During B lymphoid ontogeny, assembly of the pre–B cell receptor (BCR) is a principal developmental checkpoint at which several Src-related kinases may play redundant roles. Here the Src-related kinase Blk is shown to effect functions associated with the pre-BCR. B lymphoid expression of an active Blk mutant caused proliferation of B progenitor cells and enhanced responsiveness of these cells to interleukin 7. In mice lacking a functional pre-BCR, active Blk supported maturation beyond the pro–B cell stage, suppressed VH to DJH rearrangement, relieved selection for productive heavy chain rearrangement, and stimulated κ rearrangement. These alterations were accompanied by tyrosine phosphorylation of immunoglobulin β and Syk, as well as changes in gene expression consistent with developmental maturation. Thus, sustained activation of Blk induces responses normally associated with the pre-BCR.

Introduction

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 pro–B 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 pro–B to the pre–B cell stage of development (2).

At this developmental checkpoint, the μ chain associates with the chaperones VpreB and λ5 and is incorporated, with the accessory chains Igα and Igβ, into the pre–B 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 Igκ 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).

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, Igβ, λ5, the tyrosine kinase Syk, or the docking protein BLNK, B cell development beyond the pre–B 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).

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 Igα and Igβ. First, in RAG-deficient mice, cross-linking of Igβ induces tyrosine phosphorylation of Igα and Syk, as well as differentiation of pro–B cells to small pre–B cells (11). Second, in mice lacking the membrane-bound form of Igμ, Igβ cross-linking suppresses heavy chain rearrangement and activates light chain rearrangement (12). Third, in mice lacking the cytoplasmic domain of Igβ, development beyond the pro–B cell stage is dependent on the Igα ITAM motifs (13). Fourth, pre-BCR cross-linking is associated with an increase in the amount of tyrosine-phosphorylated Igβ that is associated with the pre-BCR in lipid rafts (14).

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 Igα and Igβ 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 Igα and Igβ ITAM motifs (16). Blk, Lyn, and Fyn play functionally redundant roles in supporting the pro–B to pre–B 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 pro–B to pre–B 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.

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.

Materials And Methods

Mice.

The transgenic line Blk(Y495F)-15 (B6 × 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-2–deficient 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.

Cell Culture.

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 β-mercaptoethanol, 10 mM Hepes, and 0.7 × MEM nonessential amino acids (GIBCO BRL) at 37°C in 5% CO2.

For thymidine uptake assays, bone marrow cell suspensions from 3–4-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.

Bone marrow cells or splenocytes from 3–5-wk-old mice were analyzed on a FACScan™ instrument (Becton Dickinson). The following mAbs were obtained from BD Biosciences: FITC-conjugated anti-CD43 (S7 clone), anti–c-kit (CD117), anti-CD24 (HSA), anti–BP-1, anti-CD22, and anti-CD2; PE-conjugated anti-CD43, anti–BP-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 anti–B220-Cy3 and anti–CD43 (S7 clone)-FITC or anti–BP-1 (BD Biosciences).

V(D)J Recombination Assays.

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 Vκ segments, for the JH3 region, and for the Jκ2 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, κ03, 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 Cκ0-Cκ2 region.

Analysis of Protein Tyrosine Phosphorylation.

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 × 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).

RNA Isolation and Analysis.

Total RNA was extracted from cell suspensions using the TRIzol reagent (Invitrogen). 1 μg total polyadenylated mRNA, isolated by adsorption to oligo-dT–coated 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).

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.

Oligonucleotide Array Hybridization and Data Analysis.

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.

Online Supplemental Material.

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 S1–S4 are available.

Results

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 3–5-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)

.

Phenotypic analysis revealed an expanded subset of B220+ bone marrow cells with a CD43-intermediate (CD43int) phenotype, reminiscent of cells at the pro–B to pre–B cell transition (Fig. 1 

A and Table I). The CD43int population was homogeneously BP-1high and CD22int and comprised 74.1 ± 6.2% of the B220+ bone marrow compartment. In contrast, B220+ CD43+ and B220+ CD43 cells comprised 24.5 ± 1.7% and 75.5 ± 9.5%, respectively, of the B220+ bone marrow compartment in nontransgenic mice. The transgenic animals also exhibited decreases in pro–B cells (B220+ CD43+ CD22lo/−) and late pre–B cells (B220+ CD43 CD22lo IgM; reference 32). In addition, the B220+ IgM+ population was relatively diminished in transgenic mice. With respect to their CD22int phenotype, these cells resemble a B progenitor population that accumulates in RAG-deficient mice bearing a μ heavy chain transgene and is expanded upon coexpression of Bcl-2 (33).

The distribution of DJH rearrangements from bone marrow cells was consistent with a polyclonal expansion of B cell progenitors in Blk(Y495F) transgenic mice (not depicted). The polyclonality of the B cell expansion in the 3–5-wk-old transgenic mice stands in contrast to the clonality of the B220+ CD43+ tumors that arise in these animals after a protracted latency period of 6–12 mo (26).

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, pro–B cells from Blk(Y495F) transgenic mice resembled μ heavy chain transgenic pro–B 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+ pro–B 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 pro–B to pre–B cell transition, Blk(Y495F) transgenic animals were crossed with RAG-2–deficient mice. A B220+ CD43int population emerged in the bone marrow of RAG-2–deficient mice bearing the Blk(Y495F) transgene, indicating developmental progression beyond the CD43+ pro–B cell stage (Fig. 2 A and Fig. S1 and Table S1, which are available). 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). Therefore, these cells resembled the expanded CD43int population we observed in Blk(Y495F) RAG-2+/+ mice.

We then asked whether Blk(Y495F) could support progression to light chain gene rearrangement in the absence of the pre-BCR. To do so, we crossed transgenic animals with μMT/μMT mice (27), in which the pre-BCR is not assembled because the μ transmembrane region is absent. In μMT/μMT mice B lymphoid development is blocked at the pro–B cell stage, although the V(D)J recombination machinery is intact (Fig. 2 B; references 27 and 35). Expression of Blk(Y495F) on the μMT/μMT background supported developmental progression beyond the block imposed by the absence of functional Ig heavy chain, as evidenced by decreased expression of CD43 and increased expression of BP-1 and CD22 (Fig. 2 B and Table S1). Thus, the more mature B progenitor population that emerged when μMT/μMT mice were supplemented with the Blk(Y495F) transgene was similar to the one that accumulated when the transgene was expressed on a wild-type or RAG-deficient background. Moreover, as we observed on the RAG-deficient background, the late pre–B cell marker CD25 (36) was not expressed in the B220+ CD43int population from Blk(Y495F) transgenic μMT/μMT mice (Fig. S1), despite the ability of B progenitors from these animals to advance to κ light chain rearrangement (see below).

Initiation of κ 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 κ rearrangement in the absence of a pre-BCR, VHDJH, DJH, and VκJκ 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 VκJκ rearrangement was observed (Fig. 3 A, top).

Detection of completed rearrangements, however, is not a direct indicator of recombinase activity at a given locus because differences in the frequency of rearrangement can be masked by differential outgrowth and survival of cells after completion of recombination. Indeed, when we examined completed VHDJH and DJH rearrangements we observed no significant difference between transgenic and wild-type bone marrow (Fig. 3 A, second and third panels from top). To evaluate recombinase activity at κ and heavy chain loci directly, we assayed signal end recombination intermediates, an indicator of ongoing V(D)J rearrangement, by ligation-mediated PCR. In the bone marrow of Blk(Y495F) transgenic μMT/μMT mice, double strand DNA breaks at the Jκ2 recombination signal, which indicate ongoing Vκ to Jκ rearrangement, were at least 25 times more abundant than in nontransgenic μMT/μMT littermates (Fig. 3 B, middle). In contrast, recombination signal ends associated with DFL16, which indicate continuing VH to DJH rearrangement, were at least five times less abundant in transgenic μMT/μMT animals than in nontransgenic μMT/μMT littermates (Fig. 3 B, top). Taken together, then, these observations provide direct evidence that the Blk(Y495F) transgene mimics the ability of the pre-BCR to activate Vκ to Jκ rearrangement and suppress VH to DJH rearrangement.

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 3–4-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.

Peripheral Accumulation of IgM B Lymphoid Progenitors in Blk(Y495F) Transgenic Mice.

B cell progenitors can accumulate in secondary lymphoid tissues under conditions of stress or polyclonal activation (37, 38), as well as in RAG- or heavy chain–deficient mice expressing activated Ras in the B lymphoid compartment (39, 40). This also occurred when Blk(Y495F) was expressed in the B lineage. Although overall splenic structure and cellularity were not significantly affected by the transgene, a substantial decrease in the percentage of B220+ cells expressing IgM was seen in spleens of 3–5-wk-old transgenic animals relative to nontransgenic littermates (16.9 ± 2.4 in transgenic animals, 51.6 ± 1.7 in nontransgenic controls; Fig. 5 

A and Table S2). Most remaining B lymphoid cells were B220lo, CD43int, CD22int, and BP-1high, suggesting that they were derived from the B progenitor population that accumulates in bone marrow. Consistent with this interpretation, splenic RNA from transgenic mice contained transcripts corresponding to RAG-2, TdT, VpreB (Fig. 5 B), and λ5 (not depicted), whose expression is characteristic of lymphoid progenitors. The B progenitors found in the spleens of young transgenic mice were polyclonal (unpublished data), in contrast to the clonal tumors that arise in these mice after 6–12 mo. These observations suggest that in Blk(Y495F) transgenic mice, B cell progenitors emigrate to peripheral lymphoid organs in the absence of BCR expression.

Consequences of Blk Activation.

The ability of Blk- (Y495F) to mimic multiple pre-BCR signals suggested a point of action near proximal targets of pre-BCR signaling. Accordingly, basal tyrosine phosphorylation of Igβ (Fig. 6 

A) and Syk (Fig. 6 B) were substantially increased in pro–B cells from transgenic mice relative to pro–B cells from nontransgenic animals.

The availability of matched, pro–B cell populations allowed us to examine the effects of Blk activation on gene expression in B lymphoid progenitors. Bone marrow cells from transgenic and nontransgenic animals were maintained with IL-7 for 10 d to obtain B220+ CD43+ cells (>98% purity), from which RNA was isolated. Preparations from three mice of each group were pooled and probes from pooled RNA samples were hybridized to arrays representing 12,488 markers. Five replicate hybridizations were performed.

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) 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 pro–B cells include CD22, CD20, Siat1 (siat1; reference 41), SHP-1 (hcp; reference 42), CCR7 (ccr7; reference 43), and Igδ, as well as Irf-4 (lsirf; pip), which stimulates germline Igκ 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).

Discussion

We have shown that a constitutively active form of Blk, expressed in the B lineage at a level comparable to that of the endogenous protein, effects a suite of responses normally stimulated by productive rearrangement and expression of the μ heavy chain, including: (a) increased proliferation of CD43int B progenitor cells, (b) enhanced responsiveness of these cells to IL-7, (c) maturation as reflected by changes in phenotype, (d) suppression of VH to DJH rearrangement, and (e) initiation of κ rearrangement.

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-7–deficient 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-BCR–expressing 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 Igβ (11), both of which support differentiation of RAG-deficient pro–B cells to pre–B 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 pre–B II cells (36), is acquired by RAG-deficient B progenitors upon the introduction of μ heavy chain (33) or cross-linking of Igβ (11), but not in the presence of Blk(Y495F). Lastly, BP-1, which is induced upon cross-linking of Igβ on RAG-deficient pro–B 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 κ 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 λ5 (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 κ locus is greatly reduced (27, 35). Cross-linking Igβ 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 Igβ cross-linking. In bone marrow B lineage cells, κ 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 κ rearrangement (54). In the T lineage, Lck can supply functions associated with the pre-TCR, including suppression of Vβ to DβJβ rearrangement and promotion of TCR-α rearrangement (55). An active Ras transgene promotes TCR-α rearrangement but fails to stimulate allelic exclusion at the TCR-β locus (56), suggesting that the ability of Lck to enforce allelic exclusion at the TCR-β locus is not exerted through Ras. The ability of activated Ras to induce κ rearrangement in JH-deficient mice (39) raises the possibility that Ras mediates the stimulatory effect of Blk on κ rearrangement.

Expression of Blk(Y495F) was associated with constitutive tyrosine phosphorylation of Igβ 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 pro–B to pre–B cell transition, accompanied by deficiencies in tyrosine phosphorylation of PKCλ and activation of nuclear factor (NF)-κB (25). In these animals the leakiness of the developmental block, as well as intact tyrosine phosphorylation of Igα/Igβ 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-κB 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-κB was similarly active in nontransgenic and Blk(Y495F) transgenic pro–B cells (unpublished data), although it remains possible that differences in NF-κB activity were masked by the conditions of ex vivo culture. Nonetheless, differential NF-κB 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.

Acknowledgments

We thank Sasha Tarakhovsky for sharing results before publication.

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.

References

References
1
Gellert, M.
2002
. V(D)J recombination: RAG proteins, repair factors and regulation.
Annu. Rev. Biochem.
71
:
101
–132.
2
Melchers, F., E. ten Boekel, T. Seidl, X.C. Kong, T. Yamagami, K. Onishi, T. Shimizu, A.G. Rolink, and J. Andersson.
2000
. Repertoire selection by pre-B-cell receptors and B-cell receptors, and genetic control of B-cell development from immature to mature B cells.
Immunol. Rev.
175
:
33
–46.
3
Reth, M., and J. Wienands.
1997
. Initiation and processing of signals from the B cell antigen receptor.
Annu. Rev. Immunol.
15
:
453
–479.
4
Stoddart, A., H.E. Fleming, and C.J. Paige.
2000
. The role of the preBCR, the interleukin-7 receptor, and homotypic interactions during B-cell development.
Immunol. Rev.
175
:
47
–58.
5
Fleming, H.E., and C.J. Paige.
2001
. Pre-B cell receptor signaling mediates selective response to IL-7 at the pro-B to pre-B cell transition via an ERK/MAP kinase-dependent pathway.
Immunity.
15
:
521
–531.
6
Rajewsky, K.
1996
. Clonal selection and learning in the antibody system.
Nature.
381
:
751
–758.
7
Cheng, A.M., R.B. Rowley, W. Pao, A. Hayday, J.B. Bolen, and T. Pawson.
1995
. Syk tyrosine kinase required for mouse viability and B cell development.
Nature.
378
:
303
–306.
8
Turner, M., P.J. Mee, P.S. Costello, O. Williams, A.A. Price, L.P. Duddy, M.T. Furlong, R.L. Geahlen, and V.L.J. Tybulewicz.
1995
. Perinatal lethality and blocked B cell development in mice lacking the tyrosine kinase Syk.
Nature.
378
:
298
–302.
9
Jumaa, H., B. Wollscheid, M. Mitterer, J. Wienands, M. Reth, and R.J. Nielsen.
1999
. Abnormal development and function of B lymphocytes in mice deficient for the signaling adaptor protein SLP-65.
Immunity.
11
:
547
–554.
10
Pappu, R., A.M. Cheng, B. Li, Q. Gong, C. Chiu, N. Griffin, M. White, B.P. Sleckman, and A.C. Chan.
1999
. Requirement for B cell linker protein (BLNK) in B cell development.
Science.
286
:
1949
–1954.
11
Nagata, K., T. Nakamura, F. Kitamura, S. Kuramochi, S. Taki, K.S. Campbell, and H. Karasuyama.
1997
. The Ig alpha/Ig beta heterodimer on mu-negative proB cells is competent for transducing signals to induce early B cell differentiation.
Immunity.
7
:
559
–570.
12
Maki, K., K. Nagata, F. Kitamura, T. Takemori, and H. Karasuyama.
2000
. Immunoglobulin β signaling regulates locus accessibility for ordered immunoglobulin gene rearrangements.
J. Exp. Med.
191
:
1333
–1340.
13
Kraus, M., L.I. Pai, A. Reichlin, Y. Hu, B. Canono, J.C. Cambier, M.C. Nussenzweig, and K. Rajewsky.
2001
. Interference with immunoglobulin (Ig)α immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation modulates or blocks B cell development, depending on the availability of the Igβ cytoplasmic tail.
J. Exp. Med.
194
:
455
–469.
14
Guo, B., R.M. Kato, M. Garcia-Lloret, M. Wahl, and D.J. Rawlings.
2000
. Engagement of the human pre-B cell receptor generates a lipid raft-dependent calcium signaling complex.
Immunity.
13
:
243
–253.
15
DeFranco, A.L.
1997
. The complexity of signaling pathways activated by the BCR.
Curr. Opin. Immunol.
9
:
296
–308.
16
Schmitz, R., G. Baumann, and H. Gram.
1996
. Catalytic specificity of phosphotyrosine kinases Blk, Lyn, c-Src and Syk as assessed by phage display.
J. Mol. Biol.
260
:
664
–677.
17
Texido, G., I.-H. Su, I. Mecklenbräuker, K. Saijo, S.N. Malek, S. Desiderio, K. Rajewsky, and A. Tarakhovsky.
2000
. The B-cell-specific src-family kinase Blk is dispensable for B-cell development and activation.
Mol. Cell. Biol.
20
:
1227
–1233.
18
Hibbs, M.L., K.W. Harder, J. Armes, N. Kountouri, C. Quillici, F. Casagranda, A.R. Dunn, and D.M. Tarlinton.
2002
. Sustained activation of Lyn tyrosine kinase in vivo leads to autoimmunity.
J. Exp. Med.
196
:
1593
–1604.
19
Nishizumi, H., I. Taniuchi, Y. Yamanashi, D. Kitamura, D. Ilic, S. Mori, T. Watanabe, and T. Yamamoto.
1995
. Impaired proliferation of peripheral B cells and indication of autoimmune disease in lyn-deficient mice.
Immunity.
3
:
549
–560.
20
Stein, P.L., H.-M. Lee, S. Rich, and P. Soriano.
1992
. pp59fynmutant mice display differential signaling in thymocytes and peripheral T cells.
Cell.
70
:
741
–750.
21
Yasue, T., H. Nishizumi, S. Aizawa, T. Yamamoto, K. Miyake, C. Mizoguchi, S. Uehara, Y. Kikuchi, and K. Takatsu.
1997
. A critical role of Lyn and Fyn for B cel responses to CD38 ligation and interleukin 5.
Proc. Natl. Acad. Sci. USA.
94
:
10307
–10312.
22
Horikawa, K., H. Nishizumi, H. Umemori, S. Aizawa, K. Takatsu, and T. Yamamoto.
1999
. Distinctive roles of Fyn and Lyn in IgD- and IgM-mediated signaling.
Int. Immunol.
11
:
1441
–1449.
23
Meng, F., and C.A. Lowell.
1997
. Lipipolysaccharide (LPS)-induced macrophage activation and signal transduction in the absence of Src-family kinases Hck, Fgr, and Lyn.
J. Exp. Med.
185
:
1661
–1670.
24
Lowell, C.A., P. Soriano, and H.E. Varmus.
1994
. Functional overlap in the src gene family: inactivation of hck and fgr impairs natural immunity.
Genes Dev.
8
:
387
–398.
25
Saijo, K., C. Schmedt, I.-H. Su, H. Karasuyama, C.A. Lowell, M. Reth, T. Adachi, A. Patke, A. Santana, and A. Tarakhovsky.
2003
. Essential role of Src-family protein tyrosine kinases in NF-κB activation during B cell development.
Nat. Immunol.
4
:
274
–279.
26
Malek, S.N., D.I. Dordai, J. Reim, H. Dintzis, and S. Desiderio.
1998
. Malignant transformation of early lymphoid progenitors in mice expressing an activated Blk tyrosine kinase.
Proc. Natl. Acad. Sci. USA.
95
:
7351
–7356.
27
Kitamura, D., J. Roes, R. Kühn, and K. Rajewsky.
1991
. A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin μ chain gene.
Nature.
350
:
423
–426.
28
Shinkai, Y., G. Rathbun, K.-P. Lam, E.M. Oltz, V. Stewart, Y. Mendelsonn, J. Charron, M. Datta, F. Young, A.M. Stall, et al.
1992
. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement.
Cell.
68
:
855
–867.
29
Schlissel, M.S., L.M. Corcoran, and D. Baltimore.
1991
. Virus-transformed pre–B cells show ordered activation but not inactivation of immunoglobulin gene rearrangement and transcription.
J. Exp. Med.
173
:
711
–720.
30
Lewis, S., N. Rosenberg, F. Alt, and D. Baltimore.
1982
. Continuing kappa-gene rearrangement in a cell line transformed by Abelson murine leukemia virus.
Cell.
30
:
807
–816.
31
Schlissel, M., A. Constantinescu, T. Morrow, M. Baxter, and A. Peng.
1993
. Double-strand signal sequence breaks in V(D)J recombination are blunt, 5′-phosphorylated, RAG-dependent, and cell cycle regulated.
Genes Dev.
7
:
2520
–2532.
32
Stoddart, A., R.J. Ray, and C.J. Paige.
1997
. Analysis of murine CD22 during B cell development: CD22 is expressed on B cell progenitors prior to IgM.
Int. Immunol.
9
:
1571
–1579.
33
Young, F., E. Mizoguchi, A.K. Bhan, and F.W. Alt.
1997
. Constitutive Bcl-2 expression during immunoglobulin heavy chain-promoted B cell differentiation expands novel precursor B cells.
Immunity.
6
:
23
–33.
34
Young, F., B. Ardman, Y. Shinkai, R. Lansford, T.K. Blackwell, M. Mendelsohn, A. Rolink, F. Melchers, and F.W. Alt.
1994
. Influence of immunoglobulin heavy- and light-chain expression on B-cell differentiation.
Genes Dev.
8
:
1043
–1057.
35
Kitamura, D., and K. Rajewsky.
1992
. Targeted disruption of the μ chain membrane exon causes loss of heavy-chain allelic exclusion.
Nature.
356
:
154
–156.
36
Rolink, A., U. Grawunder, T.H. Winkler, H. Karasuyama, and F. Melchers.
1994
. IL-2 receptor alpha chain (CD25,Tac) expression defines a crucial stage in pre-B cell development.
Int. Immunol.
6
:
1257
–1264.
37
Gartner, F., F.W. Alt, R.J. Monroe, and K.J. Seidl.
2000
. Antigen-independent appearance of recombination activating gene (RAG)-positive bone marrow B cells in the spleens of immunized mice.
J. Exp. Med.
192
:
1745
–1754.
38
Nagaoka, H., G. Gonzalez-Aseguinolaza, M. Tsuji, and M.C. Nussenzweig.
2000
. Immunization and infection change the number of recombination activating gene (RAG)-expressing B cells in the periphery by altering immature lymphocyte production.
J. Exp. Med.
191
:
2113
–2120.
39
Shaw, A.C., W. Swat, R. Ferrini, L. Davidson, and F.W. Alt.
1999
. Activated Ras signals developmental progression of recombinase-activating gene (RAG)-deficient pro–B lymphocytes.
J. Exp. Med.
189
:
123
–129.
40
Shaw, A.C., W. Swat, L. Davidson, and F.W. Alt.
1999
. Induction of Ig light chain gene rearrangement in heavy chain-deficient B cells by activated Ras.
Proc. Natl. Acad. Sci. USA.
96
:
2239
–2243.
41
Hennet, T., D. Chui, J.C. Paulson, and J.D. Marth.
1998
. Immune regulation by the ST6Gal sialyltransferase.
Proc. Natl. Acad. Sci. USA.
95
:
4504
–4509.
42
Cornall, R.J., C.C. Goodnow, and J.G. Cyster.
1999
. Regulation of B cell antigen receptor signaling by the Lyn/CD22/SHP1 pathway.
Curr. Top. Microbiol. Immunol.
244
:
57
–68.
43
Okada, T., V.N. Ngo, E.H. Ekland, R. Forster, M. Lipp, D.R. Littman, and J.G. Cyster.
2002
. Chemokine requirements for B cell entry to lymph nodes and Peyer's patches.
J. Exp. Med.
196
:
65
–75.
44
Eisenbeis, C.F., H. Singh, and U. Storb.
1995
. Pip, a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator.
Genes Dev.
9
:
1377
–1387.
45
Rao, S., S. Karray, E.R. Gackstetter, and M.E. Koshland.
1998
. Myocyte enhancer factor-related B-MEF2 is developmentally expressed in B cells and regulates the immunoglobulin J chain promoter.
J. Biol. Chem.
273
:
26123
–26129.
46
Takagaki, Y., and J.L. Manley.
1998
. Levels of polyadenylation factor CstF-64 control IgM heavy chain mRNA accumulation and other events associated with B cell differentiation.
Mol. Cell.
2
:
761
–771.
47
Shimozato, T., and P.W. Kincade.
1999
. Prostaglandin E(2) and stem cell factor can deliver opposing signals to B lymphocyte precursors.
Cell. Immunol.
198
:
21
–29.
48
Morrissey, P.J., P. Conlon, K. Charrier, S. Braddy, A. Alpert, D. Williams, A.E. Namen, and D. Mochizuki.
1991
. Administration of IL-7 to normal mice stimulates B-lymphopoiesis and peripheral lymphadenopathy.
J. Immunol.
147
:
561
–568.
49
Peschon, J.J., P.J. Morrissey, K.H. Grabstein, F.J. Ramsdell, E. Maraskovsky, B.C. Gliniak, L.S. Park, S.F. Ziegler, D.E. Williams, C.B. Ware, et al.
1994
. Early lymphocyte expansion is severely impaired in interleukin 7 receptor–deficient mice.
J. Exp. Med.
180
:
1955
–1960.
50
Marshall, A.J., H.E. Fleming, G.E. Wu, and C.J. Paige.
1998
. Modulation of the IL-7 dose-response threshold during pro-B cell differentiation is dependent on pre-B cell receptor expression.
J. Immunol.
161
:
6038
–6045.
51
Welch, P.A., P.D. Burrows, A. Namen, S. Gillis, and M.D. Cooper.
1990
. Bone marrow stromal cells and interleukin-7 induce coordinate expression of the BP-1/6/C3 antigen and pre-B cell growth.
Int. Immunol.
2
:
697
–705.
52
Spanopoulou, E., C.A.J. Roman, L.M. Corcoran, M.S. Schlissel, D.P. Silver, D. Nemazee, M.C. Nussenzweig, S.A. Shinton, R.R. Hardy, and D. Baltimore.
1994
. Functional immunoglobulin transgenes guide ordered B-cell differentiation in RAG-1-deficient mice.
Genes Dev.
8
:
1030
–1042.
53
Kitamura, D., A. Kudo, S. Schaal, W. Muller, F. Melchers, and K. Rajewsky.
1992
. A critical role of λ5 protein in B cell development.
Cell.
69
:
823
–831.
54
Corcoran, A.E., A. Riddell, D. Krooshoop, and A.R. Venkitaraman.
1998
. Impaired immunoglobulin gene rearrangement in mice lacking the IL-7 receptor.
Nature.
391
:
904
–907.
55
Anderson, S.J., K.M. Abraham, T. Nakayama, A. Singer, and R.M. Perlmutter.
1992
. Inhibition of T-cell receptor β-chain gene rearrangement by overexpression of the non-receptor protein tyrosine kinase p56lck.
EMBO J.
11
:
4877
–4886.
56
Gaertner, F., F.W. Alt, R.J. Monroe, M. Chu, B.P. Sleckman, L. Davidson, and W. Swat.
1999
. Immature thymocytes employ distinct signaling pathways for allelic exclusion versus differentiation and expansion.
Immunity.
10
:
537
–546.
57
Doi, T.S., T. Takahashi, O. Taguchi, T. Azuma, and Y. Obata.
1997
. NF-κB RelA-deficient lymphocytes: normal development of T cells and B cells, impaired production of IgA and IgG1 and reduced proliferative responses.
J. Exp. Med.
185
:
953
–961.
58
Sha, W.C., H.C. Liou, E.I. Tuomanen, and D. Baltimore.
1995
. Targeted disruption of the p50 subunit of NF-kappa-B leads to multifocal defects in immune responses.
Cell.
80
:
321
–330.

T. Tretter and A.E. Ross contributed equally to this work.

The online version of this article contains supplemental material.

Abbreviations used in this paper: BCR, B cell receptor; NF, nuclear factor.

Supplementary data