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The Journal of Immunology, 2006, 176: 4543-4552.
Copyright © 2006 by The American Association of Immunologists

Bruton’s Tyrosine Kinase and SLP-65 Regulate Pre-B Cell Differentiation and the Induction of Ig Light Chain Gene Rearrangement1

Rogier Kersseboom*, Van B. T. Ta*, A. J. Esther Zijlstra*, Sabine Middendorp*, Hassan Jumaa{dagger}, Pieter Fokko van Loo* and Rudolf W. Hendriks2,*

* Department of Immunology, Erasmus Medical Center, Rotterdam, The Netherlands; and {dagger} Institute for Biology III, Albert Ludwigs University of Freiburg, and Max Planck Institute for Immunobiology, Freiburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Bruton’s tyrosine kinase (Btk) and the adapter protein SLP-65 (Src homology 2 domain-containing leukocyte-specific phosphoprotein of 65 kDa) transmit precursor BCR (pre-BCR) signals that are essential for efficient developmental progression of large cycling into small resting pre-B cells. We show that Btk- and SLP-65-deficient pre-B cells have a specific defect in Ig {lambda} L chain germline transcription. In Btk/SLP-65 double-deficient pre-B cells, both {kappa} and {lambda} germline transcripts are severely reduced. Although these observations point to an important role for Btk and SLP-65 in the initiation of L chain gene rearrangement, the possibility remained that these signaling molecules are only required for termination of pre-B cell proliferation or for pre-B cell survival, whereby differentiation and L chain rearrangement is subsequently initiated in a Btk/SLP-65-independent fashion. Because transgenic expression of the antiapoptotic protein Bcl-2 did not rescue the developmental arrest of Btk/SLP-65 double-deficient pre-B cells, we conclude that defective L chain opening in Btk/SLP-65-deficient small resting pre-B cells is not due to their reduced survival. Next, we analyzed transgenic mice expressing the constitutively active Btk mutant E41K. The expression of E41K-Btk in Ig H chain-negative pro-B cells induced 1) surface marker changes that signify cellular differentiation, including down-regulation of surrogate L chain and up-regulation of CD2, CD25, and MHC class II; and 2) premature rearrangement and expression of {kappa} and {lambda} light chains. These findings demonstrate that Btk and SLP-65 transmit signals that induce cellular maturation and Ig L chain rearrangement independently of their role in termination of pre-B cell expansion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
During B cell development, Ig gene rearrangement is ordered such that the Ig H chain locus generally rearranges before the Ig {kappa} and {lambda} L chain loci (reviewed in Refs.1 and 2). Productive V(D)J recombination of the Ig H chain gene results in surface deposition of the precursor BCR (pre-BCR),3 which consists of µ H chain, the nonrearranging VpreB and {lambda}5 surrogate L chain (SLC) proteins, and the Ig-{alpha}/CD79a and Ig-beta/CD79b signaling components. The pre-BCR is a key checkpoint in B cell development to monitor the assembly of a functional Ig H chain and to terminate further H chain rearrangements, thus ensuring that only one functional Ig H chain is synthesized, a phenomenon referred to as allelic exclusion. Pre-BCR expression induces proliferative expansion of cytoplasmic H chain-positive pre-B cells and their progression into small resting pre-B cells, in which Ig L chain rearrangement occurs (1, 2).

In mice deficient for components of the pre-BCR complex, including Ig-{alpha}, Ig-beta, SLC, or the downstream tyrosine kinase Syk, µ H chain-positive pre-B cells are unable to proliferate (reviewed in Ref.3). Specifically, the non-Ig-like unique tail of {lambda}5 was shown to be essential for activation of downstream signal transduction pathways (4). In contrast, disruption of Bruton’s tyrosine kinase (Btk) or the adapter protein Src homology 2 domain-containing leukocyte-specific phosphoprotein of 65 kDa (SLP-65; also known as BASH or BLNK) showed that these pre-BCR signaling components are crucially involved in the termination of IL-7-driven expansion of large cycling pre-B cells (5, 6). SLP-65-deficient mice spontaneously develop pre-B cell tumors expressing high levels of pre-BCR on the cell surface (6, 7). Although Btk-deficient mice do not develop tumors, Btk cooperates with SLP-65 as a tumor suppressor, because the incidence of pre-B cell leukemia is significantly higher in SLP-65/Btk double-deficient mice than in SLP-65 single-deficient mice (8). Using transgenic (Tg) mice expressing the kinase-inactive Btk mutant K430R, we recently showed that Btk exerts its tumor suppressor function independently of its kinase activity (9).

During the transition of large cycling into small resting pre-B cells in the mouse, Btk- or SLP-65-deficient cells show defective down-regulation of SLC, the metallopeptidase BP-1 and the sialoglycoprotein CD43 as well as defective up-regulation of the adhesion molecule CD2, the IL-2R CD25, and MHC class II (5, 8). Btk-deficient cells also manifest a specific ~3-h developmental delay within the small pre-B cell compartment. Thus, it appears that in addition to their function in the termination of IL-7-driven proliferation, Btk and SLP-65 are involved in cellular maturation of cytoplasmic Ig µ+ pre-B cells.

The role of pre-BCR signaling in the induction of pre-B cell differentiation and Ig L chain rearrangement is controversial (2, 4). The hypothesis that pre-BCR signals are responsible for the redirection of V(D)J recombination activity from the Ig H chain to the L chain loci is based on observations that Ig H chain surface expression correlates with transcription of unrearranged Ig L chain gene segments. This so-called germline transcription has been implicated in the regulation of accessibility of Ig loci to the V(D)J recombinase, because it precedes or coincides with V(D)J recombination (for review, see Ref2). Second, in the absence of pre-BCR function, e.g., in mice with targeted disruption of the Ig H chain, Ig-{alpha}, Ig-beta, SLC components, or Syk, Ig {kappa} locus rearrangement is diminished. Third, the expression of an activated Ras transgene induces L chain rearrangement in JH–/– pro-B cells, which lack the ability to assemble Ig H chain variable regions (10). Fourth, the findings of reduced Ig L chain germline transcription and gene rearrangement in SLP-65-deficient pre-B cells (7, 11) and reduced {lambda} L chain usage in Btk-deficient mice (12, 13) point to important roles for these signaling molecules in the initiation of Ig L chain rearrangement. Nevertheless, evidence for a direct involvement of pre-BCR signaling in the induction of L chain V(D)J recombination is lacking. On the contrary, it has been reported that rearrangement and expression of Ig L chain genes can occur without Ig H chain expression. Ig {kappa} transcription and rearrangement are detectable in Ig µ– pro-B cells (14) and are increased by activation of NF-{kappa}B by LPS in transformed pro-B cell lines (15). Removal of IL-7 from cultured pro-B cells from JH–/– or {lambda}5–/– mice, which are incapable of expressing a proper pre-BCR, resulted in the apparent differentiation into cells that transcribe and rearrange Ig L chain loci (16). Moreover, the observations that SLC- and Ig H chain-deficient pre-B cells rearrange Ig L chain at normal frequencies and with normal kinetics argue for a model in which the initiation of Ig L chain rearrangement is independent of pre-BCR expression (4, 17).

Because a deficiency for Btk or SLP-65 affects IL-7 responsiveness, cell surface marker expression, and Ig L chain rearrangement in pre-B cells, it is possible that Btk and SLP-65 are directly involved in all three processes. Alternatively, Btk and SLP-65 signaling may only be required for the termination of IL-7-driven pre-B cell proliferation and thus the differentiation of large cycling to small resting pre-B cells. The subsequent cell surface marker changes and Ig L chain rearrangements may then be initiated independently of Btk and SLP-65 signaling. In a third model, Btk and SLP-65 also do not regulate pre-B cell differentiation or the induction of Ig L chain locus activation; they only support the survival of small resting pre-B cells.

To distinguish between these possibilities, in the current report we investigated the effects of Btk and SLP-65 deficiency on transcriptional activation of Ig L chain loci in pre-B cells. By crossing Btk/SLP-65 double-deficient mice onto a Bcl-2 Tg background (18), we determined whether providing an extended time window for Ig L chain rearrangement rescued their severe arrest at the pre-B to immature B transition. Finally, we studied Tg mice that express the constitutively active Btk mutant, E41K-Btk, under control of the CD19 promoter region (8, 19, 20, 21). The CD19 promoter region targets the expression of transgenes to all stages of B cell development, including pro-B cells (21). Therefore, the E41K-Btk Tg mice enabled us to investigate whether Btk signaling has the capacity to prematurely induce cell surface marker modulations and Ig L chain rearrangement in pro-B cells, thus independently of the developmental progression of large cycling into small resting pre-B cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and genotyping

Mice deficient for Btk (22), RAG-1 (23), or membrane µ targeted (µMT) (24) and Eµ-2–22 Bcl-2 Tg mice (18) were on the C57BL/6 background; SLP-65– mice (25) were on the BALB/c background. The VH81X-µ Tg mouse strain (26) was a gift from J. F. Kearney (Birmingham, AL). E41K-Btk mice have been described previously (8, 21). The different composite genotypes were on a mixed background, and littermates were compared in single experiments. All mice were bred and maintained at the Erasmus Medical Center animal care facility under specific pathogen-free conditions. Mice were analyzed at 8–16 wk of age. The experimental procedures used in this study were reviewed and approved by the Erasmus University committee of animal experiments. For mouse genotyping, tail DNA was analyzed by Southern blotting of BamHI digests using a partial human Btk cDNA probe (bp 133-1153), as described previously (21, 22). Alternatively, the presence of Btk transgenes, endogenous mouse Btk wild-type (WT), or Btk knockout alleles was evaluated by PCR (8, 27). PCR assays were also used to determine the genotypes for SLP-65 (8), RAG-1, and µMT mice using standard primers (<www.jax.org>) and to determine the presence of Eµ-2–22 Bcl-2 (12) and VH81X-µ (forward primer specific for framework I, 5'-CGCGCGGCCGCGTGGAGTCTGGGGGAGGCT-3'; reverse primer specific for the transgene CDR3 region, 5'-CCCAGACATCGAAGTACCAGCTACTA-CCATG-3') transgenes (26).

Flow cytometric analyses

Preparations of single-cell suspensions, flow cytometry procedures, and mAbs have been described previously (5, 22). For intracellular staining of cytoplasmic proteins, cells were first stained for cell surface markers and subsequently fixed in 2% paraformaldehyde and permeabilized using 0.5% saponin (21). The anti-SLC hybridoma LM34 (28) was provided by A. Rolink (University of Basel, Basel, Switzerland). Events (1–5 x 105) were scored using a FACSCalibur flow cytometer and were analyzed using CellQuest software (BD Biosciences).

RNA isolation and analysis

Total bone marrow (BM) cell suspensions were depleted of erythrocytes by standard ammonium chloride lysis. For mice on the µMT background, cell suspensions were enriched for B-lineage cells by magnetic sorting using anti-B220-coated microbeads (Miltenyi Biotec). Ig L chain-negative pre-B and Ig L chain-positive immature B cell fractions were obtained from in vitro BM cultures. Total BM cultures in the presence of IL-7 (Sigma-Aldrich) and quantification of IL-7-dependent proliferative responses of total BM cells have been described previously (5). Cells were labeled with biotinylated anti-Ig{kappa} (187.1) and anti-Ig{lambda} (R26-46) Abs, incubated with streptavidin-coated microbeads (Miltenyi Biotec), and subjected to cell separation by AutoMACS (Miltenyi Biotec).

Total RNA was extracted using the GenElute Mammalian Total RNA Miniprep system (Sigma-Aldrich). Two micrograms of total RNA was used as a template for double-stranded cDNA synthesis using reverse transcriptase (Superscript II; Invitrogen Life Technologies) and random hexamer primers. Samples were serially diluted 3-fold before amplification using PCR primers specific for the germline Ig L chain transcripts {lambda}10, {lambda}20, {lambda}30, V{lambda}1/2, {kappa}00.8, and {kappa}01.1; functional V{lambda}1C{lambda}1 (29, 30); RAG-1; RAG-2 (31); and beta-actin. PCR products were separated by standard agarose electrophoresis and were visualized by ethidium bromide staining. Densitometric analyses were conducted using ImageQuant analysis software (Molecular Dynamics), and statistical evaluations were performed by standard t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Reduced germline {lambda} L chain transcription in the absence of Btk

To investigate the effect of Btk signaling on transcriptional activation of Ig L chain loci, we evaluated Ig L chain germline transcription in pre-B and B cells from WT and Btk-deficient mice. Total BM cell suspensions were cultured in the presence of IL-7 for 5 days and subsequently without IL-7 for 2 days. Removal of IL-7 strongly induces pre-B cells to exit the cell cycle and to initiate stepwise activation of Ig {kappa} and {lambda} L chain gene rearrangement (17, 29, 30). We purified surface L chain-negative pre-B cell and surface L chain-positive B cell fractions (Fig. 1A) and used RT-PCR to assay the expression of germline {kappa} and {lambda} transcripts.


Figure 1
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  		FIGURE 1. Reduced germline {lambda} L chain transcription in Btk– pre-B and immature B cells. A, Flow cytometric characterization of sorted surface Ig– pre-B cell and surface Ig+ immature B cell fractions from IL-7-driven BM cultures. B, Detection of Ig L chain germline and RAG-1/2 transcripts. Ig {kappa}+{lambda} L chain-negative and -positive fractions were obtained by magnetic sorting. Total RNA from these cell fractions was reverse transcribed, serially diluted 3-fold, and used as a template for amplification of the transcripts indicated at the left. Amplification of beta-actin (bottom) was performed as a control. Products were fractionated by gel electrophoresis and detected with ethidium bromide. Data shown are representative for four mice of each genotype. Statistical evaluation of density values showed that expression levels of {lambda}10 and {lambda}20 were significantly reduced in Btk-deficient pre-B cells (p < 0.004 and p < 0.02, respectively) and immature B cells (p < 0.05 and p < 0.009, respectively) compared with WT. No significant differences were found for {kappa}00.8, {kappa}01.1, or {lambda}30 expression.

 
As shown in Fig. 1B, we found that Btk+ and Btk– pre-B cells expressed comparable levels of {kappa}00.8 and {kappa}01.1 germline transcripts, which are initiated in different regions 5' of J{kappa} and spliced to the C{kappa} region (29). In contrast, the expression levels of germline {lambda} transcripts were significantly reduced in Btk– pre-B cells compared with Btk+ pre-B cells. In particular, the levels of {lambda}10 and {lambda}20 transcripts, which initiate 5' of the J{lambda}1 and J{lambda}2 regions and are spliced to C{lambda}1 and C{lambda}2, respectively (29), were reduced in Btk-deficient pre-B cells (Fig. 1B). Interestingly, this effect of Btk appeared to be specific for the germline transcripts of the {lambda} J-C cluster, because transcripts of the V{lambda} region were present at similar levels in Btk+ and Btk– pre-B cells (Fig. 1B). Although successful VL to JL rearrangement results in the loss of germline transcription on productive alleles, transcription of unrearranged L chain alleles continues in mature B cells (32). We therefore also analyzed surface Ig+ B cells and observed a specific reduction in the expression of {lambda}10 and {lambda}20 transcripts in Btk-deficient B cells, whereas V{lambda}, {kappa}00.8, and {kappa}01.1 transcripts were present at normal levels (Fig. 1B).

Developmental progression of large to small pre-B cells is also accompanied by the up-regulation of transcription of the genes RAG-1 and RAG-2 (1). We observed similar levels of RAG-1/2 transcripts in Btk+ and Btk– pre-B cells (Fig. 1B), indicating that Btk is not involved in the initiation of RAG transcription in pre-B cells. Interestingly, RAG-1 expression was down-regulated in Btk+Ig+ B cells, but not in Btk-deficient Ig+ B cells (Fig. 1B), suggesting that termination of RAG-1 expression upon productive L chain recombination is Btk dependent.

Taken together, the finding of a specific reduction in the levels of germline {lambda}10 and {lambda}20 transcripts in Btk-deficient (pre-) B cell fractions indicates that Btk signals are needed for activation of the Ig {lambda} L chain locus for recombination by opening of {lambda} J-C clusters.

Synergistic roles of Btk and SLP-65 in the induction of L chain rearrangement

The analysis of germline transcription as a marker for L chain locus accessibility in vivo is complicated by the fact that ongoing VL to JL rearrangement will destroy its germline configuration and thereby result in the loss of germline transcription. Therefore, we bred Btk and SLP-65 single- and double-mutant mice to RAG-1-deficient mice that carry the Ig H chain transgene VH81X (26). In this background, B cell progenitors progress to the pre-B cell stage because of the presence of the prerearranged Ig H chain, which ensures pre-BCR expression and cellular proliferation (33). However, the inactivation of RAG-1 precludes any L chain gene rearrangement, and cells are arrested at the small pre-B cell stage (Fig. 2A).


Figure 2
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  		FIGURE 2. Defective pre-B cell maturation in Btk- and SLP-65-deficient pre-B cells. A, Flow cytometric characterization of the B cell population from the indicated mice on a RAG-1–/– VH81X Tg background. BM lymphoid cells were gated on the basis of forward and side scatter characteristics. The expression profile of B220 vs cytoplasmic µ H chain is plotted. B, Expression of markers that are normally down-regulated or induced during pre-B cell differentiation. B220+cy-µ+ pre-B cells were gated, and the expression data of the indicated markers and forward scatter (FSC) are shown as histograms. Numbers indicate the percentage of positive cells. Plots are representative for four mice of each genotype.

 
Detailed flow cytometric analyses of the four groups of mice (WT, Btk-deficient, SLP-65-deficient, and Btk/SLP-65 double-deficient mice) on the RAG-1–/– VH81X Tg background revealed that Btk- or SLP-65-deficient pre-B cells exhibited elevated expression of CD43 and SLC and reduced expression of CD2, CD25, and MHC class II; the defects were most pronounced in the Btk/SLP-65 double mutant (Fig. 2B). These findings show that the previously reported aberrant phenotypes of Btk and SLP-65 single- or double-deficient pre-B cells, which reflect their impaired cellular maturation (5, 8), were preserved on the RAG-1–/– VH81X Tg background. We additionally found that down-regulation of IL-7R and c-Kit expression on the surface of pre-B cells was impaired in the absence of Btk and SLP-65 (Fig. 2B).

From the four groups of mice, BM cell suspensions were analyzed for the expression of Ig {kappa} and {lambda} L chain germline transcripts. In RT-PCR experiments, we observed a specific reduction in the expression of {lambda}10 and {lambda}20 transcripts in the absence of either Btk or SLP-65. Expression levels of {lambda}3° were not significantly affected by the absence of Btk or SLP-65 (Fig. 3). We detected a modest reduction in the expression levels of the {kappa}00.8 and {kappa}01.1 germline transcripts in SLP-65-deficient pre-B cells compared with WT or Btk-deficient mice (Fig. 3). In contrast, in Btk/SLP-65 double-deficient pre-B cells, the levels of both {kappa} and {lambda} germline transcripts were severely reduced.


Figure 3
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  		FIGURE 3. Defective germline transcription of Ig L chain loci in Btk- and SLP-65-deficient pre-B cells. Expression of the indicated Ig L chain germline transcripts was analyzed by RT-PCR in the indicated four groups of mice on the RAG-1–/– VH81X Tg background. Total RNA from BM fractions was reverse transcribed, serially diluted 3-fold, and used as a template for amplification of the transcripts indicated at the left. Amplification of beta-actin (bottom) was performed as a control. Products were fractionated by gel electrophoresis and detected with ethidium bromide. Data shown are representative for three or four mice examined in each group. Statistical evaluation of density values showed t hat the expression of {kappa}00.8/{kappa}01.1 was significantly reduced in Btk/SLP-65 double-deficient pre-B cells (compared with WT, p < 0.03), and the expression of {lambda}10/{lambda}20 was significantly reduced in Btk or SLP-65 single- or double-deficient pre-B cells (compared with WT, p < 0.05). No significant differences were found for {lambda}30 expression.

 
The observed differences among the four groups of mice did not appear to result from differences in the ratio between large cycling and small resting pre-B cells, because the forward scatter profiles of the pre-B cell populations in these mice were comparable (Fig. 2B). Consistent with this, similar differences in surface marker expression profiles and Ig {kappa} and {lambda} germline transcription among the four groups of RAG-1–/– VH81X Tg mice were observed in in vitro IL-7-driven BM cultures, in which developmental progression from large cycling into small resting pre-B cells was induced by IL-7 withdrawal (data not shown).

The finding that in the absence of either Btk or SLP-65, {lambda} germline transcription is reduced, whereas {kappa} germline transcription is not dramatically affected, indicates differential regulation of {kappa} and {lambda} locus activation. Therefore, either 1) the opening of the {lambda} L chain J-C clusters is essentially dependent on Btk- and SLP-65-mediated signaling pathways; or alternatively, 2) in the absence of these signaling proteins, development does not efficiently progress to a stage critical for {lambda} activation.

Defective Ig L chain opening in Btk/SLP-65-deficient pre-B cells is not due to their reduced survival

Interestingly, the Btk/SLP-65 dependency of {kappa} and {lambda} germline transcription parallels the order of transcriptional activation of the L chain loci (29, 30): the {kappa} locus, and the {lambda} V cluster, which are only marginally affected by the absence of Btk or SLP-65, open early in B cell development, and the {lambda} J-C cluster, which is severely affected by the absence of one of the two signaling molecules, opens late in B cell development. Thus, our findings can also be explained by a model in which Btk and SLP-65 signals are mainly essential for the survival of small resting pre-B cells that initiate Ig L chain rearrangement. The almost complete arrest of B cell development in Btk/SLP-65 double-deficient mice at the pre-B cell stage (8, 34) and the absence of germline L chain transcripts in this model reflect the short life span of Btk/SLP-65 double-deficient pre-B cells.

To investigate this issue, we determined whether enforced expression of the antiapoptotic Bcl-2 gene, which is thought to provide an extended time window per cell for Ig L chain rearrangement (35), is able to rescue the severe block in B cell development in Btk/SLP-65 double-deficient mice. We crossed Btk/SLP-65 double-deficient mice with Eµ-Bcl-2 Tg mice (18) and investigated the size of the immature B cell population by flow cytometry. We found that in the presence of the Bcl-2 transgene, the size of the immature B cell population was still severely reduced (Fig. 4). Moreover, analysis of cytoplasmic µ H chain-positive pre-B cells indicated that increasing the survival of Btk/SLP-65 double-deficient pre-B cells did not rescue the defective down-regulation of SLC expression in these cells (Fig. 4). Likewise, the Bcl-2 transgene failed to correct the modulation of the CD43, CD2, and CD25 surface markers (data not shown). We verified that Tg Bcl-2 diminished apoptosis in Btk/SLP-65-deficient pre-B cells as effectively as in WT pre-B cells by analysis of cell survival of cultured BM pre-B cells upon IL-7 withdrawal (Fig. 4B).


Figure 4
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  		FIGURE 4. Bcl-2 expression does not rescue the pre-B cell arrest in Btk/SLP-65 double-deficient mice. A, Flow cytometric analysis of BM lymphoid cells from the indicated mouse groups. Expression profiles of B220 and µ H chain are displayed as dot plots. Total B220+ B-lineage fractions were gated and analyzed for the expression of {kappa} L chain on the cell surface. Next, pro/pre-B cell fractions (B220+{kappa}–) were analyzed for cytoplasmic expression of SLC. Data are shown as histograms, and numbers indicate the percentage of positive cells. Plots are representative for three to eight mice of each genotype. B, In vitro survival after IL-7 withdrawal of BM cultures from the indicated mouse genotypes.

 
Collectively, these findings demonstrate that protection of Btk/SLP-65 double-deficient pre-B cells from apoptosis does not result in the termination of SLC expression or the initiation of L chain rearrangements. Therefore, we conclude that defective Ig L chain opening in Btk- and SLP-65-deficient pre-B cells is not due to reduced survival.

E41K-Btk induces cell surface marker changes in µ– pro-B cells

Next we aimed to investigate whether Btk signaling has the capacity to induce cell surface marker modulation and Ig L chain transcription and rearrangements independently of the developmental progression of large to small pre-B cells. We used Tg mice expressing the constitutively active E41K-Btk mutant under the control of the CD19 promoter region, which were crossed onto the Btk-deficient background.

Although the expression of transgenic E41K-Btk resulted in a dose-dependent deletion of B cells at the transition of the IgMlow to the IgMhighB220low immature B cell stage in BM (8, 21), it nevertheless completely corrected the impaired modulation of pre-B cell surface markers in Btk-deficient mice (Fig. 5A). In vitro BM culture experiments showed that the expression of E41K-Btk increased the IL-7 responsiveness of pro-B cells and reduced the IL-7 responsiveness of pre-B cells (Fig. 5, B–D). E41K-Btk did not increase IL-7R {alpha}-chain transcription or surface expression in pro-B cells (data not shown), and therefore, it appears that this effect of E41K-Btk is based on the functional intersection of pre-BCR and IL-7R signaling pathways that induce pre-B cell proliferation (36).


Figure 5
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  		FIGURE 5. The effect of E41K-Btk expression on pre-B cells. A, E41K-Btk expression corrects the impaired modulation of cell surface markers in Btk-deficient pre-B cells. Cytoplasmic SLC and CD2 expression was investigated in pre-B cell fractions (B220+IgM–c-µ H chain+), whereas BP-1 and CD25 were analyzed in total pro-B/pre-B cell fractions (B220+IgM–) from the indicated mice. Data shown are representative for six to 11 animals per group. B, E41K-Btk expression corrects increased IL-7 responsiveness of Btk-deficient B-lineage cells. Analysis of proliferative responses to IL-7 of total BM fractions from the indicated mice, as determined by [3H]thymidine incorporation after 5 days of culture in the presence or the absence of 100 U/ml IL-7. Error bars are the SEMs from three to seven mice per group. C, Increased proportions of pro-B cells in BM cultures from BtkAct Tg mice. The distribution profile over the indicated B-lineage subpopulations of IL-7-driven total BM cultures of the indicated mice after 5 days of culture in the presence of 100 U/ml IL-7. Error bars are the SEMs for pro-B cells from three to seven mice per group. D, Constitutively active Btk supports expansion of pro-B cells in IL-7-driven BM cultures. The bars indicate the relative expansion of c-µ– pro-B and c-µ+ pre-B cells during culture with 10 or 100 U/ml IL-7 on day 5 compared with pro-B and pre-B cell numbers at the start of the culture, which were set at one.

 
To examine the capacity of E41K-Btk to mimic pre-BCR activation and consequently signal for premature cellular maturation of cytoplasmic µ– pro-B cells in vivo, we characterized the CD19+B220low cytoplasmic µ– pro-B cell fractions in BM from Btk+, Btk–, and E41K-Btk Tg mice (Fig. 6A). In Btk+ and Btk– mice, almost all CD19+B220+ cytoplasmic µ– pro-B cells contained SLC. In contrast, in E41K-Btk Tg mice, ~40% of the pro-B cells did not express detectable levels of SLC in the cytoplasm. Compared with Btk+ or Btk– mice, the pro-B cell fraction from E41K-Btk Tg mice contained a significantly larger subpopulation of cells expressing CD2, CD25 and MHC class II. In all groups of mice, pro-B cells were largely CD43+. To exclude the possibility that the analyses of pro-B cells were confounded by Ig µ H chain-positive pre-B cells that have subsequently lost or down-regulated Ig µ H chain expression, we crossed Btk+, Btk–, and E41K-Btk Tg mice onto the µMT background (24). In µMT mice, cells are arrested at the pro-B cell stage, because the membrane exon of the Ig H chain µ constant region is disrupted, and therefore, the Ig µ H chain cannot be expressed on the cell surface. Also on the µMT background, we found that the expression of E41K-Btk resulted in down-regulation of SLC and the induction of CD2, CD25, and MHC class II in cytoplasmic µ H chain-negative pro-B cells (Fig. 6B).


Figure 6
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  		FIGURE 6. E41K-Btk induces phenotypic changes in cytoplasmic µ– pro-B cells. Flow cytometric expression profiles of the indicated markers in pro-B cells of Btk+, Btk–, and E41K-Btk mice on a WT (A) or a µMT background (B–D). Flow cytometric analysis of surface B220 and cytoplasmic µ H chain expression on total CD19+ B-lineage cells in BM (A and B, upper parts). CD19+B220+cµ– pro-B cells were gated and analyzed for the expression of cytoplasmic SLC; surface CD43, CD2, CD25, and MHC class II; and cytoplasmic {kappa} L chain (A and B, lower parts). The results are displayed as filled histograms in which the percentages of positive cells are indicated. C, Expression profiles of cytoplasmic µ H chain and {kappa} L chain in B220+CD19+ BM fractions of the indicated mice. D, CD19+B220+cµ– pro-B cells from the indicated mice were gated and analyzed for the expression of CD25, SLC, and {kappa} L chain. Data shown are representative of four to eight animals examined within each group.

 
The phenotypic marker profiles induced by E41K-Btk in pro-B cells reflected the changes normally associated with developmental progression of large cycling into small resting pre-B cells. Therefore, we conclude that E41K-Btk expression in pro-B cells mimics pre-BCR signaling and induces cell surface marker changes in these cells even in the absence of functional Ig µ H chain proteins.

E41K-Btk induces L chain rearrangement in µ– pro-B cells

Next, we investigated the effect of Tg E41K-Btk expression on Ig L chain rearrangement in pro-B cells on both the WT background and the µMT background. Although in WT mice <5% of CD19+B220low cytoplasmic µ H chain-negative pro-B cells expressed detectable levels of Ig {kappa} L chain proteins in their cytoplasm, this proportion was significantly increased to ~10–15% of pro-B cells in E41K-Btk Tg mice (Fig. 6, A and B). In the total population of E41K-Btk µMT CD19+B220+ pro-B cells, approximately equal proportions of cells exclusively expressed Ig H or Ig L chains, whereas the fraction of H and L chain double-positive pro-B cells was low (Fig. 6C). This finding indicates that in E41K-Btk µMT pro-B cells, the majority of functional {kappa} L chain rearrangements are performed in the absence of a productive H chain rearrangement. As shown in Fig. 6D, Ig {kappa} L chain expression was mainly found in those pro-B cells in which the expression of pre-B cell-specific markers was induced. Moreover, the fractions of pro-B cells in which SLC expression was down-regulated and CD2, CD25, or MHC class II were induced were largely overlapping (Fig. 6D).

Using similar flow cytometric techniques, we were unable to detect Ig {lambda} L chain protein in E41K-Btk pro-B cells. However, when we performed RT-PCR analyses using primers specific for V{lambda}1 and C{lambda}1, rearranged {lambda} L chain transcripts were readily detectable in purified E41K-Btk µMT pro-B cell fractions (Fig. 7A). The levels of the V{lambda}1C{lambda}1 transcripts in E41K-Btk µMT pro-B cells were increased compared with control Btk+ or Btk– µMT pro-B cell fractions. Consistent with the induction of L chain rearrangement by the E41K-Btk transgene, increased levels of {kappa}00.8, {kappa}01.1, {lambda}10, and {lambda}20 germline transcripts were detected in E41K-Btk µMT pro-B cells (Fig. 7B).


Figure 7
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  		FIGURE 7. E41K-Btk induces L chain germline transcription and gene rearrangement in pro-B cells. RT-PCR analysis for the detection of {lambda} L chain transcripts from rearranged V{lambda}1-C{lambda}1 segments (A) and {kappa} and {lambda} L chain germline transcripts (B) in Btk+, Btk–, and E41K-Btk mice on a µMT background. Total RNA from the BM fraction, enriched for B220+ cells, was reverse transcribed, serially diluted 3-fold, and used as a template for amplification of the indicated transcripts. Amplification of beta-actin (bottom) was performed as a control. Products were fractionated by gel electrophoresis and detected with ethidium bromide. Data shown are representative for three mice examined in each group. Statistical evaluation showed that the expressions of {kappa}00.8, {kappa}01.1, {lambda}10, and {lambda}20 were significantly induced in E41K-Btk Tg pro-B cells (p < 0.05) compared with Btk– pro B cells. No significant differences were found for {lambda}03 expression.

 
Taken together, the findings of germline transcription and recombination of Ig L chain gene segments in E41K-Btk pro-B cells, which do not express functional µ H chain proteins, demonstrate that E41K-Btk has the capacity to transmit signals that induce Ig {kappa} and {lambda} L chain rearrangement.

Cooperation of IL-7 withdrawal and pre-BCR signaling

Pre-BCR signaling mediates selective response to IL-7 at the pro-B to pre-B cell transition via an ERK/MAPK-dependent pathway (36). However, it has been reported that removal of IL-7 induces Ig L chain rearrangement in cultured pro-B cells even in the absence of functional Ig H chain protein (16). To investigate cooperative effects of pre-BCR signaling and IL-7 withdrawal, we cultured total BM cells from Btk+, Btk–, and E41K-Btk µMT mice in the presence of IL-7 for 5 days and subsequently without IL-7 for 2 days. Consistent with reported findings (16), we observed that the Btk+ µMT pro-B cell cultures contained cytoplasmic {kappa} L chain-positive cells (~20%; Fig. 8). However, the expression of Ig {kappa} L chain was partially dependent on Btk signaling, because in Btk– µMT pro-B cell cultures, the proportions of cytoplasmic {kappa} L chain-positive cells were consistently lower (~10%; Fig. 8). Likewise, the levels of germline {lambda}01 and {lambda}02 transcripts were severely decreased in Btk– µMT pro-B cell cultures compared with Btk+ µMT pro-B cell cultures (data not shown). In E41K-Btk µMT pro-B cell cultures, the proportions of Ig {kappa}+ cells were similar to those found in Btk+ µMT cultures, and levels of {lambda}01 and {lambda}02 transcripts were slightly increased (Fig. 8 and data not shown).


Figure 8
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  		FIGURE 8. Cooperative effects of pre-BCR signaling and IL-7 withdrawal. Flow cytometric expression profiles of the indicated markers in pro-B cells of Btk+, Btk–, and E41K-Btk mice on the µMT background that were cultured in the presence of IL-7 for 5 days and for 2 additional days without IL-7. CD19+B220+ pro-B cells were gated and analyzed for the expression of the indicated markers. Data shown are representative of four to eight animals examined within each group.

 
In these IL-7-driven pro-B cell cultures, Btk activity had a more dramatic effect on the expression of the developmentally regulated markers SLC, CD2, and CD25 (Fig. 8). In E41K-Btk µMT pro-B cell cultures, SLC was expressed in only ~16% of cells compared with ~42 and ~91% in Btk+ and Btk– µMT pro-B cell cultures, respectively. Likewise, CD2 and CD25 expression was induced in ~70% of the cells in E41K-Btk µMT pro-B cell cultures, whereas the expression of these markers was very low in Btk– µMT pro-B cells (Fig. 8).

In summary, these findings indicate that in pro-B cell cultures, removal of IL-7 and Btk signaling has cooperative effects on initiation of L chain rearrangement, but modulation of SLC, CD2, and CD25 expression is essentially dependent on Btk signaling.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The pre-BCR checkpoint monitors functional Ig H chain rearrangement and triggers clonal expansion and developmental progression of Ig µ+ pre-B cells (3). Hereby the downstream signaling components SLP-65 and Btk serve as feedback inhibitors that limit pre-B cell proliferation. Mice deficient for SLP-65 or Btk show a partial block at the large cycling pre-B cell stage, whereas an almost complete arrest is observed in SLP-65/Btk double-mutant mice (6, 7, 8, 11, 12, 13, 25, 27, 34, 37). These findings suggest that Btk and SLP-65 are involved in cellular maturation of pre-B cells and the initiation of Ig L chain recombination, but evidence for their direct involvement in these processes is lacking. A possibility remains that Btk and SLP-65 are only required to terminate pre-B cell expansion and to advance pre-B cells to a stage at which Ig L chain rearrangement may be initiated in a Btk/SLP-65-independent fashion. Moreover, defective initiation of L chain rearrangements in Btk/SLP-65-deficient pre-B cells may also be explained by an essential role of these molecules in the survival of small resting pre-B cells.

In this report we show 1) that protection of Btk/SLP-65 double-deficient pre-B cells from apoptosis did not rescue modulation of pre-B cell marker expression or initiation of L chain rearrangement, and 2) that expression of the E41K-Btk mutant in pro-B cells induced the pre-B cell developmental program, including germline transcription and productive rearrangement and expression of Ig L chain as well as modulation of phenotypic markers. Therefore, we conclude that activated Btk not only acts to terminate proliferation or increase survival of pre-B cells, but has the intrinsic capacity to signal for cellular maturation and the initiation of both {kappa} and {lambda} L chain rearrangement and expression.

It is highly unlikely that E41K-Btk signals for cellular maturation and Ig L chain recombination by inducing cell cycle arrest in pro-B cells, because the expression of E41K-Btk increased IL-7 responsiveness in pro-B cells (Fig. 5). Although we observed cooperation of IL-7 withdrawal and pre-BCR signaling in vitro (Fig. 8), modulation of SLC, CD2, and CD25 expression was not induced by IL-7 withdrawal in the absence of Btk signaling.

The E41K gain of function mutation in the pleckstrin homology domain of Btk induces transformation of 3T3 fibroblasts, and this capacity is augmented by mutation of Y223, the main autophosphorylation site of Btk (19, 20). The E41K and E41K-Y223F mutants are associated with enhanced Btk membrane localization, activation, and calcium signaling in cultured cell lines (19, 20, 38, 39). Several lines of evidence show that these mutants also represent activated forms of Btk in murine B cells. Tg expression of E41K-Btk 1) enhances blast formation of splenic B cells in culture (40), 2) drives peripheral B cells efficiently into IgM plasma cell differentiation in vivo (21), and 3) induces expression of the early activation Ag CD69 on splenic B cells (R. W. Hendriks, unpublished observations). In addition, Tg E41K-Y223F-Btk manifests increased tyrosine phosphorylation in resting mature splenic B cells and has the ability to prevent tumor formation in SLP-65– pre-B cells (8). This observation indicates that not only may E41K-Y223F-Btk mimic the activation of Btk, it may also transmit signals that substitute for SLP-65 function. In this study we demonstrated that E41K-Btk was able to rescue the defects in Btk– pre-B cells and to induce premature developmental progression and Ig {kappa} and {lambda} L chain recombination and expression in Ig H chain-negative pro-B cells. Because Ig {lambda} rearrangements are normally initiated in late small resting pre-B cells when most {kappa} rearrangements cease (29, 41), expression of the E41K-Btk transgenic particularly advances Ig {lambda} L recombination. Accordingly, E41K-Btk Tg mice manifest increased Ig {lambda} usage in vivo (~15% compared with ~7% in WT littermates) (12). Similar effects were found for the E41K-Y223F Btk mutant (R. W. Hendriks, unpublished observations). We did not detect any additional in vivo effects of the Y223F mutation on the phenotype of E41K-Btk mice, supporting our previous conclusion that Y223 autophosphorylation-dependent interactions are not essential for Btk function during B cell development (27).

Approximately 10–15% of all E41K-Btk pro-B cells expressed detectable levels of {kappa} L chain proteins in the cytoplasm. Because for each {kappa} L chain allele, one of three L chain rearrangements is expected to be productive (and assuming that in the absence of Ig µ H chain protein {kappa}+ and {kappa}– pro-B cells do not manifest differences in survival, expansion, or differentiation capacities), we conclude that V to J rearrangements may be present in up to ~30% of the total pro-B cell fraction in E41K-Btk mice.

We observed that deficiency of Btk or SLP-65 in pre-B cells specifically reduced {lambda} germline transcription, whereas {kappa} germline transcription was not dramatically affected. These analyses were performed in VH81X Tg RAG-1-deficient pre-B cells (Fig. 3), which cannot perform receptor editing, a process that is usually accompanied by increased {lambda} usage (35). Therefore, our findings indicate that Btk/SLP-65 differentially regulate activation of the {kappa} and {lambda} L chain loci independently of their possible role in receptor editing (13). We conclude that the specific reduction of {lambda} germline transcription in Btk- or SLP-65-deficient pre-B cells supports our previous hypothesis that Btk transmits signals that regulate activation of the {lambda} locus for V(D)J recombination in pre-B cells (12). The finding that the absence of either Btk or SLP-65 affects germline transcription of the {lambda} locus, but not of the {kappa} locus, is consistent with various reports indicating the importance of pre-BCR signaling specifically in {lambda} locus activation. First, Ig {kappa} and {lambda} rearrangements occur independently, whereas in pro-B cells Ig {kappa} germline transcription and rearrangements are already present (albeit at low levels), and {lambda} rearrangements are only initiated in small resting pre-B cells when most k rearrangements cease (14, 29, 41). Second, Btk-deficient mice have reduced Ig {lambda} L chain usage (12). However, from the observed cooperative effects of Btk and SLP-65 on Ig {kappa} germline transcription, it is clear that in the absence of SLP-65, Btk is also required for full activation of the {kappa} locus. This would also be supported by our finding that E41K-Btk induces both Ig {kappa} and {lambda} L chain germline transcription in pro-B cells (Fig. 7). Nevertheless, this capacity of the E41K-Btk mutant may alternatively indicate that its expression not only mimics activation of Btk, but also transmits signals that substitute for SLP-65 function, analogous to our previous findings for the Y223F-E41K-Btk mutant (8).

Our experiments do not discriminate whether signals transmitted by Btk/SLP-65 directly induce opening of the Ig L chain loci for recombination or whether these signals only act indirectly by inducing the pre-B cell differentiation program. However, Ig L chain locus activation and pre-B cell maturation are probably closely connected, because transcription factors such as E2A, PU.1, Spi-B, and IFN regulatory factor-4/8 (IRF-4/8) are implicated in both the expression of, e.g., MHC class II or SLC and the opening of Ig L chain loci (42, 43, 44, 45, 46, 47). This would be consistent with recent quantitative analyses of {kappa} 3' enhancer chromatin structure and protein association, which indicate that this enhancer is activated progressively through multiple steps as cells mature (48). The transcriptional enhancers within the {kappa} and {lambda} L chain loci contain binding sites for the transcription factors E2A and EBF. Interestingly, transient transfection of E2A and EBF transcription factors into a nonlymphoid cell line was sufficient to activate V(D)J recombination at the Ig {kappa} and {lambda} loci, respectively (43). However, these transcription factors also activate transcription of the SLC genes {lambda}5 and VpreB (42), which are silenced when Ig L chain rearrangement is initiated. Moreover, the expression of Tg E41K-Btk in pro-B cells concurrently induced activation of Ig {kappa} and {lambda} L chain transcription and down-regulation of SLC expression (Figs. 6 and 7). In RT-PCR analyses, the expression of E41K-Btk in pro-B cells did not appear to result in a significant increase in the expression levels of EBF or E2A (R. W. Hendriks, unpublished observations). It remains possible, however, that Btk or SLP-65 is involved in the induction of EBF or E2A activity by stage-specific regulation of protein stability or expression of partner molecules that interfere with the actions of these transcription factors, such as Id proteins or Ikaros (47). Additional experiments are required to investigate whether Btk/SLP-65 signaling pathways converge on EBF and E2A.

Recently, the transcription factors IRF-4/8 and Spi-B were shown to be important for down-regulation of the {lambda}5 and Vpre-B genes and for the induction of germline Ig {kappa} transcription in pre-B cells (44, 45). However, Tg expression of E41K-Btk in pro-B cells did not appear to result in a significant increase in the expression levels of IRF-4 or Spi-B transcripts (R. W. Hendriks, unpublished observations), indicating that these transcription factors may not function as nuclear effectors of Btk/SLP-65 signaling. This would be supported by recent findings demonstrating that changes in PU.1 and IRF-4 association during {kappa} locus activation are independent of changes in protein expression levels; rather, they reflect changes in chromatin accessibility and histone acetylation (48). In pro-B cells intermediate levels of the transcriptional activators PU.1 and IRF-4 already associate with the {kappa} 3' enhancer, which is moderately accessible at this stage. Therefore, it is likely that pre-BCR signaling results in Ig L chain chromatin structure changes, which are linked to epigenetic marking and affect transcription factor binding site occupancy in the 3' {kappa} enhancer (48). Likewise, it is possible that pre-BCR signaling may down-regulate the expression of SLC chain genes by affecting the epigenetic status of the recently identified regulatory region within the {lambda}5-VpreB locus, which is associated with a tightly localized epigenetic mark of H3 acetylation and histone H3 K4 methylation (47).

Our finding that Btk– B cells fail to efficiently down-regulate RAG-1 expression during the pre-B to immature B cell transition in culture (Fig. 1B) implicate Btk signaling in the termination of V(D)J recombination activity upon productive Ig L chain rearrangement in immature B cells. This would be consistent with the finding that peripheral B cells from X-linked agammaglobulinemia patients, who have mutations in the Btk gene, show a distinct Ab repertoire consistent with extensive secondary V(D)J recombination (49).

In summary, we conclude that the expression of E41K-Btk, which mimics pre-BCR signaling, results 1) in the modulation of cell surface markers that signify pre-B cell maturation, and 2) in the induction and successful completion of Ig L chain rearrangements in pro-B cells. It is therefore unlikely that in pre-B cells, Btk/SLP-65 signaling is only required for the termination of pre-B cell expansion or the survival of small resting pre-B cells. Rather, we conclude that Btk and SLP-65 transmit signals that induce cellular maturation and Ig L chain rearrangement in pre-B cells.


   Acknowledgments
 
We thank K. Dahlenborg, G. Dingjan, A. Maas, and the caretakers from the Erasmus Medical Center animal facility for their assistance at various stages of the project.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by the Netherlands Organization for Scientific Research and the Association for International Cancer Research. Back

2 Address correspondence and reprint requests to Dr. Rudolf W. Hendriks, Department of Immunology, Room Ee851, Erasmus Medical Center, P.O. Box 1738, NL-3000 DR Rotterdam, The Netherlands. E-mail address: r.hendriks{at}erasmusmc.nl Back

3 Abbreviations used in this paper: pre-BCR, precursor BCR; SLC, surrogate L chain; BM, bone marrow; Btk, Bruton’s tyrosine kinase; IRF, IFN regulatory factor; SLP-65, Src homology 2 domain-containing leukocyte-specific phosphoprotein of 65 kDa; Tg, transgenic; WT, wild type; µMT, membrane µ targeted. Back

Received for publication October 18, 2005. Accepted for publication January 19, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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