Impaired V(D)J recombination and increased apoptosis among B cell precursors in the bone marrow of c-Abl-deficient mice

Previous studies on c-Abl-deficient mice have shown high post-natal mortality and lymphopenia. However, the mechanisms by which c-Abl may influence B lymphopoiesis remain obscure. In this study, we analyzed B cell sub-populations at various differentiation stages in the bone marrow (BM) of c-Abl-deficient mice. Phenotypic analyses revealed that c-Abl(-/-) pro-B cells were reduced to half of normal incidence and absolute number, while pre-B cells showed an even greater reduction. Both c-Abl(-/-) pro-B and pre-B cell populations showed considerably elevated apoptosis ex vivo and in short-term culture but their cell cycle progression was not impaired. In contrast, apoptosis of immature IgM(+)IgD(-) B lymphocytes remained at normal control levels. Inhibition of c-Abl activity by STI571 in normal BM cultures significantly increased apoptosis in B cell precursors while the survival of immature B cells was not affected. To determine whether c-Abl deficiency affects Ig heavy-chain rearrangement, we found that the frequency of V(D)J recombination was markedly reduced by 15-fold in c-Abl(-/-) pro-B cells compared with the control values. However, no perturbation in the levels of signal-end recombination intermediates was found. Taken together, we propose that c-Abl mediates a stage-specific anti-apoptotic response in precursor B cells and is required for efficient V(D)J recombination during B cell development.     

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.     

Differential regulation of mouse B cell development by transforming growth factor beta1

Transforming growth factor beta (TGFbeta) can inhibit the in vitro proliferation, survival and differentiation of B cell progenitors, mature B lymphocytes and plasma cells. Here we demonstrate unexpected, age-dependent reductions in the bone marrow (BM) B cell progenitors and immature B cells in TGFbeta1-/- mice. To evaluate TGFbeta responsiveness during normal B lineage development, cells were cultured in interleukin 7 (IL7) +/- TGFbeta. Picomolar doses of TGFbeta1 reduced pro-B cell recoveries at every timepoint. By contrast, the pre-B cells were initially reduced in number, but subsequently increased compared to IL7 alone, resulting in a 4-fold increase in the growth rate for the pre-B cell population. Analysis of purified BM sub-populations indicated that pro-B cells and the earliest BP1- pre-B cells were sensitive to the inhibitory effects of TGFbeta1. However, the large BP1+ pre-B cells, although initially reduced, were increased in number at days 5 and 7 of culture. These results indicate that TGFbeta1 is important for normal B cell development in vivo, and that B cell progenitors are differentially affected by the cytokine according to their stage of differentiation.     

Origin of CD5+ B cells and natural IgM-secreting cells: reconstitution potential of adult bone marrow, spleen and peritoneal cells.

The bulk of natural IgM secretion is currently attributed to peritoneal CD5+ B cells and their progeny, believed to be independent of adult bone marrow precursors. We have compared the capacity of peritoneal or splenic cells from normal adult mice to generate serum IgM after transfer into allotype-congenic, irradiated and bone marrow-protected mice. Recipients of either cell population produced donor-allotype IgM-secreting cells in the spleen, and had donor-derived serum IgM. In both cases as well, recipient IgM secretion recovered to control levels. Since the spleen cell-derived natural IgM production could result from expansion of CD5+ B cells present in the inoculum, we next investigated the ability of Ig- bone marrow (BM) cells (Ig- BM) to reconstitute natural IgM secretion in irradiated mice. This cell population was most efficient in reconstituting donor-derived IgM secretion. The origin and phenotype (IgM, CD5) of B cells present in spleen and peritoneum of recipient mice were also analyzed. In agreement with the high level of donor IgM-secreting cells, transfers of splenic and Ig- BM cells fully reconstitute donor B cells in spleen and peritoneum and inhibit reconstitution from host origin. In contrast, donor peritoneal cells reconstitute B cells very poorly in spleen and allow for reconstitution by host cells. Furthermore, Ig- BM cells as well as splenic or peritoneal donor cells, all reconstitute CD5+ B cells in the peritoneum of recipient mice. Interestingly, the fraction of IgM+ cells of each allotype that differentiate to IgM secretion varies widely, but normal levels of IgM are established even when the number of donor B cells present in the animal is very limited.     

GL7 defines the cycling stage of pre-B cells in murine bone marrow.

We have identified a novel subset of early B lineage cells in the mouse bone marrow (BM) by GL7 expression on cell surface. GL7(+)B220(low) BM cells have a large cell size and are CD43(-to low), CD95(-), Sca-1(-), I-A(low), IgM(-) and IgD(-), suggesting that they are large pre-B cells. These BM cells express lambda5 and VpreB but not terminal deoxytransferase (TdT) and Bcl-2, and approximately 50 % of them are in cell cycle. This fraction was not detected in BM cells of Rag-1-deficient and Scid mice, supporting that GL7(+)B220(low) BM cells belong to fraction C' and D according to Hardy's criteria or to an early large pre-B-II fraction according to Melchers-Rolink's criteria. Furthermore, GL7(+)B220(low) BM cells can differentiate into IgM(+) immature B cells in co-culture with stromal cells. These results suggest that B lymphocytes pass through the GL7(+) pre-B cell stage during differentiation in the BM. Thus, GL7 is the critical marker to define the proliferation stage of large pre-B cells.     

Independent homeostatic regulation of B cell compartments

In the present study we used mice with a developmental arrest of B cell production to study the ability of a limited number of normal B cell precursors to populate peripheral B cell pools. In chimeras reconstituted with mixtures of bone marrow (BM) cells from normal and B cell-deficient donors, we show that the rate of BM B cell production is a constant function of the number of BM pre-B cells and is not modified by the peripheral B cell pool size, i.e. there is no feedback regulation of the central pre-B cell compartment by the number of mature B cells. We also show that the physiological number of peripheral B cells requires a minimum continuous input of newly formed cells, but is not determined by the number of B cell precursors. Chimeras with a threefold reduced rate of BM B cell production have normal numbers of peripheral B cells. Parabiosis between normal and B cell-deficient mice showed that the BM B cell production of one mouse suffices to replenish the B cell pool of three mice. Finally, we show that the compartment of activated IgM-secreting B cells is homeostatically autonomous since the number of cells it comprises is regulated independently of the size of the mature B cell pool. The results presented here support a model of the immune system in which the size of the different B cell compartments, i.e. pre-B, resting B and IgM-secreting, is autonomously regulated.     

The B cell receptor, but not the pre-B cell receptor, mediates arrest of B cell differentiation

B cell development in organ cultures of fetal liver from mice at day 14 of gestation resembles in kinetics and cell numbers generated the one observed in vivo. This development in vitro can be blocked by an IL-7 receptor-specific monoclonal antibody. Monoclonal antibodies specific for the pre-B cell receptor, i. e. for VpreB, lambda5, or muH chains, do not perturb B cell development in these organ cultures up to and including the CD25+ small pre-BII cell stage. However, muH chain-specific antibodies inhibit the appearance of the subsequent surface IgM+ immature B cells. In organ cultures of muH chain allotype heterozygous (muHa x muHb)F1 fetal livers a dose-dependent inhibition by allotype-specific monoclonal antibodies of sIgM+ immature B cells expressing the corresponding, but not the other, allotype was observed. By combining cell sorting with limiting dilution analysis of lipopolysaccharide-reactive cells, the probable target cell of this muH chain-specific inhibition was identified as an IgM+, CD23-immature B cell. Hence, engagement of the pre-B cell receptor by specific antibodies does not influence B cell development, while engagement of the B cell receptor on immature B cells does.     

Cyclin D2 controls B cell progenitor numbers

Cyclin D2 affects B cell proliferation and differentiation in vivo. It is rate-limiting for B cell receptor (BCR)-dependent proliferation of B cells, and cyclin D2-/- mice lack CD5+(B1) B lymphocytes. We show here that the bone marrow (BM) of cyclin D2-/- mice contains half the numbers of Sca1+B220+ B cell progenitors but normal levels of Sca1+ progenitor cells of other lineages. In addition, clonal analysis of BM from the cyclin D2-/- and cyclin D2+/+ mice confirmed that there were fewer B cell progenitors (B220+) in the cyclin D2-/- mice. In addition, the colonies from cyclin D2-/- mice were less mature (CD19lo) than those from cyclin D2+/+ mice (CD19Hi). The number of mature B2 B cells in vivo is the same in cyclin D2-/- and cyclin D2+/+ animals. Lack of cyclin D2 protein may be compensated by cyclin D3, as cyclin-dependent kinase (cdk)6 coimmunoprecipitates with cyclin D3 but not cyclin D1 from BM mononuclear cells of cyclin D2-/- mice. It is active, as endogenous retinoblastoma protein is phosphorylated at the cdk6/4-cyclin D-specific sites, S807/811. We conclude that cyclin D2 is rate-limiting for the production of B lymphoid progenitor cells whose proliferation does not depend on BCR signaling.     

Monoclonal antibodies that block adhesion of B cell progenitors to bone marrow stroma in vitro prevent B cell differentiation in vivo

B cell differentiation requires adhesion of B cell progenitors to bone marrow (BM) or fetal liver stroma. We show that B lymphoid cells can adhere to the BM stroma cell line CS 1.3, in vitro. Two monoclonal antibodies, SAB-1 and SAB-2, inhibited the adhesion of a B220+ progenitor B cell line but did not interfere with the binding of cytoplasmic mu chain-positive pre-B cells or mature B cells to the BM stromal cell line. Injection of both SAB-1 and SAB-2 antibodies into pregnant mice reduced by 90% the number of B220+n B lineage cells in the livers of their embryos. Livers from such embryos also were virtually devoid of cells able to give rise to B cell colonies in soft agar cultures (CFU-preB). Either antibody separately had no effect. Flow cytometry analysis show that SAB-1 is present on CS 1.3 stroma cells and on a pre-B cell line while SAB-2 is present on pro-B and pre-B cell lines, but not on CS 1.3 stromal cells. SAB-1 and SAB-2 react with different molecules and neither antibody seems to recognize CD44, and adhesion molecule that may also participate in B cell differentiation. Proteinase K and trypsin can digest both SAB-1 and SAB-2 antigens from viable cells suggesting that both are cell surface proteins. We propose that antibodies SAB-1 and SAB-2 probably recognize novel cell-cell adhesion molecules, and that these molecules are involved in the interactions between B cell progenitors and stroma cells.     

IFN-alpha/beta enhances BCR-dependent B cell responses

Type I interferon (IFN-I) is constitutively produced in the bone marrow (BM), and induced at sites of inflammation and following infection by viruses or microorganisms. We have previously shown that IFN-I regulates the generation and selection of normal B cell populations in the BM. In the present work, we assess the effects of IFN-I on mature B cell function by monitoring the responses of IFN-alpha/beta-treated murine splenic B cells to apoptotic, mitogenic and activating stimuli. A similar analysis is performed on BM mature B cells obtained from wild-type or IFN-I receptor-deficient mice. IFN-alpha/beta is shown to induce B cells to a state of partial activation characterized by the up-regulation of CD69, CD86 and CD25 molecules in the absence of either proliferation or terminal differentiation. B cells treated with IFN-alpha/beta show an increased survival and resistance to Fas-mediated apoptosis. IFN-alpha/beta also enhances B cell responses to BCR ligation such as calcium fluxes, IgM internalization, induction of activation markers and proliferation. These results indicate that in addition to its inhibitory effect on viral replication and T cell apoptosis, IFN-alpha/beta plays an essential role during an inflammatory response by lowering the threshold for B cell induction, thereby promoting fast and polyclonal antibody responses.     

CD72-deficient mice reveal nonredundant roles of CD72 in B cell development and activation.

CD72, a B cell surface protein of the C-type lectin superfamily, recruits the tyrosine phosphatase SHP-1 through its ITIM motif(s). Using CD72-deficient (CD72-/-) mice, we demonstrate that CD72 is a nonredundant regulator of B cell development. In the bone marrow of CD72-/- mice, there was a reduction in the number of mature recirculating B cells and an accumulation of pre-B cells. In the periphery of CD72-/- mice, there were fewer mature B-2 cells and more B-1 cells. In addition, CD72 is a negative regulator of B cell activation, as CD72-/- B cells were hyperproliferative in response to various stimuli and showed enhanced kinetics in their intracellular Ca2+ response following IgM cross-linking.     

The effect of an immunoglobulin mu transgene on B cell maturation.

In mu 17.2.25-transgenic (M54) mice the absolute number of surface IgM (sIgM) B cells in lymphoid organs is drastically reduced compared to normal C57BL/6 mice and a high frequency of B cells express the immunoglobulin (Ig) encoded by the transgene rather than endogenous Ig on the surface. To determine the effect of a mu transgene on B cell development, adoptive cell transfers were performed using mu transgenic (M54) bone marrow and fetal liver cells. The data presented support the following conclusions: (a) adult transgenic bone marrow contains functional B cell precursors able to mature and repopulate the spleen and peritoneum of recipient mice. The relative frequency of transgene (sIgMa) and endogenous (sIgMb) surface sIgM-positive B cells reconstituted by transgenic bone marrow in allotype-matched C57BL/6 recipients is the same as in the M54 donors; (b) serum analysis indicates that transgenic bone marrow donor cells can reconstitute B cells in congenic and severe combined immunodeficient (SCID) recipient mice; (c) transgenic fetal liver cells are not a richer source of precursors for B cells expressing endogeneous Ig; (d) in transgenic mice sIgM+ B cells are not restricted to the CD5+ phenotype, however, the relative frequency of sIgMb B cells that are CD5+ is higher in transgenic than normal mice; and (e) bone marrow cells from adult normal and transgenic mice are able to generate CD5+ B lymphocytes in the spleen and peritoneum of allotype-congenic and neonatal SCID recipient mice. The results indicate that the presence of a complete mu heavy chain transgene does not result in a selective developmental block of "conventional" bone marrow-derived pre-B and B cells.     

Regulation of pre-B cell proliferation in bone marrow: immunofluorescence stathmokinetic studies of cytoplasmic mu chain-bearing cells in anti-IgM-treated mice, hematologically deficient mutant mice and mice given sheep red blood cells

To identify factors influencing the in vivo proliferate activity of bone marrow pre-B cells, the metaphase-blocking drug, vincristine sulfate, was injected into (a) mice depleted of B lymphocytes by treatment with anti-mouse IgM antibodies from birth; (b) hematologically deficient W/Wv and Sl/Sld mutants, and (c) mice injected with a foreign agent, sheep red blood cells (SRBC). Subsequently, a quantitative measure of pre-B cell proliferation was provided by examining marrow cells by immunofluorescence labeling for the absolute number of pre-B cells, identified by the presence of cytoplasmic mu chains (c mu) without surface mu (s mu), which had been arrested in metaphase. In anti-IgM-treated mice, some changes were observed in the size of the large pre-B cell population and in the incidence of mitotic cells after vincristine administration, but the overall production rate of pre-B cells did not differ from that in controls given normal rabbit serum. Pre-B cell kinetics in W/Wv and Sl/Sld mice also generally resembled those in homozygous controls. In contrast, after SRBC injection, there was an increase in the rate at which large pre-B cells entered mitosis. Thus, the proliferation of c mu + s mu- bone marrow pre-B cells shows no evidence of feedback control from the mature B lymphocyte pool, as indicated by lack of stimulation of pre-B cell production in anti-IgM-treated mice, and is independent of the hemopoietic defects of W/Wv or Sl/Sld mutants. On the other hand, the increased bone marrow pre-B cell proliferation after SRBC injection demonstrates that the magnitude of B cell genesis in the bone marrow can be influenced by extrinsic agents and thus may be influenced by environmental stimuli.     

Autoimmune bone marrow environment severely inhibits B cell development by inducing extensive cell death and inhibiting proliferation

The spontaneous scurfy (sf) mutation in mice results in a complete loss of Tregs, leading to a lethal, multi-system autoimmune syndrome. We have carefully examined B lymphopoiesis in sf mice. Paradoxically, the B cell numbers at all developmental stages including pro-B, pre-B, immature and mature B cells are significantly decreased in the BM and spleen of sf mice, compared to that of wild-type littermate controls. The developing B cells in sf mice exhibit profoundly elevated cell death induced by down-regulation of Bcl-XL expression and up-regulation of Fas expression. In addition, the clonal expansion of pre-B and immature B cells in sf mice is significantly reduced compared to wild-type controls. Foxp3 expression is not detectable in all stages of developing B cells in wild-type mice, indicating that the defects are B-cell extrinsic, which is further supported by the recovery of B cell maturation in BM chimeric mice. Remarkably, IFN-γ production is significantly elevated in numerous cell types in the BM of sf mice. Taken together, these results indicate that the autoimmune inflammatory marrow environment has dramatic inhibitory effects on B cell development by inducing apoptosis and suppressing proliferation of developing B cells.     

Maternal IgG stimulates B lineage cell development in the progeny

To examine the physiological role of maternal natural IgG antibodies on the development of B lineage cells of the progeny, we have bred homozygous μMT/μMT or heterozygous μMT/+ females to μMT/μMT or μMT/+ males, respectively. We could thus compare normal or B cell-deficient mice born from Ig-deprived (Ig^?) or phenotypically normal mothers (Ig⁺). B cell-deficient progeny of heterozygous mothers contain no detectable serum IgA or IgM, but IgG concentrations that peak at 2 mg/ml by 7–21 days of age, decay after weaning with a half-life of 7 days, and remain detectable for 2 months after birth. At 7 days after birth, μMT/+ progeny born of Ig⁺ mothers contain two- to threefold higher numbers of bone marrow (BM) pre-B and B cells, and of splenic B cells, compared to mice of the same age born from Ig^? mothers. In contrast, the former progeny exhibit two to four times lower numbers of Ig-secreting plasma cells in spleen and thymus, and contain sixfold lower serum IgM concentrations. A similar maternal IgG-dependent stimulation of BM B cell precursors is also observed in μMT/μMT progeny. No significant differences were detected between the groups on day 3 after birth, suggesting the requirement for a minimal IgG concentration in the serum.     

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.     

IL-7 sensitizes human pre-B cells but not pro-B cells to Fas/APO-1 (CD95)-mediated apoptosis

Homeostasis of human B cell development is maintained by a complex network of cytoplasmic and surface expressed molecules. Abnormalities in this process may result in the expansion of malignant B cell precursors in B lineage acute lymphoblastic leukaemia (ALL). ALL cells share surface antigens with normal early precursor B cells. We have studied here the role of Fas/APO-1 (CD95) antigen on leukaemic precursor B cell line growth and survival, and the modulation of its effects by signals involved in normal early B cell development. Four ALL cell lines representative of the early steps of B cell differentiation are shown to express surface Fas/APO-1 (CD95) antigen and to undergo apoptosis in the presence of anti-Fas cross-linking antibodies. This effect is strongly enhanced when pre-B, but not pro-B cells, are pretreated with IL-7 but not with IL-2, IL-3, IL-4 or IL-10. Furthermore, pre-B cell death induced by anti-Fas antibodies in combination with IL-7 is increased upon pre-B receptor but not CD19 cross-linking. Bcl-2 and Bax protein expression is not influenced by IL-7 or pre-BR stimulation in either pro-B or pre-B cell lines. These results indicate that signals involved in normal early B cell development can modulate the Fas (CD95)-mediated apoptosis of leukaemic precursor B cells.     

IL-10 production in a CD5+ B cell lymphoma arising in a CD4 monoclonal antibody-treated SJL mouse.

A majority of SJL mice develop spontaneous reticulum cell sarcomas (RCS) at about 1 year of age which can be transplanted into young SJL recipients. Previous studies have shown that RCS tumors are of B cell lineage, and that the development of these lymphomas and their subsequent growth depends upon host-derived T helper cell factors. In vivo treatment of SJL mice with anti-CD4 monoclonal antibody (mAb) prevents the development of the characteristic B lymphomas. Most of the mAb-treated animals were tumor free and had a significantly prolonged life span. However, one such CD4 mAb-treated mouse developed a transplantable IgM+ CD5+ B cell lymphoma (designated NJ101), which has not previously been described in SJL/J mice. NJ101 is clonal on the basis of a discrete non-germ line Ig heavy chain gene rearrangement by Southern blot analysis. Unlike the sIg- CD5- transplantable RCS-X cell line, the IgM+ CD5+ NJ101 lymphoma cells will grow in immuno-compromised hosts, such as irradiated recipients or in recipients treated with CD4 mAb in vivo. The RCS (B cell) lymphoma requires CD4+ T cells for progressive growth, whereas the growth of the CD5+ B lymphoma cells is enhanced by the removal of such cells. Thus, CD5+ B cell clonal development may be aided by the removal of regulatory T cells and/or the malignant CD5+ B cells may produce their own growth factors in an autocrine manner. Examination of IL-10 message by quantitative polymerase chain reaction techniques indicate that the CD5+ B lymphoma cells produce increased levels of IL-10 relative to normal LN cells or purified RCS lymphoma cells. These results suggest that two different types of B cell tumors, both of which can develop in SJL mice, have different growth requirements. Furthermore, treatment to prevent the occurrence of the characteristic RCS malignancy of SJL mice may lead to the development of another form of B cell neoplasia.