB cells after functional immunoglobulin heavy chain rearrangement. Immunity 1995, 3:601-608.
13. Kitamura D, Kudo A, Schaal S, Muller W, Melchers F, Rajewsky K: A critical role of λ5 protein in B cell development. Cell 1992, 69:823-831.
30. ten Boekel E, Melchers F, Rolink A: The status of Ig loci rearrangements in single cells from different stages of B cell development. Int Immunol 1995, 7:1013-1019.
14. Kitamura D, Roes J, Kuhn R, Rajewsky K: A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin µ chain gene. Nature 1991, 350:423-426.
31. Yamagami T, ten Boekel E, Schaniel C, Andersson J, Rolink A, κ–/– small pre-BII cells • Melchers F: Four of five RAG-expressing JCκ have no L chain gene rearrangements: detection by highefficiency single cell PCR. Immunity 1999, 11:309-316. These two papers [31•,32•] describe the immunoglobulin light chain (IgL) rearrangements in small pre-B II, immature and mature B cells at the singlecell level. The accumulation of cells having undergone several κL chain rearrangements in the small pre-B II compartment is described. Moreover, it is shown that the high κ : λ ratio observed in mice is already established at the small pre-B II cell stage.
15. Gong S, Nussenzweig MC: Regulation of an early developmental β. Science 1996, checkpoint in the B cell pathway by Igβ 272:411-414. 16. Cheng AM, Rowley B, Pao W, Hayday A, Bolen JB, Pawson T: Syk tyrosine kinase required for mouse viability and B-cell development. Nature 1995, 378:303-306.
Selection events in B cell development Rolink et al.
32. Yamagami T, ten Boekel E, Andersson J, Rolink A, Melchers F: • Frequencies of multiple IgL chain gene rearrangements in single normal or κL chain-deficient B lineage cells. Immunity 1999, 11:317-327. See annotation to [31•]. 33. Engel H, Rolink A, Weiss S: B cells are programmed to activate κ • and λ for rearrangement at consecutive developmental stages. Eur J Immunol 1999, 29:2167-2176. In this study, the accessibility of the κ and λ loci during mouse B cell development is analyzed. The findings clearly indicate that the κ locus opens at an earlier developmental stage than the λ locus and thus offer an explanation for the high κ : λ ratio found in mouse B cells. 34. Tiegs SL, Russell DM, Nemazee D: Receptor editing in self-reactive bone marrow B cells. J Exp Med 1993, 177:1009-1020. 35. Gay D, Saunders T, Camper S, Weigert M: Receptor editing: an approach by autoreactive B cells to escape tolerance. J Exp Med 1993, 177:999-1008. 36. Radic MZ, Erikson J, Litwin S, Weigert M: B lymphocytes may escape tolerance by revising their antigen receptors. J Exp Med 1993, 177:1165-1173. 37.
Dorner T, Foster SJ, Farner NL, Lipsky PE: Immunoglobulin κ chain receptor editing in systemic lupus erythematosus. J Clin Invest 1998, 102:688-694.
38. Osmond DG: Proliferation kinetics and the lifespan of B cells in central and peripheral lymphoid organs. Curr Opin Immunol 1991, 3:179-185. 39. Rolink AG, Andersson J, Melchers F: Characterization of immature B cells by a novel monoclonal antibody, by turnover and by mitogen reactivity. Eur J Immunol 1998, 28:3738-3748. 40. Turner M, Gulbranson-Judge A, Quinn ME, Walters AE, MacLennan IC, Tybulewicz VL: Syk tyrosine kinase is required for the positive selection of immature B cells into the recirculating B cell pool. J Exp Med 1997, 186:2013-2021. 41. Torres RM, Flaswinkel H, Reth M, Rajewsky K: Aberrant B cell development and immune response in mice with a compromised BCR complex. Science 1996, 272:1804-1808. 42. Torres RM, Hafen K: A negative regulatory role for Ig-alpha during • B cell development. Immunity 1999, 11:527-536. In contrast to the prediction that the cytoplasmic region of Igα plays a positive role in BCR signaling, the authors show for the first time that the developmental arrest in Igα∆c/∆c mice may be due to hyperactivation of this mutant BCR complex. α cytoplasmic 43. Kraus M, Saijo K, Torres RM, Rajewsky K: Igα • truncation renders immature B cells more sensitive to antigen contact. Immunity 1999, 11:537-545. Using the hen egg lysozyme (HEL) system, effects of the cytoplasmic domain of Igα on negative selection are reported. In contrast to anergy in wild-type mice harboring double transgenes (BCRs against HEL and soluble HEL), Igα∆c/∆c mice harboring the double transgenes show rearrangements of endogenous κL chain, presumably reflecting receptor editing and/or apoptosis. Furthermore, because BCR-mediated calcium mobilization and tyrosine phosphorylation are augmented in the mutant mice, the authors suggest that this increased signaling strength leads to selection of editing/apoptosis rather than anergy. 44. Rolink AG, Brocker T, Bluethmann H, Kosco-Vilbois MH, Andersson J, Melchers F: Mutations affecting either generation or survival of cells influence the pool size of mature B cells. Immunity 1999, 10:619-628.
207
45. Schubart DB, Rolink A, Kosco-Vilbois MH, Botteri F, Matthias P: B-cell-specific coactivator OBF-1/OCA-B/Bob1 required for immune response and germinal centre formation. Nature 1996, 383:538-542. 46. Schubart K, Massa S, Schubart D, Corcoran LM, Rolink AG, Matthias P: B cell development and immunoglobulin gene transcription in the absence of Oct-2 and OBF-1. Nat Immunol 2001, 2:69-74. 47. •
Schubart DB, Rolink A, Schubart K, Matthias P: Lack of peripheral B cells and severe agammaglobulinemia in mice simultaneously lacking Bruton’s tyrosine kinase and the B cell-specific transcriptional coactivator OBF-1. J Immunol 2000, 164:18-22. The authors analyze OBF/btk double-mutant mice. They find that B cell development in the bone marrow is unaffected but that mature peripheral B cells are absent, which points at the very important role for these two molecules in the transition of immature B cells from the bone marrow to the spleen. 48. Loder F, Mutschler B, Ray RJ, Paige CJ, Sideras P, Torres R, Lamers MC, Carsetti R: B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals. J Exp Med 1999, 190:75-89. 49. Shachar I, Flavell RA: Requirement for invariant chain in B cell maturation and function. Science 1996, 274:106-108. 50. Chang CH, Guerder S, Hong SC, van Ewijk W, Flavell RA: Mice lacking the MHC class II transactivator (CIITA) show tissuespecific impairment of MHC class II expression. Immunity 1996, 4:167-178. 51. Gosgrove D, Gray D, Dierich A, Kaufman J, Lemeur M, Benoist C, Mathis D: Mice lacking MHC class II molecules. Cell 1991, 66:1051-1066. 52. Markowitz JS, Rogers PR, Grusby MJ, Parker DC, Glimcher LH: B lymphocyte development and activation independent of MHC class II expression. J Immunol 1993, 150:1223-1233. 53. Madsen L, Labrecque N, Engberg J, Dierich A, Svejgaard A, Benoist C, Mathis D, Fugger L: Mice lacking all conventional MHC class II genes. Proc Natl Acad Sci USA 1999, 96:10338-10343. 54. Gu H, Tarlinton D, Muller W, Rajewsky K, Forster I: Most peripheral B cells in mice are ligand selected. J Exp Med 1991, 173:1357-1371. 55. Lam KP, Kuhn R, Rajewsky K: In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death. Cell 1997, 90:1073-1083. 56. Takeda S, Rodewald HR, Arakawa H, Bluethmann H, Shimizu T: MHC class II molecules are not required for survival of newly generated CD4+ T cells, but affect their long-term life span. Immunity 1996, 5:217-228. 57. ••
Hayakawa K, Asano M, Shinton SA, Gui M, Allman D, Stewart CL, Silver J, Hardy RR: Positive selection of natural autoreactive B cells. Science 1999, 285:113-116. In this study, the authors elegantly show that natural autoreactive B ells can be positively selected in the B-1 compartment in the presence of the autoantigen. 58. Kouskoff V, Lacaud G, Nemazee D: T cell-independent rescue of B lymphocytes from peripheral immune tolerance. Science 2000, 287:2501-2503. 59. Andersson J, Melchers F, Rolink A: Stimulation by T cell independent antigens can relieve the arrest of differentiation of immature auto-reactive B cells in the bone marrow. Scand J Immunol 1995, 42:21-33.