Developmental regulation of the Bcl-2 protein and susceptibility to cell death in B lymphocytes.

Cell death is a prominent feature of B cell development. For example, a large population of B cells dies at the pre-B cell stage presumably due to the failure to express a functional immunoglobulin receptor. In addition, developing B cells expressing antigen receptors for self are selectively eliminated at the immature B cell stage. The molecular signals that control B cell survival are largely unknown. The product of the bcl-2 proto-oncogene may be involved as its overexpression inhibits apoptotic cell death in a variety of biological systems. However, the physiological role of the endogenous Bcl-2 protein during B cell development is undetermined. Here we show a striking developmental regulation of the Bcl-2 protein in B lymphocytes. Bcl-2 is highly expressed in CD43+ B cell precursors (pro-B cells) and mature B cells but downregulated at the pre-B and immature B cell stages of development. We found that Bcl-2 expressed by B cells is a long-lived protein with a half-life of approximately 10 h. Importantly, susceptibility to apoptosis mediated by the glucocorticoid hormone dexamethasone is stage-dependent in developing B cells and correlates with the levels of Bcl-2 protein. Furthermore, expression of a bcl-2 transgene rescued pre-B and immature B cells from dexamethasone-induced cell death, indicating that Bcl-2 can inhibit the apoptotic cell death of progenitors and early B cells. Taken together, these findings argue that Bcl-2 is a physiological signal controlling cell death during B cell development.     

Expression of human complement receptor type 2 (CD21) in mice during early B cell development results in a reduction in mature B cells and hypogammaglobulinemia

Complement receptor (CR) type 2 (CR2/CD21) is normally expressed only during the immature and mature stages of B cell development. In association with CD19, CR2 plays an important role in enhancing mature B cell responses to foreign Ag. We used a murine Vlambda2 promoter/Vlambda2-4 enhancer minigene to develop transgenic mice that initiate expression of human CR2 (hCR2) during the CD43(+)CD25(-) late pro-B cell stage of development. We found peripheral blood B cell numbers reduced by 60% in mice expressing high levels of hCR2 and by 15% in mice with intermediate receptor expression. Splenic B cell populations were altered with an expansion of marginal zone cells, and basal serum IgG levels as well as T-dependent immune responses were also significantly decreased in transgenic mice. Mice expressing the highest levels of hCR2 demonstrated in the bone marrow a slight increase in B220(int)CD43(+)CD25(-) B cells in association with a substantial decrease in immature and mature B cells, indicative of a developmental block in the pro-B cell stage. These data demonstrate that stage-specific expression of CR2 is necessary for normal B cell development, as premature receptor expression substantially alters this process. Alterations in B cell development are most likely due to engagement of pre-B cell receptor-mediated or other regulatory pathways by hCR2 in a CD19- and possibly C3 ligand-dependent manner.     

Functional characterization of lymphoid cells generated in serum-deprived culture stimulated with stem cell factor and interleukin 7 from normal and autoimmune mice

We have investigated the phenotypic and functional characteristics of murine pre-B cells obtained in semisolid and liquid culture with stem cell factor (SCF) and interleukin 7 (IL-7). Both serum-supplemented and serum-deprived culture conditions were used. The source of bone marrow cells was either normal mice (CD1 and C3H) or the lupus strain of mice MRL/Ipr and its congenic strain MRL/+. SCF (100 ng/ml) and IL-7 (250 ng/ml) supported murine B cell proliferation in vitro from all the murine strains analyzed both in serum-supplemented and serum-deprived conditions. Maximal colony growth was observed in both cases when the factors were used in combination. The growth factors alone induced some colony growth in serum-supplemented cultures but were either ineffective or had modest activity in serum-deprived cultures. Cells harvested from the colonies or generated in liquid cultures and stimulated with SCF + IL-7 in the absence of serum had almost exclusively a pre-B cell phenotype (BP-1+, B220+, slg-, CD4-, CD8-, Mac-1, RB-6-). Both the maximal colony growth in semisolid culture and the maximal number of cells in liquid culture were observed at day 12–14. At this time, the pre-B cells failed to differentiate further and started to die. Pre-B cells generated in vitro were, however, capable of differentiating in vivo. SCID mice injected with 2 × 10⁶ pre-B cells had readily detectable serum levels of IgM (54 ± 26 m?g/ml) and IgG (60 ± 95 m?g/ml) at 4 weeks and 6 weeks posttransplantation, respectively. Mature B and T cells of the donor major histocompatibility complex type were detected in the SCID mice at sacrifice 14 weeks posttransplantation. These data indicate that purified (>80% BP-1+) populations of functional pre-B cells can be grown from murine bone marrow of normal mice as well as of lupus mice in serum-deprived cultures stimulated with SCF and IL-7. These cultures, therefore, provide a highly enriched source of pre-B cells but also contain T cell precursors that differentiate upon adoptive transfer into SCID mice. © 1995 Wiley-Liss, Inc.     

Cellular maturation defects in Bruton's tyrosine kinase-deficient immature B cells are amplified by premature B cell receptor expression and reduced by receptor editing

In the mouse, Bruton's tyrosine kinase (Btk) is essential for efficient developmental progression of CD43(+)CD2(-) large cycling into CD43(-)CD2(+) small resting pre-B cells in the bone marrow and of IgM(high) transitional type 2 B cells into IgM(low) mature B cells in the spleen. In this study, we show that the impaired induction of cell surface changes in Btk-deficient pre-B cells was still noticeable in kappa(+) immature B cells, but was largely corrected in lambda(+) immature B cells. As lambda gene rearrangements are programmed to follow kappa rearrangements and lambda expression is associated with receptor editing, we hypothesized that the transit time through the pre-B cell compartment or receptor editing may affect the extent of the cellular maturation defects in Btk-deficient B cells. To address this issue, we used 3-83 mu delta transgenic mice, which prematurely express a complete B cell receptor and therefore manifest accelerated B cell development. In Btk-deficient 3-83 mu delta mice, the IgM(+) B cells in the bone marrow exhibited a very immature phenotype (pre-BCR(+)CD43(+)CD2(-)) and were arrested at the transitional type 1 B cell stage upon arrival in the spleen. However, these cellular maturation defects were largely restored when Btk-deficient 3-83 mu delta B cells were on a centrally deleting background and therefore targeted for receptor editing. Providing an extended time window for developing B cells by enforced expression of the antiapoptotic gene Bcl-2 did not alter the Btk dependence of their cellular maturation. We conclude that premature B cell receptor expression amplifies the cellular maturation defects in Btk-deficient B cells, while extensive receptor editing reduces these defects.     

Human B cell development. II. Subpopulations in the human fetus

In man, during fetal development the B cell populations show distinct phenotypes at different tissue sites. The pre-B and B lymphocytes of the fetal liver and bone marrow express IgM and B cell markers, B1 (CD20) and BA-1 (CD24). These "early" cells are negative with a number of other reagents, anti-IgD, RFB4 (CD22), RFB6 (CD21), and RFA-2, which on the other hand recognize peripheral B cells. These peripheral B lymphocytes in the developing fetus are heterogeneous. The diffusely distributed B cells in the earliest lymph node samples, 16 to 17 wk of gestational age, and from 16 to 21 wk in the spleen, are strongly IgM+ (IgD+,RFB4+,RFB6+, and RFA-2+) but lack T cell-associated markers such as T1 (CD5, p 67,000 dalton equivalent of murine Ly-1) and Tü-33. In fetal lymph nodes, primary nodules develop around the follicular dendritic (FD) cells from 17 wk onward, and contain a virtually pure population of B cells; B1+,BA1+,RFB4+,RFB6+,RFA-2+, which simultaneously express IgM,IgD together with T1 (CD5), a T cell-associated antigen. A sizeable subpopulation of these IgM+,T1+ cells are also positive for Tü-33, another T cell-associated marker. In the spleen, the B cells of the IgM+,IgD+,T1+ type appear in smaller numbers and only relatively late around wk 22. These cells are diffusely distributed at first, and start accumulating around the small FD cell clusters as soon as these emerge about the 23rd gestational wk. At that time, the IgM+,T1+B cells can also be washed out from the peritoneal and pleural cavities. The T1+,IgM+B cells may represent the normal equivalent cells of B chronic lymphoid leukemia and centrocytic lymphoma, and appear to be the counterpart of Ly-1+,IgM+B cells in the mouse.     

Differential expression of the c-myb proto-oncogene marks the pre-B cell/B cell junction in murine B lymphoid tumors

A series of murine B lymphoid tumor cell lines which are representative of the pre-B cell, immature and mature B cell, and plasma cell stages of B cell development have been examined for expression of c-myb proto-oncogene mRNA. The pre-B cell lymphoma cell lines express equivalent high steady state levels of c-myb mRNA. In contrast, the B cell lymphoma and plasmacytoma cell lines express steady state c-myb mRNA at levels which are 0.005 to 0.1 times that of the pre-B cell lymphoma lines. These results correlate high levels of c-myb mRNA expression with the pre-B cell stage of development. Subclones of the 1881 pre-B cell lymphoma which express K light chain and are surface IgM-positive as well as two types of hybrid B lymphoid cell lines have been used to demonstrate that surface immunoglobulin expression is not sufficient to result in the down-regulation of c-myb mRNA levels or changes in the expression N-myc mRNA, lambda 5 mRNA, or the BP-1 surface antigen which are markers of the pre-B cell stage of development. Thus, changes in the expression of genes which are independent of immunoglobulin expression are associated with transition from the pre-B cell to the immature B cell stage of development.     

Constitutive versus cell cycle regulation of c-myb mRNA expression correlates with developmental stages in murine B lymphoid tumors.

The expression of c-myb mRNA is differentially regulated in murine B lymphoid tumors such that B cell lymphomas and plasmacytomas contain significantly less c-myb mRNA than pre-B cell lymphomas. To examine the low level of c-myb mRNA expression in the murine B cell lymphoma cell line BCL1, nonessential amino acid starvation was used to block these cells in a G1 state. When BCL1 cells were released from this block, a 7- to 10-fold increase in c-myb mRNA was detected in late G1 and S phase cells relative to that detected in exponentially growing BCL1 cells. This increase was not inhibited by aphidicolin. To determine whether cell cycle regulation of c-myb mRNA expression occurred during exponential growth in both murine pre-B cell lymphoma and B cell lymphoma cell lines, elutriation was used to separate exponentially growing cell populations. An increase in c-myb mRNA expression was seen in late G1 and S phase fractions from B cell lymphoma cell lines. In contrast, c-myb mRNA levels remained constant in elutriation fractions isolated from pre-B cell lymphoma cell lines. Expression of c-myb mRNA was not detected in exponentially growing or in Go serum-stimulated murine fibroblasts. These results indicate that constitutive vs cell cycle regulation of c-myb mRNA expression is related to the state of differentiation in murine B lymphoid tumors and suggest that a switch in regulation may occur during normal B cell development.     

The CD24 antigen discriminates between pre-B and B cells in human bone marrow.

When bone marrow (BM) lymphoid cells from 12 adult healthy donors were labeled by CD24 antibodies and analyzed by flow cytometry, two positive populations of cells were demonstrated in each sample (by a separated bimodal specific immunofluorescence). One population had intermediate CD24-Ag density (termed CD24+ cells) whereas the other had high CD24-Ag density (termed CD24(2+) cells). CD24+ cells represented 5.8 +/- 2.7% of the total lymphoid BM cells and CD24(2+) cells 5.6 +/- 2.5%. Using dual fluorescence analysis on eight samples, all CD24+ cells expressed the CD21 and CD37 mature B cell Ag and also surface IgM (sIgM), but this population lacked CD10 Ag. These cells also expressed CD19 Ag, and at a higher density than CD24(2+) cells. They were also positive for HLA-DR Ag. Conversely, CD24(2+) cells were shown to be early cells of the B cell lineage. While all the CD24(2+) cells were HLA-DR+ and CD19+, 64 +/- 16% of them expressed CD20 Ag (at a lower density than CD24+ cells), 65 +/- 21% CD10 Ag, and 22 +/- 8% were positive for cytoplasmic mu-chains (c mu). None of these cells expressed the CD21 and CD37 mature B cell Ag or sIgM. Additional experiments on four different healthy donors demonstrated that 30 +/- 9% of the CD24(2+) cells expressed the CD34 Ag and that the CD24+ cells did not express it. Thus, the CD24 Ag permits discrimination between two populations of the B cell lineage present in adult BM: 1) A CD24(2+) cell population including "pre" pre-B cells (HLA-DR+, CD19+, CD10+/-, CD20-, CD21-, CD34+, CD37-, c mu-), "intermediate" pre-B cells (HLA-DR+, CD19+, CD10+, CD20+, CD21-, CD34-, CD37-, c mu-), and "true" pre-B cells (HLA-DR+, CD19+, CD10+, CD20+, CD21-, CD34-, CD37-, c mu+). 2) A CD24+ cell population including B cells of the standard phenotype (HLA-DR+, CD19+, CD10-, CD20+, CD21+, CD34-, CD37+, c mu-, sIgM+).     

Two types of mu chain complexes are expressed during differentiation from pre-B to mature B cells.

Immunoglobulin mu chains synthesized in murine pre-B cells are known to be associated with surrogate light chains designated as omega (omega), iota (iota) and B34. In addition to these molecules, we identified the complexes of polypeptides (50, 40, 27 and 15.5 kd) associated with surface or intracellular mu chains of pre-B cell lines. Most of these polypeptides were continuously synthesized and associated with mu chains in virgin B cells lines, although some of them scarcely bound to the mu kappa dimer or mu 2 kappa 2 tetramer concomitantly present in the same clone or population. However, in mature B cells they were no longer detectable except B34. Cross-linking of micron chains on the surface of pre-B cells resulted in an increase in intracellular free Ca2+, indicating that the micron chain complex on the surface of pre-B cell lines acted as a signal transduction molecule. However, the receptor cross-linkage of pre-B cell lines did not induce the increased inositol phospholipid metabolism usually observed in virgin and mature B cell lines. These results suggest that, during the differentiation from pre-B to mature B cells, the cells express two types of mu chain complexes which exhibit different structures as a whole and possess different signal transducing capacities.     

Essential role of phospholipase C gamma 2 in early B-cell development and Myc-mediated lymphomagenesis

Phospholipase Cgamma2 (PLCgamma2) is a critical signaling effector of the B-cell receptor (BCR). Here we show that PLCgamma2 deficiency impedes early B-cell development, resulting in an increase of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B cells. PLCgamma2 deficiency impairs pre-BCR-mediated functions, leading to enhanced interleukin-7 (IL-7) signaling and elevated levels of RAGs in the selected large pre-B cells. Consequently, PLCgamma2 deficiency renders large pre-B cells susceptible to transformation, resulting in dramatic acceleration of Myc-induced lymphomagenesis. PLCgamma2(-/-) Emu-Myc transgenic mice mainly develop lymphomas of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B-cell origin, which are uncommon in wild-type Emu-Myc transgenics. Furthermore, lymphomas from PLCgamma2(-/-) Emu-Myc transgenic mice exhibited a loss of p27Kip1 and often displayed alterations in Arf or p53. Thus, PLCgamma2 plays an important role in pre-BCR-mediated early B-cell development, and its deficiency leads to markedly increased pools of the most at-risk large pre-B cells, which display hyperresponsiveness to IL-7 and express high levels of RAGs, making them prone to secondary mutations and Myc-induced malignancy.     

Characterization of CD45 and CD45R monoclonal antibodies using transfected mouse cell lines that express individual human leukocyte common antigens

Five cell lines that express one of five isoforms of the human leukocyte common Ag (CD45) were established by transfecting a murine pre-B cell line with leukocyte common Ag cDNA constructs. Using these cell lines, the specificities of CD45 and CD45R mAb were examined. Of the 43 mAb tested, 25 antibodies recognized sequences common to all five isoforms, 16 antibodies recognized isoforms that include exon A encoded sequences, one antibody recognized isoforms that include exon B encoded sequences, and one antibody recognized only the isoform that does not include either exon A, B, or C encoded sequences.     

BP-3 alloantigen. A cell surface glycoprotein that marks early B lineage cells and mature myeloid lineage cells in mice

To explore the cell surface molecules expressed on pre-B cells we have produced a panel of alloantibodies against transformed pre-B cells from BALB/c mice by immunizing a wild mouse, Mus spretus. One of these antibodies, BP-3, recognized glycoproteins of Mr 38,000 to 48,000 on pre-B cells transformed either by the Abelson murine leukemia virus or an erb B oncogene construct. Removal of N-linked oligosaccharides from the BP-3 Ag revealed a single core protein of Mr 32,000. The Ag was expressed by bone marrow cells in all but one (A/J) of the inbred mouse strains tested and in wild mice of biochemical groups Mus-1 and Mus-2. Analysis of the tissue distribution revealed expression of the BP-3 reactive molecule on normal pre-B and B cells in the bone marrow, 35% of B cells in the circulation, 30% of the B cells in the spleen, and less than or equal to 20% of B cells in lymph nodes, peritoneal cavity, and Peyer's patches. The subpopulation of BP-3+ B cells in bone marrow and peripheral tissues displayed an immature phenotype (IgM IgD +/- ). Examination of a panel of transformed B lineage cells confirmed the early stage-specific expression of the BP-3 alloantigen. In addition, a myeloid cell line and normal myeloid cells were found to express the BP-3 alloantigen. In contrast to B lineage cells, the level of BP-3 expression increased as a function of myeloid cell differentiation. Myeloid cells in the bone marrow expressed relatively little Ag, whereas circulating neutrophils and peritoneal macrophages expressed relatively high levels of the BP-3 alloantigen with Mr 38,000, 41,000, and 46,000. The data suggest that this variably glycosylated cell surface protein could play different roles in the differentiation of B lineage and myeloid lineage cells. The BP-3 alloantigen appears to be a useful marker for virgin B cells that have recently migrated from the bone marrow to the periphery.     

B lymphopoiesis in the thymus

The thymus has been regarded as the major site of T cell differentiation. We find that in addition to alphabeta and gammadelta T cells, a significant number (approximately 3 x 104 per day) of B220+IgM+ mature B cells are exported from the thymus of C57BL/6 mice. Of these emigrating B cells, we estimate that at least approximately 2 x 104 per day are cells which developed intrathymically, whereas a maximum of approximately 0.8 x 104 per day are cells which circulated through the thymus from the periphery. The thymus possesses a significant number of pro-B and pre-B cells that express CD19, VpreB, lambda5, and pax-5. These B cell progenitors were found in the thymic cortex, whereas increasingly mature B cells were found in the corticomedullar and medullary regions. Other lymphoid cells, including NK cells and lymphoid dendritic cells, are not exported from the thymus at detectable levels. Thus, the thymus contributes to the formation of peripheral pools of B cells as well as of alphabeta and gammadelta T cells.     

Tyrosine phosphorylation of CD19 in pre-B and mature B cells.

Cross-linking of B cell surface immunoglobulins (sIg) results in activation of mature B cells and stimulates a molecular signaling mechanism for antigen-specific B cell expansion and differentiation. This signaling pathway is dependent on tyrosine (Tyr) phosphorylation and results in the activation of sIg-associated src family kinases and p72SYK. Rapid Tyr phosphorylation occurs on multiple protein substrates. Here we show that activation of B cells by cross-linking sIg results in an increase in Tyr phosphorylation of the lineage-restricted B cell surface antigen CD19, and show that it is a major substrate of activated Tyr kinase following sIg stimulation. Lower levels of constitutive CD19 Tyr phosphorylation occurred in most sIg+ mature B cell lines examined and in normal dense tonsillar B cells. We also find that when CD19 is Tyr-phosphorylated it becomes competent to interact with SH2 domains suggesting a mechanism whereby, following B cell activation, CD19 could be linked to intracellular signaling pathways. In sIg- pre-B cell lines, CD19 was expressed but was not constitutively phosphorylated on tyrosine. Upon CD19 cross-linking, Tyr phosphorylation of CD19 was induced in sIg- pre-B cell lines. CD19 cross-linking also directly induced Tyr phosphorylation of CD19 and other substrates in mature B cells. The ability of CD19 to signal in the absence of sIg expression may provide important stimulation in pre-B cell development.     

Expression, distribution and specificity of Fc receptors for IgM on murine B cells

Subpopulations of normal adult murine splenic B cells and a panel of murine B cell tumors were examined for their ability to bind murine IgM specifically. By using two-color flow cytometric analyses, we have demonstrated that 90 to 95% of surface (s)IgD+ B cells express surface membrane receptors for IgM (Fc mu R). The binding of pentameric murine IgM to splenocyte Fc mu R was IgM-specific since it was totally inhibited by other polymeric IgM proteins, but not by Ig of other H chain classes or by mAb specific for the murine IgG or IgE FcR. Binding of IgM to splenic cells was saturable. Fc mu R were co-expressed with the Fc gamma R as well as the Fc epsilon R on the majority of splenic B cells. Minor populations of splenic mononuclear cells expressed only an Fc mu R, Fc gamma R or Fc epsilon R. In a survey of B tumor cell lines representing different stages of B cell development, we observed that the Fc mu R was expressed on pre-B cell lines and that Fc mu R detection was maximal on immature B cell lines that expressed sIgM and low amounts of sIgD and Ia. Fc mu R were not detected on cell lines that had switched from sIgM to the expression of another sIg, or on plasmacytomas and hybridomas. The studies with normal splenocytes establish that the majority of sIgD+ B lymphocytes in adult BALB/c mice express surface membrane receptors that specifically bind IgM. The studies with B lineage tumor cells suggest that the expression of Fc mu R on B cells is developmentally regulated and that the pattern of expression exhibited by Fc mu R during B cell ontogeny differs from the patterns that have been previously found for IgG and IgE FcR. These observations raise the possibility that Fc mu R might have a functional significance in some aspect of B cell maturation and activation. By using a family of IgM H chain constant region domain deletional mutants, we have further demonstrated that, like the T cell Fc mu R, the B cell Fc mu R also requires a C mu 3 domain for binding to occur, raising the possibility that the T and B cell Fc mu R in mice may be structurally related molecules.     

Impaired precursor B cell differentiation in Bruton's tyrosine kinase-deficient mice

Bruton's tyrosine kinase (Btk) is a cytoplasmic signaling molecule that is crucial for precursor (pre-B) cell differentiation in humans. In this study, we show that during the transition of large cycling to small resting pre-B cells in the mouse, Btk-deficient cells failed to efficiently modulate the expression of CD43, surrogate L chain, CD2, and CD25. In an analysis of the kinetics of pre-B cell differentiation in vivo, Btk-deficient cells manifested a specific developmental delay within the small pre-B cell compartment of about 3 h, when compared with wild-type cells. Likewise, in in vitro bone marrow cultures, Btk-deficient large cycling pre-B cells showed increased IL-7 mediated expansion and reduced developmental progression into noncycling CD2(+)CD25(+) surrogate L chain-negative small pre-B cells and subsequently into Ig-positive B cells. Furthermore, the absence of Btk resulted in increased proliferative responses to IL-7 in recombination-activating gene-1-deficient pro-B cells. These findings identify a novel role for Btk in the regulation of the differentiation stage-specific modulation of IL-7 responsiveness in pro-B and pre-B cells. Moreover, our results show that Btk is critical for an efficient transit through the small pre-B cell compartment, thereby regulating cell surface phenotype changes during the developmental progression of cytoplasmic mu H chain expressing pre-B cells into immature IgM(+) B cells.