Dmu expression causes enrichment of MZ B cells, but is non permissive for B cell maturation in Rag2-/- mice even if combined with Bcl-2

Rearrangements in reading frame 2 promote the expression of a truncated heavy chain, the Dmu protein. Dmu can assemble into a pre-B cell receptor like complex that appears to induce a subset of signals elicited by full length mu, but cannot promote the pro-B to pre-B cell transition of Rag-/- B cells. In order to determine if this could stem from an impaired survival signal not properly induced by the Dmu protein, we introduced Bcl-2 into Dmu-transgenic, Rag2-/- mice. Despite the fact that the Bcl-2 transgene expression promoted some increase in the fraction of CD43- B cells, an identical increase was also observed in Rag2-/- mice. Moreover, whereas in mu-transgenic Rag2-/-Bcl-2+ mice, CD2 and CD25 expression were up regulated and c-Kit was down regulated, these markers were unaltered in Dmu-transgenic Rag2-/- Bcl-2+ mice compared to Rag2-/- Bcl-2+ mice, indicating that Dmu cannot support pre-B cell maturation despite extended survival of B cell precursors by Bcl-2. In addition, we observed that in Dmu-transgenic recombination competent mice, the Dmu induced partial block is permissive for marginal zone B cell development whereas the formation of follicular B cells is severely reduced. While the Dmu protein is expressed in peripheral B cells escaping the block, only a minor fraction of Dmu is exposed to the outer cell surface.     

Distinct signaling requirements for Dmu selection,IgH allelic exclusion,pre-B cell transition,and tumor suppression in B cell progenitors

The pre-B cell receptor triggers expansion and differentiation of pre-B cells (the pre-B cell transition), as well as inhibition of V(H) to DJ(H) recombination (allelic exclusion). The latter also accounts for counter-selection of pro-B cells expressing Dmu protein (Dmu selection). However, the signaling pathways responsible for these events remain poorly defined. Here we show complete arrest of B cell development at the pre-B cell transition in BASH/CD19 double mutant mice, indicating partial redundancy of the two B cell-specific adaptors. Allelic exclusion remained intact in the double mutant mice, whereas Dmu selection was abolished in BASH mutant mice. Thus, distinct signals are required for these events. In addition, both mutant mice succumbed to pre-B cell leukemia, indicating that BASH and CD19 contribute to tumor suppression.     

Lambda5 is required for rearrangement of the Ig kappa light chain gene in pro-B cell lines.

Lambda5 associates with V(pre-B) to form the surrogate light (L) chain. The phenotype of lambda5 knockout mice showed severe impairment of B cell development from pro-B to immature B cell stages. To investigate the function of the surrogate L chain at this stage, we restored expression of lambda5 to lambda5-deficient pro-B cell lines which were established from bone marrow cells of lambda5 knockout mice in the presence of IL-7 and a stromal cell line. Some of these lines are severely impaired in B cell development from pro-B to immature B cell stages as is seen in vivo in lambda5 knockout mice. Restoration of lambda5 protein by retroviral-mediated gene transfer into established lambda5-deficient pro-B cell lines induced rearrangement of the Ig kappa L chain genes after removal of IL-7 from the culture. Immunoprecipitation revealed that the restored lambda5 in the cell line is coupled with V(pre-B) to form the surrogate L chain. The results demonstrate that formation of a complete surrogate L chain, consisting of both lambda5 and V(pre-B), stimulates efficient rearrangement of the kappa L chain genes.     

Early arrest of B cell development in nude, X-linked immune-deficient mice

Mice simultaneously expressing the nude and xid mutations have a severe deficit of both mature T and B cells. We now report studies designed to determine at which point in B cell differentiation development is arrested. Nude-xid mice have normal numbers of hematopoietic colony forming units (CFU-s) but lack two early pre-B cell markers: susceptibility to transformation by Abelson murine leukemia virus (A-MuLV) and production of cytoplasmic mu (C mu) heavy chain. Thus, there is a defect in lymphocyte development prior to or early in pre-B cell differentiation but after hematopoietic stem cell formation. The monoclonal reagents DNL 1.9, 14.8 and RA3-3A1/6.1, which react with the surface protein B220 (Ly5) on pro-B, C mu- pre-B, C mu+ pre-B, and surface Ig+ B cells, revealed the presence of positive cells in nude-xid mice. The bone marrow of nude-xid mice contains more B220+ cells than C mu+ cells. Our data suggest that the developmental block in these mice occurs at the earliest identifiable step in the B lymphocyte lineage, after the appearance of B220+ C mu- pro-B cells, but before the differentiation of C mu-bearing (pre-B) cells.     

Unexpected requirement for ZAP-70 in pre-B cell development and allelic exclusion

ZAP-70, a member of the Syk family of tyrosine kinases, has been reported to be expressed exclusively in T and NK cells. We show here that it is expressed throughout B cell development and that it plays a role in the transition of pro-B to pre-B cells in the bone marrow, a checkpoint controlled by signals from the pre-B cell receptor (pre-BCR), which monitors for successful rearrangement of immunoglobulin heavy chain genes. Whereas mice deficient in Syk show a partial block at this step, mice mutant in both Syk and ZAP-70 show a complete block at the pro-B cell stage and a failure of heavy chain allelic exclusion, hallmarks of defective pre-BCR signaling.     

Conventional light chains inhibit the autonomous signaling capacity of the B cell receptor

Signals from the B cell antigen receptor (BCR), consisting of mu heavy chain (muHC) and conventional light chain (LC), and its precursor the pre-BCR, consisting of muHC and surrogate light chain (SLC), via the adaptor protein SLP-65 regulate the development and function of B cells. Here, we compare the effect of SLC and conventional LC expression on receptor-induced Ca(2+) flux in B cells expressing an inducible form of SLP-65. We found that SLC expression strongly enhanced an autonomous ability of muHC to induce Ca(2+) flux irrespective of additional receptor crosslinking. In contrast, LC expression reduced this autonomous muHC ability and resulted in antigen-dependent Ca(2+) flux. These data indicate that autonomous ligand-independent signaling can be induced by receptor forms other than the pre-BCR. In addition, our data suggest that conventional LCs play an important role in the inhibition of autonomous receptor signaling, thereby allowing further B cell differentiation.     

Complete arrest from pro- to pre-B cells in a case of B cell-negative severe combined immunodeficiency (SCID) without recombinase activating gene (RAG) mutations

The B-cell lineage in a patient with B-cell-negative severe combined immunodeficiency (SCID) was analysed by using antisurrogate light chain (SL) MoAbs. Peripheral CD3(+) T cells and CD19(+) B cells were absent in the patient. The common gamma (gamma c) chain was expressed normally on the patient's peripheral NK cells and his peripheral mononuclear cells did not possess any mutations in recombinase activating gene (RAG)-1, 2. Normal levels of expression of Ku70 and Ku80 protein were found by Western blot analysis. The patient did, however, display an increase in fibroblast sensitivity to irradiation. Furthermore, flow cytometric analyses of bone marrow cells showed that surface IgM and cytoplasmic mu positive cells were absent and that CD19(+) B cells were composed of only CD34(+) terminal deoxynucleotidyl transferase (TdT)(+) SL(+) pro-B cells. The complete arrest of pro- to pre-B cell development in the SCID patient's bone marrow suggests that some genes involved in V(D)J recombination, excepting the RAG gene, may play a causative role in the immunodeficiency.     

The pre-B cell receptor and its function during B cell development

The process of B cell development in the bone marrow occurs by the stepwise rearrangements of the V, D, and J segments of the Ig H and L chain gene loci. During early B cell genesis, productive Ig H chain gene rearrangement leads to assembly of the pre-B cell receptor (pre-BCR), which acts as an important checkpoint at the pro-B/preB transitional stage. The pre-BCR, transiently expressed by developing precursor B cells, comprises the Ig muH chain, surrogate light (SL) chains VpreB and lambda5, as well as the signal-transducing heterodimer Ig alpha/Ig beta. Signaling through the pre-BCR regulates allelic exclusion at the Ig H locus, stimulates cell proliferation, and induces differentiation to small post-mitotic pre-B cells that further undergo the rearrangement of the Ig L chain genes. Recent advances in elucidating the key roles of pre-BCR in B cell development have provided a better understanding of normal B lymphopoiesis and its dysregulated state leading to B cell neoplasia.     

CD79 alpha/CD79 beta heterodimers are expressed on pro-B cell surfaces without associated mu heavy chain

During B cell development, the surface expression of CD79 alpha/CD79 beta heterodimers had been thought to begin in the pre-B cell stage where the heterodimers constitute pre-B cell receptors together with mu heavy and surrogate light chains. Thereafter, in mature B cells, CD79 alpha/CD79 beta associates with surface Ig to form B cell antigen receptors. In this study, we revealed by using newly established mAb that CD79 beta was expressed on the surface of pro-B cells which had not undergone the productive Ig gene rearrangement. Biochemical analysis showed that CD79 beta on pro-B cells existed either as monomers or as disulfide-linked heterodimers with CD79 alpha, non- covalently associated with four unidentified membrane molecules. Our finding that CD79 beta is expressed on earlier B-lineage cells than previously expected coincides with the recent study in which CD79 beta- deficient mice exhibit a blockade of B cell differentiation at the pro- B cell stage. Thus, it is speculated that the CD79 beta-containing complexes on pro-B cell surfaces may function to induce early B cell differentiation.      

Powered by pairing: the surrogate light chain amplifies immunoglobulin heavy chain signaling and pre-selects the antibody repertoire

Selective expansion of functional pre-B cells is accomplished by the assembly of a signaling-competent pre-B cell receptor (pre-BCR) consisting of immunoglobulin mu heavy chains (muHC), surrogate light chains (SLC) and Igalpha/Igbeta. Here, we review recent data showing that muHCs, in the absence of SLC, deliver autonomous differentiation signals. However, enhanced signaling necessary for pre-B cell expansion requires cross-linking of pre-BCRs via the non-immunoglobulin tail of SLC's subunit lambda5. We also discuss how SLC's ability to modulate the strength of pre-BCR signals is controlled by a muHC's idiotype and its affinity to the chaperone BiP. In this model, BiP in concert with SLC functions as a pre-selector of the antibody repertoire.     

Bruton's tyrosine kinase is required for signaling the CD79b-mediated pro-B to pre-B cell transition

Formation of the pre-BCR complex is a critical check point during B cell development and induces the transition of pro-B to pre-B cells. CD79b (Igbeta) is a signaling component in the pre-BCR complex, since differentiation to the pre-B phenotype is induced by cross-linking the CD79b expressed on developmentally arrested pro-B cells from recombination-activating gene (RAG)-2-deficient mice. Bruton's tyrosine kinase (BTK) plays important roles in B cell development. However, its molecular mechanisms in early B cell development are not fully understood. To examine whether BTK functions in CD79b-mediated signaling for the pro-B/pre-B transition, we utilized RAG2/BTK double-knockout (DKO) mice. Pro-B cells from RAG2/BTK-DKO mice did not differentiate into pre-B cells following CD79b cross-linking, although tyrosine phosphorylation of cellular proteins including Erk1/2 and phospholipase C-gamma2 was induced in the same manner as RAG2-KO mice. BTK is phosphorylated after cross-linking of CD79b on RAG2-deficient pro-B cells. These findings suggest that BTK-dependent pathways downstream of CD79b are critical for the pro-B/pre-B transition and BTK-independent signaling pathways are also activated via the pre-BCR complex.     

The variable,C(H)1, C(H)2 and C(H)3 domains of Ig heavy chain are dispensable for pre-BCR function in transgenic mice

The pre-BCR consists of Ig micro protein, the product of a heavy chain gene assembled by V(D)J recombination in pro-B cells, the surrogate light chains V(pre-B) and lambda 5, and the signaling chains Ig alpha and Ig beta. Signaling by the pre-BCR is a checkpoint required for further maturation of pro-B cells in the adult bone marrow. However, it is currently not known whether an extracellular ligand is required to initiate pre-BCR signaling. We reasoned that if the ectodomain of the pre-BCR is required to interact with a ligand, then a truncated heavy chain protein would not support B cell development. To test this notion, we produced transgenic mice expressing a heavy chain protein whose extracellular domains except for C(H)4 were replaced by an irrelevant Ig superfamily ectodomain from the human CD8 alpha protein. This transgene resulted in pre-BCR-like signaling since it rescued development of pre-B cells in recombinase-activating gene (RAG)1-deficient mice and resulted in allelic exclusion of the endogenous Ig heavy chain gene in RAG-proficient mice. These findings lead us to suggest that the majority of the extracellular region of the pre-BCR is not required for pre-BCR function and, thus, ligand binding is unlikely to be required for pre-BCR function.     

Regulation of cell survival during B lymphopoiesis: suppressed apoptosis of pro-B cells in P53-deficient mouse bone marrow.

B cell development in mouse bone marrow (BM) is subject to quality controls that eliminate aberrant cells by apoptosis, but the intrinsic cellular mechanisms that mediate this negative B cell selection remain unclear. The p53 tumor suppressor transduces signals resulting in apoptosis and cell cycle arrest in cells that sustain DNA damage. Faulty V(D)J recombination in scid lymphocyte precursors activates a p53-dependent DNA damage checkpoint. In the present study, we have examined whether p53 is involved in apoptotic selection of normally developing B cells in BM. Double immunofluorescence labeling and flow cytometry were used to quantitate phenotypically defined B cell populations and their apoptotic rates in BM of homozygous p53-deficient mice. B220(+) mu(-) and terminal deoxynucleotidyl transferase (TdT)(+) pro-B cells were increased in both incidence and absolute number to controls. In contrast, pre-B cells were only slightly increased and the sIgM(+) B lymphocyte compartment remained essentially normal. The incidence of apoptosis among p53(-/-) pro-B cells was greatly reduced, both ex vivo and in short-term culture, whereas, apoptosis of pre-B cells and B lymphocytes was not significantly different from normal. The results indicate that p53 is actively involved as an apoptosis inducer at an early quality control checkpoint in B lymphopoiesis.