Maintenance of Surrogate Light Chain Expression Induces Developmental Delay in Early B Cell Compartment¹

Development of B lineage cells is a carefully regulated process, characterized by specific gene expression patterns and circumscribed cellular events. The pre-BCR functions in a unique capacity in the developmental paradigm, facilitating a variety of programs that direct the B cell toward maturity. In this study, we investigate the requirements for the regulation of the pre-BCR using a system that allows us to temporally extend the expression of the pre-BCR.

In the fetal liver and in postnatal bone marrow, a number of cell surface markers and genetic events have been used to identify discrete stages along the developmental pathway (1, 2, 3, 4, 5). The work of a number of groups has shown a correlation among expression levels of specific genes (such as RAG1/2, the surrogate L chain, and TdT), the cell surface presentation of molecules (such as c-kit, CD43, heat-stable Ag, and CD25), the cell cycle status of the cell, and the rearrangement status of the H and L chain loci (1, 5, 6).

Subsequent to commitment to the B lineage, lymphoid progenitors must successfully navigate the pro-B cell/pre-B cell transition. This key stage of development, which can be delineated by expression of an epitope of leukosialin (CD43) and the high m.w. form of CD45 (B220), is a critical checkpoint that plays a role in determining the shape of the mature B cell repertoire (1, 3, 7, 8). The fate of cells at this point is in large part decided by productive rearrangement of the Ig H chain V, D, and J gene segments (9). This rearrangement generates a gene element coding for the V region of the μ H chain (10). Upon completion of this crucial objective, assembly of the pre-BCR can ensue. In order for a cell to progress through the pre-BCR-positive stage, there must be a suitable fit of the μ H chain protein and components of the surrogate L chain (11). It is generally accepted that once a competent, membrane-bound complex can be assembled, signals downstream of this receptor facilitate B cell differentiation (12). The pre-BCR has been implicated in a number of functions, including allelic exclusion, clonal expansion, suppression of reading frame 2 rearrangements, and selection of Ig H chains (11, 13, 14, 15, 16, 17). However, the mechanisms through which all of these processes are regulated, as well as regulation of pre-BCR expression, remain unclear.

The many roles attributed to the pre-BCR have been elucidated primarily by studies in mutant mice. Model systems that are lacking the Igα/β signaling components of the pre-BCR exhibit a defect in the transition of cells from the pro-B cell compartment to the pre-B cell compartment (18, 19). In the μMT mouse model, the inability to deposit a pre-BCR on the cell surface prevents entry into the proliferating pre-B cell pool (20). The absence of surrogate L chain in the mouse model with a targeted deletion of λ5 drastically reduces the efficiency with which cells progress into the pre-B cell compartment (21). Transgenic mice that produce H chains incapable of pairing with surrogate L chain show defects in proceeding through the pre-BCR checkpoint and production of a normal mature B cell compartment (11). All of these models indicate that the pre-BCR has a very specific function in the development of B cells and plays a pivotal role in determining a cell’s outcome. The pre-BCR appears to function in a unique capacity in the developmental paradigm, facilitating a variety of programs that direct a cell toward maturity. The data also indicate that there are few, if any, mechanisms available to fully compensate for the pre-BCR. The significance of this receptor predicts that the timing of its expression would be subject to very fine regulation; moreover, the timing of receptor expression would be crucial to receptor function.

To investigate the validity of these predictions and the requirements for precise regulation of the pre-BCR, we have developed a system that allows us to temporally extend the expression of one component of the pre-BCR, λ5. Our studies reveal that in the condition of extended expression, the pre-BCR can delay the progression of maturing B lineage cells as they move through development toward the stage of Ig secretion. Moreover, maintaining λ5 expression can influence the regulation of other developmental events, including the accessibility of various signaling molecules and cell cycle status. These studies are crucial in furthering our understanding of how these receptors function in development and the significance of fine regulatory control in this system. These data support the concept of a crucial role for the pre-BCR in B cell development and extend the unique function of the surrogate L chain within the B cell developmental paradigm.

The monoclonal B (MB)³ mouse has been previously described (22). In brief, the MB mouse carries a functionally rearranged V_H17.2.25 exon targeted to the IgH locus, a λ1 L chain transgene, and is RAG2^−/−. Further Ig rearrangements are prevented by the absence of RAG2. Fetal liver cells from C57BL/6 mice were taken at day 16 or 17 of gestation. Bone marrow, spleen, and peripheral blood cells from MB and C57BL/6 mice were taken from animals 8–12 wk old. All mice are maintained in specific pathogen-free conditions in our animal colony at the Ontario Cancer Institute.

Single-cell suspensions were prepared from spleen, bone marrow, peripheral blood, and fetal liver of relevant mice. In all cases, RBC were lysed with ACK (0.155 M NH₄Cl, 0.1 mM EDTA, and 0.01 M KHCO₃). For phenotypic analysis of developmental compartments, cells were stained with a variety of Abs from BD Pharmingen, including FITC-conjugated anti-B220 (mAb RA3-6B2), PE-conjugated anti-CD43 (mAb S7), PE-conjugated anti-CD23 (mAb B3B4), PE-conjugated anti-CD22 (mAb Cy34.1), biotinylated anti-CD24 (mAb M1/69), and biotinylated anti-CD25 (mAb 7D4). Also used was a biotinylated anti-μ IgH (mAb 33-60). Staining with biotinylated Abs was revealed with the secondary reagent streptavidin Quantum Red (Sigma-Aldrich). FACS analysis was performed using the FACSCalibur (BD Biosciences).

Bone marrow cells were isolated from MB and C57BL/6 mice to analyze stage-specific expression of developmentally regulated transcripts. Cells were stained with FITC-conjugated anti-c-kit (mAb ACK4; gift from S. Berger, University of Toronto, Toronto, Ontario, Canada), PE-conjugated anti-B220 (mAb RA3-6B2; BD Pharmingen), and biotinylated anti-CD25 (mAb 7D4; BD Pharmingen). Two populations were isolated. Fractions consisted of either B220⁺c-kit⁺CD25⁻ cells or large B220⁺c-kit⁻CD25⁺ cells.

Cells were also sorted subsequent to retroviral infection. Bone marrow and fetal liver cells from C57BL/6 mice were stained with FITC-conjugated anti-B220 (mAb RA3-6B2; BD Pharmingen), PE-conjugated anti-CD43 (mAb S7; BD Pharmingen), and biotinylated anti-μ IgH (mAb 33-60). Sorted cells were B220⁺CD43⁺μ⁻, representing a population transitioning from the pro- to pre-B cell compartment. All cells were sorted using FACStar^Plus (BD Biosciences) and MoFlo (Cytomation) instrumentation.

Total RNA was isolated from B220⁺c-kit⁺CD25⁻ and B220⁺c-kit⁻CD25⁺ sorted cells using Tri Reagent (Molecular Research Center). First-strand cDNA was synthesized using Superscript II (Invitrogen Life Technologies). From each population, transcripts were amplified for λ5 (component of the surrogate L chain). λ5 cDNA was amplified using 5′ LAM5, 5′-CTTGAGGGTCAATGAAGCTCAGAAGA-3′ and 3′ LAM5, 5′-CTTGGGCTGACCTAGGATTG-3′ (1). Hypoxanthine guanine phosphoribosyltransferase (HPRT) cDNA was amplified as a control for the presence and quality of amplifiable template in each reaction. β-actin cDNA was amplified to normalize the amount of transcript present. A small fraction of each primary PCR (λ5 surrogate L chain amplifications) was used to provide HPRT and β-actin templates. HPRT was amplified using 5′ HPRT, 5′-GTAATGATCAGTCAACGGGGGAC-3′ and 3′ HPRT, 5′-CCAGCAAGCTTGCAACCTTAACCA-3′. β-actin was amplified using 5′ β actin, 5′-CCTAAGGCCAACCGTGAAAAG-3′ and 3′ β-actin, 5′-TCTTCATGGTGCTAGGAGCCA-3′. All primers were designed to amplify regions that spanned introns to distinguish the amplification of DNA from cDNA.

PCR products were electrophoresed through 1.0% agarose gels and transferred to Hybond nylon membranes (Amersham Biosciences). Blots were hybridized with the following relevant ³²P-radiolabeled probes: λ5 INT probe, 5′-CCAGGCAGTGTGAAGTTCTCCTC-3′; HPRT probe, 5′-GTGGAGATGATCTCTCAACTT-3′; and β-actin probe, 5′-GTTTGAGACCTTCAACACCCCAGCCATGTA-3′. Hybridized blots were exposed to a phosphor screen (Molecular Dynamics/Amersham Biosciences), and subsequently, radioactivity was visualized and quantified with a Storm 860 PhosphorImager.

Abelson cell lines were derived, as previously described (23). Briefly, bone marrow cells extracted from MB mice were plated at 2 × 10⁶ cells/ml and seeded with Abelson viral stocks (provided by S. Lewis, University of Toronto, Toronto, Ontario, Canada). For infection, supernatants were combined with 4 μg/ml polybrene (Sigma-Aldrich). Following infection and extended culture, cells were diluted in single-cell suspensions of 0.3% soft agarose (Difco-Bacto Agar) in RPMI 1640 medium supplemented with 10% FCS, 2 mM l-glutamine, and 50 μM 2-ME. Colonies arising from single clones in soft agar were expanded in liquid medium.

Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen). mRNA was reverse transcribed into cDNA using Enhanced Avian Reverse Transcriptase (Sigma-Aldrich), according to the manufacturer’s instructions. cDNA was amplified and measured using TaqMan primers and probes (Applied Biosystems) on the ABI Prism 7700 Sequence Detection System (Applied Biosystems). Transcripts from the following genes were amplified: Btk, Lambda5, Blnk, Syk, and Lyn. All samples were run in triplicate and normalized to 18S. Primers and probes were designed to span introns to eliminate the amplification of genomic DNA. Data were analyzed with Prism 7700 Sequence Detector version 1.9.1 software.

The MIEV vector (gift from R. Hawley, American Red Cross, Rockville, MD) was constructed from the murine stem cell virus-based internal ribosome entry site-neovirus retroviral vector, as described (24). It contains the EGFP gene from the pEGFP-1 plasmid (BD Clontech) (25). The coding sequence for the murine λ5 gene was cloned into the XhoI-SalI multiple cloning site of MIEV. Due to the presence of the internal ribosome entry site in MIEV, inserted sequences and the EGFP gene are assembled into a bicistronic transcript such that the presence of GFP indicates the expression of the cloned gene. Retroviral constructs were transfected into BOSC 23 packaging cells using Effectene reagent (Qiagen). GP + E cells were infected with 48-h BOSC 23 supernatants, and GFP expression was verified by FACS analysis. GFP⁺ cells were sorted using the FACStar^Plus (BD Biosciences) and used to infect C57BL/6 B220⁺CD43⁺μ⁻ B cells. Cells were infected in a coculture system for 72 h, followed by sorting of GFP⁺ B lymphocytes that were then plated at limiting dilution. Lymphocytes were cultured in OPTI-mem (Invitrogen Life Technologies) with 10% FCS, 10 ng/ml rIL-7 (R&D Systems), and 8 μg/ml polybrene.

Tissue culture grade 96-well plates were seeded with the S17 cell line (gift from K. Dorshkind, University of California, Los Angeles, CA) at a density of 1500 cells/well. After 24 h, cells were irradiated at 2000 rad and used as the feeder layer for infected B cells. B cells were infected by virus carrying either MIEV-LAM5 or MIEV-empty (EMP). Lymphocytes were plated at limiting dilution with 32 wells for each cell density. Medium was supplemented with 10 μg/ml LPS (Difco Laboratories).

Immunosorbent 96-well plates were coated with goat anti-mouse IgM (Jackson ImmunoResearch Laboratories). Secreted IgM from each well of the limiting dilution assay was detected with HRP-conjugated goat anti-mouse IgM (Sigma-Aldrich). Plates were developed with the TMB Microwell detection system (Kirkegaard & Perry Laboratories). Color development was limited by addition of 1 N sulfuric acid. Each plate was standardized with known titrated quantities of Ab. OD₄₀₅ values were read by an OPTI-Max spectrophotometer (Molecular Dynamics).

B cells carrying either the MIEV-LAM5 or the MIEV-EMP retroviral vector were plated in 96-well plates at a density of 3000 cells/well. Cells were grown in OPTI-mem (Invitrogen Life Technologies) with 10% FCS and 10 ng/ml rIL-7 (R&D Systems). Each well was supplemented with 1 μCi of [³H]thymidine and incubated at 37°C, 5% CO₂ for 9 h. Cells were then lysed with the Filtermate cell harvester (Packard Bioscience), and levels of incorporated [³H]thymidine were measured on a TopCount NXT (Packard Bioscience). Cells were pulsed with [³H]thymidine, and proliferation was assayed at very early time points to assess proliferation differences due only to differential receptor expression.

B cell clonal expansion is observed in early B cell compartments, during the pro- and pre-B cell stages of development (14, 26, 27, 28). The proliferation at this stage of development is thought to enrich the population for cells that have assembled a productive VDJ rearrangement at the H chain locus. To assess whether pre-BCR expression affects the proliferative capacity of pre-B cells, we devised a retroviral expression system to increase and maintain expression of the surrogate L chain component λ5. The availability of λ5 protein has been shown to stabilize VpreB protein and would therefore augment the assembly of the surrogate L chain and pre-BCR complexes (15, 29). In addition, VpreB deficiency, unlike λ5 or μHC deficiency, does not markedly affect B cell development in mice, suggesting that the VpreB proteins are present in excess over the components of the pre-BCR. Detailed analysis revealed that VpreB1 protein is present in excess, and VpreB2 is not, because haplosufficency (VpreB1^+/−VpreB2^+/− and VpreB1^+/−VpreB2^−/−) does not impair B cell development (30, 31). cDNA coding for λ5 was inserted into the XhoI-SalI site of the MIEV vector (32). As described in Materials and Methods, B220⁺CD43⁺μ⁻ B cells harvested from C57BL/6 wild-type bone marrow expressing either the MIEV-EMP or MIEV-LAM5 construct (Fig. 1) were plated in 96-well plates and pulsed immediately with tritiated [³H]thymidine to assess proliferation. This assay is only 9 h; thus, cell division per se would not be expected: the assay solely discerns the relative DNA synthesis activity of the cells. As measured by [³H]thymidine incorporation, GFP⁺ cells carrying MIEV-EMP demonstrated proliferation levels similar to levels observed in GFP⁻ (uninfected) cells (Fig. 2). This result indicated that expression of the vector does not mediate a proliferative advantage and that cells behave similarly to those that have not been infected. GFP⁺ cells carrying the MIEV-LAM5 vector, however, showed a 3-fold increase in proliferation as compared with GFP⁻ cells and cells infected with the empty vector MIEV-EMP (Fig. 2). This result demonstrates that expression of the pre-BCR can have a direct effect on proliferation and hence the growth of pre-B cell clones, and provides evidence of a role for the pre-BCR in expanding the developing B cell pool.

The BCR and the pre-BCR play essential roles in the fate and function of a developing B cell. The expression of these molecules determines the activities of the cell, whereas the strict regulation of each of its parts ensures adherence to a precise developmental program. Despite being responsible for a variety of functions, the BCR and pre-BCR are associated with similar signaling molecules (33, 34). The expression and compartmentalization of these molecules contribute to their participation in different tasks (35, 36).

To determine whether maintained expression of λ5 and the pre-BCR could have an effect on the intracellular activities of B cells, we examined the expression patterns of molecules known to be involved in signaling pathways downstream of the BCR and pre-BCR. Using B cell lines derived from MB mouse bone marrow (a mouse model carrying a preformed IgH and IgL on a RAG2-deficient background), we examined the expression of molecules associated with signaling through these receptors by comparing cells expressing either the mature BCR, the pre-BCR, or both.

Cell line MB1P2 is a clone representing a B cell expressing only the mature BCR, cell line MB1P4 is a clone representing a cell expressing a pre-BCR only, and MB1P5 is a clone expressing a mixed cell surface phenotype.

To verify the cell line phenotypes and their use as models, the transcript levels for the surrogate L chain component λ5 were measured. As shown in Fig. 3 A, only lines MB1P4 and MB1P5 express detectable levels of λ5, in which the MB1P4 line appears to have 2× the levels found in the MB1P5 line. As expected of a mature B cell model, MB1P2 has no detectable transcript for the surrogate L chain component λ5. All cell lines were derived in the same manner from genetically identical animals, providing an ideal system for comparisons.

Fig. 3,B shows the comparison of the expression levels of the nonreceptor protein tyrosine kinase lyn. Lyn is a src family kinase associated with the phosphorylation of ITAMs in the Igα/β subunits of the BCR, as well as other signaling molecules (37). It is thought to have positive and negative influences on BCR signaling, and although it has some role in early B cell development, it appears to make a more significant contribution in the later stages of development and B cell function (38, 39, 40, 41, 42, 43, 44). Transcript levels for lyn are shown to be significantly higher in the mature B cell line MB1P2 as compared with the pre-B cell line MB1P4. The mixed receptor line MB1P5 also expresses lower levels of lyn, more closely resembling expression levels in MB1P4 (Fig. 3,B, right panel). Syk is a nonreceptor tyrosine kinase thought to be involved in pre-BCR signaling and with variable signaling contributions in immature and mature B cells (39, 45, 46, 47). Transcript levels of syk were negligible in cell lines MB1P2 and MB1P4 (Fig. 3 C), whereas they were low, but detectable in the MB1P5 line. This may reflect the regulation of syk, which appears to be carefully regulated at specific stages in development to allow differentiation and reduce proliferation (48). MB1P5, however, exhibits an expression pattern that is distinct from these more conventional cell lines, indicating that cells with simultaneous and continuous expression of both forms of the BCR may have dysregulated gene expression and, therefore, unique expression profiles.

Fig. 3,D compares the expression pattern of btk in each of the cell lines. Btk is the gene associated with the xid disease in mice and X-linked agammaglobulinemia in humans (49, 50, 51, 52). It has been shown to have a very prominent role in early B cell development as well as functioning in general B cell signaling (53, 54, 55, 56). These data show comparable levels of btk expression in lines MB1P4 and MB1P5 (a 2-fold difference), whereas the detectable levels in line MB1P2 were negligible (Fig. 3 D, left panel). This result is consistent with previous findings that mature B cells can have low or undetectable levels of btk (57).

Fig. 3 E shows the levels of BLNK expressed in each of the cell lines. BLNK (also known as SLP-65 or BASH) is an adaptor molecule thought to interact with syk and btk in BCR signaling and to play an integral role in calcium mobilization (58, 59, 60, 61). These results show that whereas the mature MB1P2 line expresses a 4-fold greater amount of BLNK as compared with MB1P5, there is only a 2-fold difference in expression between MB1P5 and the pre-B cell line MB1P4.

Taken together, these data demonstrate that overall, cell lines expressing both the mature and pre-BCR present a gene expression profile that is more similar to a pre-B cell than a mature cell. These data indicate that extending expression of λ5 and the pre-BCR can skew the intracellular profile of a cell to that of a less mature state even in the presence of the mature BCR. These findings illustrate the significance of the regulation of λ5 expression with respect to developmental progression and the ability of a cell to implement programs consistent with B lineage maturation.

Observations in cell lines with differential expression of λ5 and the pre-BCR provide evidence for some of the consequences of receptor expression, persistence of λ5 on the cell surface, and a representation of how this may function in B cell development. To extend these findings, we investigated the expression of these receptors in a primary cell system. The gene for the λ5 component of the surrogate L chain, among others, is tightly regulated through B cell development (1, 62). It is unclear, however, precisely how its expression is controlled. A decrease in λ5 transcript levels coincides with the small pre-B cell stage of development, the onset of L chain gene rearrangement, and, of course, a decline in pre-BCR expression. As described above, our investigations with cell lines correlate receptor expression patterns with distinct gene regulation profiles that have implications for cell function. Temporally extending receptor expression in a primary cell system will determine whether the predictions from the cell line data are fulfilled. To determine whether prolongation of λ5 expression would retard or interfere with B cell differentiation, the retroviral construct encoding λ5 was expressed in wild-type pro-B cells, following which B cell progression was monitored.

As previously described, B220⁺CD43⁺μ⁻ pro-B cells were sorted from C57BL/6 bone marrow or fetal liver (see Fig. 1) and infected with retrovirus carrying either the empty construct MIEV-EMP or the λ5-expressing construct MIEV-LAM5. The infection rate ranged from 60 to 80%. Following the infection, cells were sorted for GFP⁺ expression and plated at limiting dilution with a stromal cell layer and LPS. The limiting dilution assay used at least six cell concentrations. Supernatant from each well was sampled and analyzed for the presence of IgM. The frequency of B cell precursors capable of secreting Ig was calculated based on the Poisson distribution. In the condition of extended pre-BCR expression, maintained by continuous expression of λ5, there was a lower frequency of maturing B cells as compared with cells without extended pre-BCR expression (Fig. 4). Based on six independent experiments, cells capable of achieving Ig secretion were on average 8-fold less frequent under the condition of modified pre-BCR presentation. In the examples shown, mature B cells were ∼6× more frequent (1 in 290 vs 1 in 1,600) and 16× more frequent (1 in 2,300 vs 1 in 37,000) in the absence of manipulated pre-BCR expression (Fig. 4, A and B, respectively). These data indicate that the kinetics of B cell differentiation are delayed upon maintenance of pre-BCR expression.

The delay in B cell maturation was also evident upon analysis of phenotypic markers. As cells move through the pre-B cell compartment, expression of surface μ H chain and the α-chain of the IL-2R (CD25) begins to increase. When compared with cells infected with the empty MIEV vector, fewer MIEV-LAM5 infectants were expressing increased levels of both μ and CD25 (Fig. 5). The population of cells expressing higher levels of μ H chain shifted to 22% in cells carrying MIEV-EMP, whereas a shift of only 14% was seen in cells carrying MIEV-LAM5. Increased CD25 expression was observed in 93% of MIEV-EMP-expressing cells as compared with 84% in MIEV-LAM5-expressing cells. These results indicate that cells that do not shut off components of the surrogate L chain are less efficient at completing their developmental programs.

The regulated pattern of gene expression during B cell differentiation ensures that the appropriate signals, tests, and programs are implemented to produce a diverse, mature repertoire. The MB mouse, which expresses a single IgH molecule and a single IgL molecule during development of the B lineage, exhibits a marked reduction in the number of mature B cells. The splenic compartment has 65% fewer B cells, on average, than a normal (C57BL/6) spleen.

As many studies have shown, early events in B cell development are crucial to allow efficient B cell progression. In this context, and in light of our findings with retroviral extension of λ5 expression, we investigated the regulation of λ5 in bone marrow cells from C57BL/6 and MB mice. Bone marrow cells were sorted into early and late B cell fractions. The early stage cells are B220⁺c-kit⁺CD25⁻, whereas late-stage cells are characterized by the absence of c-kit and the presence of CD25 (B220⁺c-kit⁻CD25⁺). λ5 transcripts were evaluated by PCR and normalized with β actin. Comparison of λ5 transcript expression levels in early and late developmental fractions of C57BL/6 B lineage cells revealed that as B cells mature, the quantity of transcript decreases ∼40-fold (Fig. 6, A and C). This result is consistent with studies that have shown a decline in pre-BCR expression and related transcripts as B cells progress (1). In MB B lineage cells, however, this reduction is not seen. Levels of λ5 transcripts remain stable in later stage B cells, i.e., transcript levels are similar at both stages (Fig. 6, B and C). The levels of HPRT transcripts were unchanged. The HPRT measurements verify the assay, representing a gene expected to have an unaltered expression pattern in these two compartments. Examination of the peripheral B cell compartment based on cell surface markers revealed that in the MB system, the development of mature B lymphocytes was severely compromised. Cell surface expression of CD45R, CD22, and CD23, known to increase with B cell maturation, was appreciably diminished in the MB system (Fig. 7). The reduced expression of the differentiation-specific markers CD25, CD43, and heat-stable Ag, normally observed at this stage, was also not observed. B lineage cells expressing high levels of μHC and low levels of B220 are normally not found in the peripheral compartment; however, a substantial population was present in MB mice (Fig. 7, first row). The lack of a substantial mature B cell compartment in the MB system was consistent with the developmental delay seen upon extension of λ5 expression in our retroviral system. These results suggest that maintaining expression of components of the pre-BCR, with their related signaling and developmental functions, has consequences for normal B cell progression.

Collectively, these data indicate that expression of the pre-BCR is associated with specific and distinct cellular programs that play a direct role in how cells expressing these receptors respond to internal and external signals. Altering the regulation of a single component of the pre-BCR is sufficient to modify cellular function, and thereby underscores the significance of strict regulatory programs in B cell development.

The successful completion of the B cell developmental program depends on a fine balance of signals, thresholds, and gene regulation (1, 5). The various cell surface markers and expressed receptors associated with a particular stage of development are most likely modulated by events taking place inside the cell, but also play a role themselves in modifying the operations of the cell. Our studies reveal that maintained expression of the pre-BCR can alter the progress of a developing B cell. By controlling the expression of the pre-BCR, we identified a direct role for this receptor in the proliferation of pre-B cells in the presence of IL-7. These findings confirm the significance of this receptor in development and demonstrate that its function is distinct.

The relationship between the pre-BCR and clonal expansion is a concept based on observations of the behavior of pre-BCR-positive cells (6, 26). Cells that are unable to form a competent pre-BCR do not enter the proliferating pre-B cell compartment (8, 14, 63). However, it has been unclear whether deposition at the cell surface is directly involved in mediating enhanced proliferation or whether it merely provides a signal for survival of the cell (64). In the system presented in this study, the enhanced expression of pre-BCR complexes, as established by the maintenance of λ5, confers a proliferative advantage to the B cell. This result is consistent with the findings that de novo synthesis of the pre-BCR can induce proliferation in pre-B cells (65). The observations by this group indicate that formation of pre-BCR complexes in the presence of IL-7 or stromal cells is sufficient to promote clonal expansion. The signals and events associated with the pre-BCR are, in some fashion, responsible for the proliferative burst associated with this stage of development, and our data support and extend these findings. Although stromal cells were not present in our system, and thus could not contribute to the observed proliferative expansion, the role of pre-BCR ligands such as heparin or heparan sulfate (present in FCS) cannot be excluded (66). Others have observed that cells can enter cycle at an early pro-B cell stage before expression of the pre-BCR; however, these studies did not address whether pre-BCR can still function to augment this proliferation (67). In fact, the authors note that the pre-BCR-positive pool is enriched for productively rearranged IgH loci, an observation that instead suggests that the pre-BCR imparts a proliferative advantage. The mechanism of this B cell expansion has not been clearly defined, because it remains uncertain whether and how a ligand is involved in this pre-BCR function (26, 68, 69, 70, 71, 72).

In the model we propose, namely that increased pre-BCR expression mediates a higher rate of cell division, cells other than pre-B cells are not required to enhance this proliferation. It is also notable that the increase in proliferation is only observed in the cells with augmented expression of the pre-BCR and not in those cultured alongside them that do not express this receptor. This conclusion is illustrated by comparable proliferative behavior in uninfected cells isolated from both conditions (i.e., uninfected cells cultured alongside either MIEV-EMP infectants or MIEV-LAM5 infectants) (Fig. 2). This evidence suggests different outcomes for a cell according to the number of receptors that it expresses. The positive selection of B cells and preferential clonal expansion are concordant with these data, in which the competence of the H chain/surrogate L chain interaction will determine how efficiently pre-BCRs are deposited on the surface (reviewed in Ref. 73). Therefore, B cells that express H chains that interact well with surrogate L chain will be more likely to expand and survive in the periphery.

The developmental stage of a cell and its expression of the mature or precursor BCR have implications for the expression of signaling components that are integral to their function. These signaling molecules, which are essential elements in the control of gene transcription and cell function, have the ability to alter cellular outcomes depending on their localization and level of expression (48, 74). These results indicate that B cells, at different stages of development and cell surface phenotypes, are associated with a distinct pattern of molecules that are crucial to differentiation and function. This would have implications for the transduction of signals and cell functions that are affected by these molecules. Additionally, this interpretation is borne out in the distinct calcium flux profiles observed downstream of signaling in B cell lines as a result of activation through either the pre-BCR, the mature BCR, or both on cells possessing a mixed cell surface phenotype (our unpublished observations). Studies indicate that the spatiotemporal characteristics of calcium signals provide important information regarding the determination and regulation of transcription factors (75). Evidence has also shown that the function of the various forms of the BCR is determined by the context of its expression (76). Receptors expressed on cells with incongruous differentiation states reveal differences that can include the species of molecules that are involved, as well as quantitative differences in the recruitment of various signaling elements (76). Taken together, the modified regulation of surrogate L chain components and the alteration of various signaling-related elements point to a unique environment in which differentiation signals can be modified and cellular outcomes altered. These findings have implications for the nature of the signals that must coalesce in order for normal B cell development to occur, as well as the distinct characteristics associated with the receptors that are central to normal B cell function.

The developmental delay observed in the maintenance of pre-BCR expression points to a distinct role for the receptor in B cell development. The continued presence of the receptor appears to abrogate assembly of a mature receptor or undermine its function with respect to progression. A potential mechanism of this abeyance involves the augmented proliferative capacity imparted by the pre-BCR. The continued expression of the pre-BCR, without dilution or down-regulation, may function to maintain the cycling status of the cells. Maintenance of a high level of proliferation may inhibit the onset of cellular events that coincide with an exit from cycle. Studies have shown that the increased proliferative capacity of BLNK^−/− and double-deficient BLNK/btk pre-B cells is linked to the increased expression of the IL-7R and the pre-BCR, leading to inefficient differentiation (77, 78, 79). The stability of RAG2 protein is dependent upon the cycle status of the cell (80). If the cell remains in cycle, functional RAG2 protein may not accumulate to suitable levels and the efficiency of recombination at the L chain locus will not increase. This situation, of course, will affect the rate at which cells attain a mature phenotype and progress to the stage of LPS responsiveness. An alternative hypothesis is that the pre-BCR acts to improve survival of the developing B cell, and that maintaining pre-BCR expression allows cells to survive that normally would not. In this scenario, increased survival dilutes cell numbers and reduces the frequencies of cells that achieve IgM secretion. This scenario, however, is inconsistent with our findings that cells carrying the MIEV-EMP vector do not undergo spontaneous apoptosis more rapidly than cells carrying MIEV-LAM5 (our unpublished observations).

The signals and functions that are unique to the pre-BCR may hinder progression due to their incompatibility with other maturation events. The increased and continuous presence of pre-BCR complexes may alter the accessibility to certain molecules and/or allow a particular signaling pathway to dominate. Our studies have shown that molecules thought to play a role in transmitting pre-BCR and BCR signals are differentially expressed depending on the differentiation state of the cell. These differences may account for the differential recruitment and employment of various components in the signaling pathway, such as distinctive phosphorylation patterns and subcellular protein localization (76, 81). We and others have found distinct calcium mobilization profiles upon aggregation of the pre-BCR, contrasting its downstream effects with those of the BCR (35, 76, 82, 83). Unique proteins have also been detected following pre-BCR signaling, but have yet to be characterized (70). All of these data point to particular signaling functions of this receptor that contribute specifically to development.

The promoter for the λ5 component of the surrogate L chain is B cell stage specific, and function of the pre-BCR is precisely delineated (62). Continuous expression of λ5 from a retroviral promoter in this system would undoubtedly affect the perceived operations of a cell. As it has been shown in this study, interference with the precise regulation of λ5 disrupts the normal sequence of later maturation events and the onset of other crucial gene activities. Contributing to the developmental delay may be the inability of mature L chain to displace surrogate L chain. Indeed, we have in this study provided support for the concept that pre-BCR-associated signals and cellular activities have exclusive characteristics, and our observations in animals with preformed IgL chain genes indicate that there are further changes beyond L chain rearrangement that must take place in order for a cell to achieve maturity.

The persistence of λ5 expression, and its ability to impede B cell maturation, provides novel insight into the function of the pre-BCR. The inefficient generation of mature B cells in the MB system may wholly, or in part, be attributable to dysregulation of the pre-BCR. Pre-BCR function is virtually indispensable in the generation of a normal B cell compartment, providing signals that shape the repertoire and proportions of the mature pool. Our studies demonstrate for the first time that temporal modification of pre-BCR expression can impact on the scheme of differentiation, emphasizing the importance of its regulation for normal function. Evidence provided in this study serves to delineate that the participation of various molecules throughout development is affected by the differentiation state of the cell. These data also clearly indicate that dysregulation of λ5 expression is sufficient to alter developmental progress. In this system, with the ability to alter pre-BCR regulation and analyze developmental events at various time points, we attain the crucial ability to further dissect pre-BCR function to establish the critical aspects of its role in progression.

We are grateful to the members of the Wu and Paige laboratories for helpful discussions and to Lesley Cunningham for editorial assistance. We thank Claude Cantin for excellent assistance with flow cytometry and cell sorting, and Stacy Hirano and Tara Collins for expert technical assistance. We thank Dr. Robert Hawley for the gift of the MIEV vector and Dr. Stuart Berger for the ACK4 Ab.

The authors have no financial conflict of interest.