Address correspondence to Stephen Desiderio, Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, PCTB 701, Baltimore, MD 21205. Phone: (410) 955-4735; Fax: (410) 955-9124; email: sdesider{at}jhmi.edu
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Abstract |
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Key Words: B cell development signal transduction allelic exclusion V(D)J recombination Src kinases
At this developmental checkpoint, the µ chain associates with the chaperones VpreB and
Although the molecular details of signaling through the pre-BCR are poorly understood, genetic approaches have identified several critical components of the signaling machinery. In mice lacking Igµ, Igß,
The intracellular signaling events after appearance of the pre-BCR are not clearly defined, but several lines of evidence indicate a role for phosphorylation of Ig
The Src-related tyrosine kinases Blk, Lyn, and Fyn associate with the BCR, and current models of BCR or pre-BCR signaling propose that one or more of these Src family kinases, possibly in combination with Syk, participate in phosphorylation of the Ig
Although loss of function mutations have revealed a redundant requirement for Blk, Lyn, or Fyn in B cell development, gain of function mutations, by revealing the consequences of kinase activation, would be expected to provide additional mechanistic insight. To this end we asked whether a constitutively active form of Blk, the only member of the group expressed preferentially in B cells, could provide some or all of the functions associated with the pre-BCR. Our results indicate that in B cell progenitors, active Blk mimics several consequences of pre-BCR signaling.
Cell Culture.
For thymidine uptake assays, bone marrow cell suspensions from 34-wk-old mice were cultured with 10 ng/ml rIL-7 for 5 d, by which time the nonadherent cell population contained >95% B220+ cells, >80% of which were also CD43+. Triplicate samples of B220+ CD43+ cells were separated from dead cells by ficoll centrifugation, washed three times with PBS, and incubated with rIL-7 or media alone in 96-well plates at 105 cells per well. Thymidine incorporation was assayed after 3 d of culture after the addition of [3H]thymidine (1 µCi/well) 16 h before assay.
For isolation of RNA or protein, B220+ CD43+ cells were cultured as described above, but for 10 d in the presence of 10 ng/ml IL-7 added every 3 d of culture. Cell concentration was maintained below 106/ml.
Flow Cytometric Analysis.
V(D)J Recombination Assays.
Analysis of Protein Tyrosine Phosphorylation.
RNA Isolation and Analysis.
For RT-PCR, 1 µg total RNA was synthesized using reverse transcriptase (Superscript II; Invitrogen) and random hexameric primers. Reverse transcripts were amplified by PCR. Sequences of oligonucleotide primers are provided in Table S4, available at http://www.jem.org/cgi/content/full/jem.20030729/DC1.
Oligonucleotide Array Hybridization and Data Analysis.
Online Supplemental Material.
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Igµ heavy chain genes are assembled from discrete segments by V(D)J recombination, a process initiated by RAG-1 and RAG-2 (1). The joining of coding segments is random with respect to reading frame, and most primary products of V(D)J recombination are nonproductive. Heavy chain gene assembly begins in proB cells with the formation of DJH joints on both alleles. VH to DJH joining is then activated sequentially at the two alleles. Productive assembly of a heavy chain gene and expression of an intact µ chain marks the transition from the proB to the preB cell stage of development (2). 5 and is incorporated, with the accessory chains Ig
and Igß, into the preB cell receptor (BCR). The pre-BCR signals several cellular responses, including: (a) cessation of further VH to DJH joining, (b) increased sensitivity to IL-7, (c) cell proliferation, (d) suppression of apoptosis, (e) developmental progression, and (f) activation of rearrangement at the Ig
locus (for review see references 3 and 4). The combined proliferative and antiapoptotic pre-BCR signals contribute to the expansion of Igµ-expressing clones. Increased responsiveness to IL-7, by allowing proliferation and survival at diminished cytokine concentrations, may function in the positive selection of cells that have undergone productive heavy chain rearrangement and developmental progression to light chain rearrangement (4, 5).
5, the tyrosine kinase Syk, or the docking protein BLNK, B cell development beyond the preB cell stage is impaired, as evidenced by a marked reduction in the number of peripheral B cells and an increase in the proportion of B220+ CD43+ progenitor B cells in the bone marrow relative to the more mature B220+ CD43- cells (610).
and Igß. First, in RAG-deficient mice, cross-linking of Igß induces tyrosine phosphorylation of Ig
and Syk, as well as differentiation of proB cells to small preB cells (11). Second, in mice lacking the membrane-bound form of Igµ, Igß cross-linking suppresses heavy chain rearrangement and activates light chain rearrangement (12). Third, in mice lacking the cytoplasmic domain of Igß, development beyond the proB cell stage is dependent on the Ig
ITAM motifs (13). Fourth, pre-BCR cross-linking is associated with an increase in the amount of tyrosine-phosphorylated Igß that is associated with the pre-BCR in lipid rafts (14).
and Igß ITAM motifs (for review see reference 15). Blk and Lyn prefer similar consensus substrate sequences, distinct from that of Src and resembling sites in the Ig
and Igß ITAM motifs (16). Blk, Lyn, and Fyn play functionally redundant roles in supporting the proB to preB cell transition. Single or pairwise deficiencies of these or other Src-like kinases have little or no effect on early B cell development (1724). In contrast, the proB to preB cell transition is attenuated in triply mutant Blk-/- Lyn-/- Fyn-/- mice (25). Thus, any one of these three kinases is essential for effective pre-BCR signaling.
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Mice.
The transgenic line Blk(Y495F)-15 (B6 x SJL), bearing a cDNA encoding Blk(Y495F) under control of the H-2k promoter and the Igµ intronic enhancer, has been described (26). µMT mice (C57BL/6 background; reference 27) were obtained from The Jackson Laboratory. RAG-2deficient mice (129S6 background; reference 28) were obtained from Taconic Laboratories. Blk(Y495F) transgenic mice deficient in membrane-bound Igµ or in RAG-2 were generated by interbreeding Blk(Y495F) transgenic and µMT or RAG2-deficient mice, respectively.
Cells were maintained in RPMI 1640 supplemented with 10% FCS, 50 u/ml penicillin/streptomycin, 1 mM sodium pyruvate (Sigma-Aldrich), 2 mM L-glutamine (GIBCO BRL), 50 µM ß-mercaptoethanol, 10 mM Hepes, and 0.7 x MEM nonessential amino acids (GIBCO BRL) at 37°C in 5% CO2.
Bone marrow cells or splenocytes from 35-wk-old mice were analyzed on a FACScanTM instrument (Becton Dickinson). The following mAbs were obtained from BD Biosciences: FITC-conjugated anti-CD43 (S7 clone), antic-kit (CD117), anti-CD24 (HSA), antiBP-1, anti-CD22, and anti-CD2; PE-conjugated anti-CD43, antiBP-1, anti-CD22, anti-CD25, and anti-B220; and Cy3-conjugated anti-B220. FITC-conjugated anti-IgM and PE-conjugated anti-IgD were obtained from Southern Biotechnology Associates, Inc. Cells stained with PKH26 (Sigma-Aldrich) or annexin V (BD Biosciences) were counterstained with antiB220-Cy3 and antiCD43 (S7 clone)-FITC or antiBP-1 (BD Biosciences).
DNA from B220+ CD43- or unsorted bone marrow cells was assayed for rearrangement by PCR as previously described, using primers specific for the VH VJ558 family, for the DFL16 and DSP2 families, for V segments, for the JH3 region, and for the J
2 region (29). Products were detected by hybridization to 32P-labeled probes (30). The ligation-mediated PCR assay for signal end breaks was performed using the linker-specific primer BW-1 and one of the locus-specific primers µ02,
03, or DFL16.1B (31). Products were detected by hybridization to 32P-labeled probes specific for the germline Cµ0-JH3 region, the region 5' of DFL16.1, or the germline C
0-C
2 region.
Cells were lysed in a buffer containing 50 mM TrisCl, pH 8.0, 150 mM NaCl, 1% NP-40, 1% deoxycholic acid, 0.1% SDS, 1 mM NaVO3, 1 mM PMSF, and 10 µg each leupeptin, aprotinin, and pepstatin. Antibodies against Syk (Santa Cruz Biotechnology, Inc.) or CD79b (Southern Biotechnology Associates, Inc.) were affixed to protein A/G agarose. 10 µg antibody was incubated overnight with cell lysate (5 x 107 cell equivalents) at 4°C. Beads were collected by centrifugation and washed in lysis buffer. Immunoprecipitates were fractionated by SDS-PAGE and phosphotyrosine was detected by immunoblotting with antibody 4G10 (Upstate Biotechnology).
Total RNA was extracted from cell suspensions using the TRIzol reagent (Invitrogen). 1 µg total polyadenylated mRNA, isolated by adsorption to oligo-dTcoated beads (Oligotex; QIAGEN), was used as a template for synthesis of double stranded cDNA using reverse transcriptase (Superscript II; Invitrogen) and a T7-(dT)24 primer. Biotin-labeled cRNA probes for array hybridization were transcribed from cDNA templates using T7 RNA polymerase (Enzo Biochem).
Biotin-labeled cRNA probes were hybridized to oligonucleotide microarrays (mouse U74Av2; Affymetrix, Inc.) containing 12,488 probe sets. Transcripts that were scored by Affymetrix Microarray Suite as present on at least one array were analyzed using GeneSpring 4.0 (Silicon Genetics). The signal intensity of each probe set was normalized to the median value of all intensities measured in the corresponding array, and then further normalized to the median of all array-normalized intensities determined for that gene over all hybridizations. Three sets of genes were selected for further study. A gene was included in the first set if (a) its normalized expression in Blk(Y495F) transgenic samples deviated from expression in nontransgenic controls by at least twofold and (b) its expression deviated from that of the control samples with a significance cutoff of P < 0.01 (Welch's approximate t test). A gene was included in the second set if it was (a) absent from all control arrays but present in all transgenic arrays or (b) present in all control arrays but absent from all transgenic arrays. A gene was included in the third set if its normalized expression differed by more than fivefold between transgenic and nontransgenic samples. Hierarchal clustering was performed using the standard correlation coefficient as a distance metric.
Fig. S1 supplements Fig. 2 and shows the effect of Blk (Y495F) on expression of the B cell developmental markers CD24, CD25, CD2, and c-kit in RAG-deficient or µMt/µMT mice, as assessed by fluorescence cytometry. Table S1 shows the distribution of B220 CD43+ and B220 CD43- cells in the bone marrow of RAG-deficient or µMT/µMT mice expressing the Blk (Y495F) transgene. Table S2 shows the distribution of B220 IgM+ and B20 IgM- B cells in the spleens of Blk (Y495F) transgenic mice. Table S3 supplements Fig. 6 C and assigns the differently expressed genes of known function to functional categories. Table S4 depicts the primer pairs used for RT-PCR, whose results are shown in Fig. 6 D. Fig. S1 and Tables S1S4 are available at http://www.jem.org/cgi/content/full/jem.20030729/DC1.
Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Expansion of B Cell Progenitors in Bone Marrow of Blk(Y495F) Transgenic Mice.
We first considered whether active Blk might deliver proliferative signals independent of pre-BCR expression using a line of transgenic mice, Blk(Y495F)-15, in which a constitutively active Blk mutant is expressed specifically in the B lymphoid lineage (26). Bone marrow B lymphoid progenitors from 35-wk-old transgenic mice and nontransgenic littermates were examined. Blk(Y495F) transgenic and nontransgenic mice showed similar bone marrow cellularity. Transgenic mice, however, exhibited a slight increase in the percentage of B220+ bone marrow cells (Table I).
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Increased IL-7 Responsiveness of B Cell Progenitors from Blk(Y495F) Transgenic Mice.
The accumulation of B220+ CD43int cells in the bone marrow suggested that Blk(Y495F) had stimulated cellular proliferation. To examine this, bone marrow cells were loaded ex vivo with PKH-26 and cultured with IL-7 at 20 ng/ml in the presence of autologous stromal cells. After 3 d, the PKH-26 signal remained undiluted in 22 and 69% of the nontransgenic B220+ CD43+ and B220+ CD43- populations, respectively (Fig. 1 B). In contrast, only 8% of viable B220+ CD43int cells from Blk(Y495F) mice had not undergone cell division by 3 d, as evidenced by PKH-26 fluorescence (Fig. 1 B). Gating of transgenic samples on B220+ CD43+ and B220+ CD43- populations yielded similar results, as expected because the majority of B220+ cells in transgenic bone marrow are homogeneously B220+ CD43int (unpublished data).
To determine whether the Blk(Y495F) transgene could confer hypersensitivity to IL-7, we cultured bone marrow from transgenic or nontransgenic mice for 5 d under conditions that favor outgrowth of B220+ CD43+ cells. Nonadherent cells were stimulated with increasing amounts of IL-7 in the absence of stromal cells. Thymidine incorporation was measured after 3 d of restimulation (Fig. 1 C). The maximal proliferative response of cells from Blk(Y495F) transgenic mice was more than twice that of cells from nontransgenic animals (17,457 ± 1,501 cpm vs. 8,434 ± 2,079 cpm) and we observed a shift in IL-7 sensitivity. Cells from transgenic mice responded to IL-7 at concentrations as low as 20 pg/ml, whereas cells from nontransgenic animals required a 10-fold higher concentration for a similar response. In this respect, proB cells from Blk(Y495F) transgenic mice resembled µ heavy chain transgenic proB cells, which also exhibit a lower threshold for responsiveness to IL-7 (5).
We asked whether differences in the frequency of apoptosis might contribute to the outgrowth of B cell progenitors in the transgenic animals. In freshly isolated B lymphoid progenitors from bone marrow of Blk(Y495F) transgenic mice, we observed slight decreases in the apoptotic fraction, as defined by annexin V staining, relative to wild-type. This held whether we gated on B220+ CD43+ (16.1 ± 1.5 in transgenic vs. 22.8 ± 2.7 in wild-type) or B220+ CD43- (15.8 ± 1.6 in transgenic vs. 18.1 ± 1.4 in wild-type) cells. These observations suggest that the accumulation of B cell progenitors expressing Blk(Y495F) results primarily from increased proliferation.
Constitutively Active Blk Overcomes Developmental Blocks in RAG-2-/- and µMT/µMT Mice.
In mice lacking RAG-2, B lymphoid development is arrested at the CD43+ proB cell stage (Fig. 2
A; reference 28). This block can be overcome by introduction of a µ transgene (34). To test the ability of the Blk(Y495F) mutant to bypass the requirement for µ heavy chain in signaling the proB to preB cell transition, Blk(Y495F) transgenic animals were crossed with RAG-2deficient mice. A B220+ CD43int population emerged in the bone marrow of RAG-2deficient mice bearing the Blk(Y495F) transgene, indicating developmental progression beyond the CD43+ proB cell stage (Fig. 2 A and Fig. S1 and Table S1, which are available at http://www.jem.org/cgi/content/full/jem.20030729/DC1). The appearance of the differentiation markers BP-1 and CD22, increased expression of CD24 (HSA) and CD2, and decreased expression of c-kit were all consistent with this interpretation (Fig. 2 A and Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20030729/DC1). Therefore, these cells resembled the expanded CD43int population we observed in Blk(Y495F) RAG-2+/+ mice.
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Initiation of Rearrangement in Blk(Y495F) Transgenic µMT/µMT Mice.
Light chain rearrangement is suppressed in homozygous µMT mice and productive rearrangement of a µMT allele fails to enforce allelic exclusion (35). To determine whether active Blk could drive developmental progression to rearrangement in the absence of a pre-BCR, VHDJH, DJH, and V
J
rearrangements were assayed in the bone marrow of Blk(Y495F) transgenic µMT/µMT mice and nontransgenic µMT/µMT littermates at 4 wk of age (Fig. 3
A). In Blk(Y495F) transgenic mice, a striking increase in completed V
J
rearrangement was observed (Fig. 3 A, top).
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Relief of Selection for Functional Rearrangement at the Heavy Chain Locus in Blk(Y495F) Transgenic Mice.
B cell progenitors that do not assemble a functional heavy chain gene are eliminated by apoptosis. We wished to test whether the Blk(Y495F) transgene relieves selection for functional heavy chain gene rearrangement at this checkpoint. VHDJH joints were amplified by PCR from sorted B220+ CD43- bone marrow cells of three 34-wk-old Blk(Y495F) transgenic mice and three nontransgenic littermates (Fig. 4)
. Of 42 VHJ558-D-JH3 rearrangements obtained from nontransgenic mice, 6 (14.3%) were found to be nonproductive, whereas of 45 VHJ558-D-JH3 rearrangements isolated from Blk(Y495F) transgenic mice, 30 (66.7%) were nonproductive. The increased representation of nonproductive rearrangements in transgenic mice is highly significant (P < 0.000001) and approximates the level expected for random rearrangement in the absence of selection. Similar results were obtained when rearrangements were amplified from unsorted bone marrow cells (unpublished data). These results are consistent with the interpretation that constitutively active Blk relieves the selection for functional heavy chain rearrangement at the pro-B to pre-B transition by enabling cellular survival in the absence of the pre-BCR.
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Of the genes assayed, 51 were scored as differentially expressed and of these, the 35 genes of known or inferred function (Fig. 6 C) were assigned to 9 categories (Table S3, available at http://www.jem.org/cgi/content/full/jem.20030729/DC1) as defined by the Gene Ontology Consortium (www.geneontology.org). More than one third (13/35) of these genes encode markers or regulators of B lymphoid development. Those up-regulated in transgenic proB cells include CD22, CD20, Siat1 (siat1; reference 41), SHP-1 (hcp; reference 42), CCR7 (ccr7; reference 43), and Ig, as well as Irf-4 (lsirf; pip), which stimulates germline Ig
transcription (44), Mef2C (mef2c), which stimulates expression of J chain (45), and CstF1 (cstf1), part of an RNA processing complex that generates Igµ secretory transcripts (46). Consistent with the ability of Blk(Y495F) to suppress heavy chain rearrangement, VH transcripts (IgH V) were diminished in transgenic B220+ CD43+ cells. Down-regulation of transcripts for the prostaglandin E2 receptor (ptgerep4), a positive regulator of apoptosis in B lymphoid cells (47), and the IL-3 receptor (il3r) are also in agreement with the maturation-promoting effects of active Blk. The results obtained by microarray were confirmed by RT-PCR for five markers that were overexpressed (ccr7, CD22, irf4, gem, and ifi203) and four markers that were underexpressed (clf1, semB, tnfc, and il3r) in transgenic cells (Fig. 6 D).
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IL-7 may limit expansion of B cell progenitors in the bone marrow. Exogenous IL-7 provokes an overexpansion of B cell precursors (48), whereas in IL-7deficient mice the transition from the pro-B to pre-B stage is partially impaired (49). Assembly of a pre-BCR is associated with increased responsiveness to IL-7 (50), perhaps reflecting convergence of pre-BCR and IL-7 signals at the level of MAP kinase activation (5). A similar increase in IL-7 sensitivity was seen in progenitor B cells from Blk(Y495F) transgenic mice. Increased responsiveness of pre-BCRexpressing cells to IL-7 would present a selective growth advantage when the availability of IL-7 is reduced, as may occur in some stromal microenvironments (4). The increase in IL-7 sensitivity conferred by Blk(Y495F) may contribute to the overrepresentation of B cell progenitors and the apparent lack of selection for µ heavy chain expression observed in Blk(Y495F) transgenic mice. As BP-1 is induced by IL-7 (51), increased sensitivity to this lymphokine may in part explain the BP-1high phenotype of B220+ CD43int transgenic B progenitors.
Expression of active Blk in the B lineage of µMT/µMT or RAG-2-/- mice advances development past the blocks induced by these deficiencies. This is evident from decreased expression of CD43 and c-kit, increased expression of CD24, and appearance of BP-1, CD2, and CD22. This action of Blk resembles the effects of transgenic µ heavy chain (33, 52) or cross-linking of Igß (11), both of which support differentiation of RAG-deficient proB cells to preB cells, with concomitant down-regulation of c-kit and CD43 and increased expression of CD24 and CD2.
In several ways, however, Blk(Y495F) and µ heavy chain transgenes differ in the extent to which they support B cell development. In the RAG-deficient setting, µ heavy chain drives the emergence of B220+ CD43- cells (33, 34, 52), whereas active Blk supports accumulation of a B220+ CD43int population. Moreover, CD25, a marker characteristic of preB II cells (36), is acquired by RAG-deficient B progenitors upon the introduction of µ heavy chain (33) or cross-linking of Igß (11), but not in the presence of Blk(Y495F). Lastly, BP-1, which is induced upon cross-linking of Igß on RAG-deficient proB cells (11), is expressed in the predominant B progenitor population in Blk(Y495F) transgenic RAG-2-/- mice but not in the progenitors that accumulate in µ transgenic RAG-/- animals (11).
Thus, the predominant B progenitor phenotype in Blk(Y495F) transgenic RAG-deficient animals is phenotypically identical to the B progenitor population that is expanded in recombination-competent, Blk(Y495F) transgenic mice, but less mature than the most advanced progenitors observed in µ transgenic RAG-deficient mice. This distinction suggests that the pre-BCR delivers additional signals, perhaps supplied by activation of other BCR-associated kinases, which effect further developmental progression.
The pre-BCR stimulates gene rearrangement and suppresses heavy chain rearrangement. A functional pre-BCR, however, is not essential for activation of light chain rearrangement, which occurs at a low level in the absence of membrane-bound µ chain or
5 (35, 53). Nonetheless, in bone marrow B cell precursors from µMT/µMT mice, Ig light chain gene rearrangement is attenuated and the incidence of specific DNA cleavage at the
locus is greatly reduced (27, 35). Cross-linking Igß reverses this attenuation and suppresses ongoing V(D)J rearrangement at the heavy chain locus (12).
With respect to V(D)J recombination, the effects of Blk(Y495F) in a µMT/µMT background are similar to those of Igß cross-linking. In bone marrow B lineage cells, rearrangement is stimulated, whereas the yield of VH to DJH recombination intermediates is reduced. The effects of the Blk(Y495F) transgene on heavy and light chain rearrangement are likely not related to increased IL-7 sensitivity, which would have been expected to promote VH to DJH recombination and suppress
rearrangement (54). In the T lineage, Lck can supply functions associated with the pre-TCR, including suppression of Vß to DßJß rearrangement and promotion of TCR-
rearrangement (55). An active Ras transgene promotes TCR-
rearrangement but fails to stimulate allelic exclusion at the TCR-ß locus (56), suggesting that the ability of Lck to enforce allelic exclusion at the TCR-ß locus is not exerted through Ras. The ability of activated Ras to induce
rearrangement in JH-deficient mice (39) raises the possibility that Ras mediates the stimulatory effect of Blk on
rearrangement.
Expression of Blk(Y495F) was associated with constitutive tyrosine phosphorylation of Igß and Syk, suggesting that the most proximal sequels of pre-BCR signaling are mimicked by Blk activation. A comparison of gene expression in Blk(Y495F) transgenic and nontransgenic B cell progenitors was used to identify direct or indirect targets of pre-BCR signaling. Fewer than 1% of expressed markers exhibited significant differences in levels of expression. Of the 21 annotated genes whose expression increased in transgenic cells, markers associated with development beyond the pro-B stage were disproportionately represented (>41%), validating the expression screen and providing further evidence that active Blk promotes developmental progression.
A recent report demonstrates that mice triply deficient in Blk, Lyn, and Fyn suffer an attenuation of the proB to preB cell transition, accompanied by deficiencies in tyrosine phosphorylation of PKC and activation of nuclear factor (NF)-
B (25). In these animals the leakiness of the developmental block, as well as intact tyrosine phosphorylation of Ig
/Igß and Syk, may reflect the action of residual tyrosine kinases such as Hck, Fgr, and Lck. In this light, our studies of Blk(Y495F) transgenic animals are consistent with and complementary to those obtained with the triple mutant mice.
Impaired activation of the p50-p65 NF-B heterodimer by the pre-BCR (25) seems unlikely to account for the developmental defect seen in animals lacking Blk, Lyn, and Fyn because B cell development is unimpaired in mice deficient in p50 or p65 (RelA; references 57 and 58). NF-
B was similarly active in nontransgenic and Blk(Y495F) transgenic proB cells (unpublished data), although it remains possible that differences in NF-
B activity were masked by the conditions of ex vivo culture. Nonetheless, differential NF-
B activity is not essential for maintaining the differences in proliferation and developmental maturity that we observed between transgenic and nontransgenic cell populations.
Despite their partial redundancy in supporting the pro-B to pre-B transition, the functions of Blk may differ in detail from those of other Src-related kinases expressed in B lymphoid cells. For example, a constitutively active form of Lyn, unlike the Blk(Y495F) mutant, affects neither the proliferation of B progenitors nor their responsiveness to IL-7 (18). Although such differences are consistent with nonequivalent roles for Blk and other Src-related kinases in early B lymphoid development, the ability of Blk, Lyn, or Fyn to sustain B cell development in the absence of the other two kinases suggests considerable functional overlap.
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Acknowledgments |
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This work was supported by the Howard Hughes Medical Institute and by grant CA16519 from the National Cancer Institute. T. Tretter is a postdoctoral fellow of the Deutsche Forschungsgemeinschaft and A.E. Ross is a predoctoral fellow of the Medical Scientist Training Program of the National Institutes of Health.
Submitted: May 5, 2003
Accepted: October 8, 2003
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Footnotes |
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The online version of this article contains supplemental material.
Abbreviations used in this paper: BCR, B cell receptor; NF, nuclear factor.
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