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The Journal of Immunology, 2006, 177: 2242-2249.
Copyright © 2006 by The American Association of Immunologists

Pre-B Cell Receptor Assesses the Quality of IgH Chains and Tunes the Pre-B Cell Repertoire by Delivering Differential Signals1

Yohei Kawano, Soichiro Yoshikawa, Yoshiyuki Minegishi and Hajime Karasuyama2

Department of Immune Regulation, Tokyo Medical and Dental University Graduate School, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
It is well understood how a variety of Ig H and L chains, components of BCR, are generated in the DNA level during B cell development. However, it has remained largely unknown whether and how each component is monitored for its quality and selected before the assembly into the BCR. Here we show that µH chains produced by pre-B cells display a wide spectrum of ability to form the pre-BCR, which is composed of µH and surrogate light (SL) chains and is crucial for B cell development. The level of surface pre-BCR expression varies among pre-B cells, depending on the ability of their µH chains to pair with SL chains. The higher the level of pre-BCR expression by pre-B cells, the stronger their pre-BCR signaling, and the better they proliferate and differentiate. Thus, the extent of survival, proliferation, and differentiation of individual pre-B cells is primarily determined by the SL-pairing ability of their µH chains. Furthermore, IgH chains with higher potential to assemble with IgL chains appear to be positively selected and amplified through the assessment of their ability to pair with SL chains at the pre-BCR checkpoint before the assembly into the BCR. These results indicate that the pre-BCR assesses the quality of µH chains and tunes the pre-B cell repertoire by driving the preferential expansion and differentiation of cells with the higher quality of µH chains.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The immune system can respond to and eliminate a vast array of potential pathogens by virtue of the diverse repertoire of Ag receptors expressed on B and T cells. The huge repertoire of BCRs in the B cell population is generated through recombination of the V, D, and J segments of Ig genes during B cell differentiation in the fetal liver and adult bone marrow (1, 2). A drawback of this strategy is that it inevitably produces harmful autoreactive B cells. However, these cells are eliminated by means of receptor editing or deletion (3). Thus, the formation of the BCR is an important checkpoint for selecting appropriate B cells in the course of their development.

Another important checkpoint has been identified at the pre-B cell stage before BCR formation. The rearrangement at the IgH locus usually takes place before that at the IgL locus during B cell development (1, 2). Once a productive rearrangement occurs at the IgH locus at the pro-B cell stage, µH chains are produced and assembled with invariant VpreB/{lambda}5 surrogate light (SL)3 chains to form the pre-BCR (4, 5). A deficiency in pre-BCR formation or signaling results in severe impairment of B cell differentiation in both humans and mice (6). Thus, the formation of the pre-BCR functions as a checkpoint to positively select the pro-B cells that have succeeded in a productive rearrangement of the IgH chain gene (7, 8, 9). Two different transgenic mice were established, both carrying a productively rearranged µH transgene; one showed intact B cell development, but the other showed a complete blockade of B cell development at the pro-B cell stage (10, 11). µH chains expressed in the former paired with SL chains to form the pre-BCR, whereas those in the latter did not. Approximately half of the µH chains produced by early pre-B cells in the adult bone marrow and up to 80% of those produced in the fetal liver are incapable of pairing with SL chains to form pre-BCRs (12, 13, 14). These impotent µH chains are also deficient in their ability to pair with conventional IgL chains (10). Thus, the formation of the pre-BCR functions as a checkpoint to verify not merely the production of µH chains but also their functionality in advance of their association with conventional L chains (15).

It remains to be answered whether all of the SL-pairing µH chains can associate with conventional L chains and whether µH chains with better fitness to L chains are selected before they are assembled into the BCR. SL chains are invariant in their structure, whereas µH chains expressed in individual pre-B cells differ in their variable region. If there is a gradient in the avidity of interaction between µH chains and SL chains (16), further questions are raised, including the following. What are the functional consequences of the difference in the avidity? Does it affect surface pre-BCR expression, the strength of pre-BCR signaling, and/or the proliferation and differentiation of pre-B cells that are induced by pre-BCR signaling?

Bone marrow pro-B cells from normal mice have been shown to divide 2–5 times when cultured in vitro without added cytokines such as IL-7 (17). No pro-B cells from {lambda}5–/– mice divide under the same culture conditions, suggesting that the cell division depends on the expression of the pre-BCR. One hypothetical model has been proposed in which the variation in the extent of cell division observed in individual pro-B cells is determined by the avidity of their µH chains for SL chains, independent of cytokines or stromal cells (15, 17). However, this possibility has not yet been proven. Another group has demonstrated a collaborative interaction between the pre-BCR- and the IL-7R-signaling pathways that promotes the proliferation of pre-B cells (18, 19). According to the model proposed in these reports, the threshold for pre-B cell proliferation in limiting concentrations of IL-7, as is expected to be the case in vivo, can only be reached by a combination of signals derived from both the pre-BCR and IL-7R. This provides pre-BCR+ pre-B cells with a proliferative advantage over pre-BCR– pre-B cells (20). The discrepancy between these two models with regard to the requirement for IL-7 for pre-B cell proliferation remains to be resolved.

We previously identified two distinct subpopulations among early pre-B cells expressing both µH and SL chains in the bone marrow and fetal liver; one produces SL-pairing µH chains, and the other produces SL-nonpairing µH chains (14). A comparative analysis demonstrated that the subpopulation expressing SL-pairing µH chains was positively selected during B cell development in fetal liver as well as in bone marrow. Here, we extended this study and demonstrated that the SL-pairing ability of µH chains was much more diverse than previously thought and that there were qualitative differences among the SL-pairing µH chains in terms of the pre-BCR formation and signaling. The quality of µH chains produced by individual pro-B cells determines the fate of these cells at the transition from the pro-B cell stage to the pre-B-cell stage via the differential pre-BCR signaling that affects cell survival, proliferation, and differentiation. Therefore, this mechanism impacts the establishment of the primary B cell repertoire even before L chains are produced. These findings provides new insights into the mechanism whereby the IgH chains, components of the BCR, are assessed for their quality to be selected before the assembly into the BCR.


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

Mice deficient for the membrane exon of the µH chain gene (µMT mice) (21) were bred and maintained under specific pathogen-free conditions in our animal facility. All of the experiments in this study were performed according to the guidelines for animal use and experimentation as set forth by the Tokyo Medical and Dental University (Tokyo, Japan).

Cell lines

The pro-B cell line 38B9 (22) was grown in IMDM supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/ml penicillin-streptomycin, and 5 x 10–5 M 2-ME at 37°C in 5% CO2. The retroviral packaging cell line Plat-E (23) was cultured in DMEM supplemented with 10% FCS, 100 U/ml penicillin-streptomycin, 5 mg/ml puromycin (Sigma-Aldrich), and 5 mg/ml blasticidin (Invitrogen Life Technologies).

Antibodies

Abs specific to {lambda}5 and VpreB (M-17; catalog no. sc-25014) were purchased from Santa Cruz Biotechnology. Phospho-specific Abs to c-Abl (Tyr245), STAT5 (Tyr694), ERK (Thr202/Tyr204), Syk (Tyr525/Tyr526), B cell linker (BLNK) (Tyr96), and Akt (Ser473) were from Cell Signaling. Abs specific to c-Abl and {alpha}-tubulin were from Oncogene Research Products and Sigma-Aldrich, respectively. The HRP-conjugated anti-mouse µH chain was from Southern Biotech. The allophycocyanin-labeled Ab to the µH chain, CD19, the biotin-labeled Ab to c-kit, CD2, and the PE-labeled Ab to {kappa}L chain were from BD Pharmingen. The pre-BCR-specific mAb (SL156) (24) was biotinylated in our laboratory.

Construction of and infection with retroviral vectors

VHDHJH-rearranged DNA fragments were isolated from C57BL/6 fetal liver and adult bone marrow cells and subcloned into pBS-µH (14) to obtain cDNAs encoding a membrane form of µH chains, which were then inserted into the retroviral vector pMX-IRES-GFP (23) as described previously (14). Plat-E cells were cultured in Plat-E medium for 24 h and then infected with pMX-IRES-GFP carrying the indicated cDNA using FuGENE (Roche). The culture supernatants were collected 48 h later. The infection of 38B9 pro-B cells with retroviral vectors was performed as described (14). To infect the bone marrow pro-B cells, B220+ pro-B cells were enriched from the bone marrow cells of µMT mice using B220 MACS beads (Miltenyi Biotec). Cells were suspended in complete IMDM and prestimulated with recombinant mouse stem cell factor (50 ng/ml; Peprotech) and mouse IL-7 (50 ng/ml, PeproTech) for 24 h. After being washed, 3 x 106 cells in complete IMDM per well in 24-well plates were infected with retroviral supernatants in the presence of 8 µg/ml Polybrene (Sigma-Aldrich), mouse stem cell factor (50 ng/ml), and mouse IL-7 (50 ng/ml) for 24 h. Following several rounds of washes, cells were suspended and cultured in complete IMDM. Under these experimental conditions, we found no apparent toxic effect of Polybrene and no significant difference between infections with and without the empty vector in terms of cell survival and proliferation.

RT-PCR

Total RNA was prepared from cells by using ISOGEN (Toyobo). cDNAs were synthesized with an oligo(dT) primer (Amersham Biosciences) and then amplified by PCR with primers specific for germline {kappa} transcript and beta-actin (25). The PCR consisted of 30 cycles for the germline {kappa} transcript or 24 cycles for beta-actin of 94°C for 30 s, 58°C for 30 s, and 72°C for 45 s.

ELISA

GFP+ 38B9 transfectants (1 x 107) were lysed with 500 µl of lysis buffer (1% Nonidet P-40, 10 mM Tris-HCl (pH7.5), 150 mM NaCl, and 1 mM EDTA) supplemented with complete protease inhibitor (Roche). To detect the total µH chains and the µH chains associated with {lambda}5 in cell lysates, flat-bottom ELISA plates (Sumilon) were coated with 2 µg/ml anti-µH chain mAb (M41) (26) or 3 µg/ml anti-{lambda}5 mAb (LM34) (27), respectively. The ELISA was performed as described (28). In brief, a series of diluted cell lysates (50 µl) were applied to the coated wells and incubated for 4 h, and the plates were then incubated with 2 µg/ml HRP-conjugated goat-anti-mouse µH Ab (Southern Biotech) for 2 h, followed by incubation with 300 µg/ml ABTS diammonium salt (Wako Biochemicals) in 100 mM citric acid (pH 4.35).

Immunoprecipitation and immunoblotting

GFP+ 38B9 transfectants were lysed with 0.5% Nonidet P-40 lysis buffer (10 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1.5 mM MgCl2, 10 mM NaF, 1 mM Na3VO4, 5 nM calyculin A (Calbiochem) and a protease inhibitor mixture (Roche). Cell lysates were reacted with µH chain-specific mAb M41, and immunoprecipitates or whole cell lysates were resolved by electrophoresis on 8 or 13% SDS-polyacrylamide gels. The proteins were electrotransferred to polyvinylidene difluoride membranes, which were blocked with 5% skim milk, probed with the indicated Abs (Figs. 1C and 3) followed by HRP-conjugated secondary Abs, and developed with SuperSignal substrate (Pierce). To detect Igbeta phosphorylation, cell lysates were reacted with the anti-Igbeta mAb HM79 (29) followed by immunoblotting with the anti-phosphotyrosine mAb PY-20 (Santa Cruz Biotechnology).


Figure 1
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  		FIGURE 1. µH chains expressed in early pre-B cells exhibit great variability in their pre-BCR-forming capacity. A, Productively rearranged µH genes were isolated from early pre-B cells in adult bone marrow and fetal liver (14 ), inserted into the retroviral vector pMX-IRES-GFP, and then used to infect the µH–SL+ pro-B cell line 38B9. Two days after the infection, the cells were subjected to cytoplasmic staining with the µH chain-specific mAb or cell surface staining with either the µH chain-specific mAb or the pre-BCR-specific mAb (SL156). The staining profiles of the GFP+ infected cells (thick histogram) and the GFP– uninfected cells (thin histogram) were overlaid. The data shown are for an empty vector (mock) transfectant and four representative µH-transfectants designated µHUnd, µHLow, µHMed, and µHHigh according to their surface pre-BCR expression. The mean fluorescence intensity in the GFP+ cells is indicated in each panel in which the intensity of background staining of the GFP– cells for cytoplasmic µH, surface µH, and surface pre-BCR was 3.4, 2.3, and 2.6, respectively. Similar results were obtained in three independent experiments. B, GFP+ infected cells were sorted with a cell sorter from 38B9 cells infected with each µH clone. Cell lysates prepared from the sorted cells were subjected to sandwich ELISA to determine the total amounts of µH chains as well as the amounts of µH chains associated with {lambda}5 proteins. The data show the absorbance at 405 nm, which reflects the proportion of µH chains associated with {lambda}5. C, Cell lysates prepared as in B were reacted with the anti-µH mAb. Immunoprecipitates (IP) were resolved on SDS-PAGE, followed by immunoblotting (IB) with Abs specific to the indicated molecules (upper panel). In parallel, whole cell lysates (WCL) were immunoblotted in the same way (lower panel). The data are representative of three independent experiments.

 

Figure 3
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  		FIGURE 3. Stronger pre-BCR signals are produced with higher levels of surface pre-BCR expression. Each of the sorted GFP+ 38B9 transfectants was cultured without (left half) or with 0.1 µM STI571 (right half). One day later, cell lysates were prepared and subjected to SDS-PAGE followed by immunoblotting (IB) with Abs specific to the indicated molecules (p stands for phosphorylated). To detect phosphorylated Igbeta, the cell lysates were first reacted with the anti-Igbeta mAb, and the immunoprecipitates were subjected to SDS-PAGE followed by immunoblotting with the anti-phosphotyrosine Ab. The data are representative of five independent experiments.

 
Flow cytometric analysis

Cells were surface stained with fluorescence-labeled mAb or biotinylated mAbs followed by streptavidin-PE (BD Pharmingen). For intracellular staining, cells were fixed and permeabilized with Cytofix/Cytoperm (BD Pharmingen) before staining. Stained cells were analyzed with FACSCalibur (BD Biosciences).

Cell survival and proliferation assay

GFP+ 38B9 transfectants were sorted with FACSVantage (BD Bioscience) and cultured at 2 x 105 cells/1 ml/well in the presence or absence of the indicated concentration of STI571 (Fig. 2) (Novartis Pharmaceuticals). The viable cells were identified using trypan blue staining and counted at the indicated time points (Fig. 2). In parallel, cells were suspended in FACS buffer containing 0.1% Triton X-100, 200 µg/ml RNase A, and 25 µg/ml propidium iodide and analyzed with FACSCalibur to determine their apoptotic state. Bone marrow pro-B cells were cultured at 1–2 x 105 cells/1 ml/well after the infection, the viable cells were counted as for 38B9 cells. To measure the cell divisions, cells were labeled with PKH26 (Sigma-Aldrich) (30) 1 day after retroviral infection according to manufacturer’s protocol and cultured for 48 h. The number of cell divisions was determined by analyzing the red fluorescence intensity of GFP+CD19+ cells with the Proliferation Wizard module in ModFit LT 3.0 software (Verity Software House) (31).


Figure 2
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  		FIGURE 2. Higher level of pre-BCR expression confers better survival and proliferation on 38B9 pro-B cells when cultured with an Abl inhibitor. A, GFP+ 38B9 cells infected with each µH clone as shown in Fig. 1 were sorted and cultured in the absence (upper panels) or presence (lower panels) of the Abl inhibitor STI571 (0.1 µM). Twenty-four hours later, the cells were analyzed for the presence of subdiploid DNA as an indication of apoptosis. The number in each panel gives the percentage of cells carrying subdiploid DNA in each transfectant. The data are representative of three repeated experiments. B, The sorted GFP+ transfectants (2 x 105 cells/1 ml/well) were cultured with 0.1 µM STI571, and the percentage of viable cells before and after 1 day and 2 days of culture was plotted for each transfectant: mock (x), µHUnd ({diamond}), µHLow ({square}), µHMed ({triangleup}), and µHHigh ({circ}). Viable cells were defined as cells possessing DNA above the subdiploid area in the assay shown in A. The data are representative of three repeated experiments. C, The sorted GFP+ transfectants (2 x 105 cells/1 ml/well) were cultured without (left panel) or with 0.1 µM STI571 (right panel). The cells were harvested at the indicated time points, and the number of viable cells was counted using the dye exclusion test. The symbols representing each transfectant are the same as in B. The data are representative of two independent experiments.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Great variability in pre-BCR-forming capacity among µH chains expressed in early pre-B cells

An array of µH clones isolated from pre-B cells was individually introduced into the µH– SL+ pro-B cell line 38B9 (22) to examine the ability of the clones to form pre-BCRs in association with SL chains. We aimed to analyze "naive" µH clones that had not yet passed through the pre-BCR checkpoint. Therefore, all of the µH clones analyzed were isolated from early pre-B cells in fetal liver and adult bone marrow (14) but not from established pre-B and B cell lines, because the latter might have selected via the pre-BCR and therefore could be biased. We noticed that the surface expression of pre-BCR was not an all-or-nothing phenomenon. Instead, the levels of surface pre-BCR expression that we detected with anti-µH chain or anti-pre-BCR mAbs varied greatly among the µH clones, even though the amount of total µH chains per cell was almost the same among the clones as assessed by cytoplasmic staining (some examples are shown in Fig. 1A). The characteristics of the 28 µH clones analyzed in the present study are summarized in Table I, showing the usage of the VH, DH, and JH segments, the CDR3 sequences, and the level of surface and cytoplasmic µH chains expressed in 38B9 transfectants. The higher level of surface µH chain expression could not be attributed to the higher level of cytoplasmic µH chain expression. In the following experiments, we analyzed four representative µH clones that displayed different levels of surface pre-BCR, µHUnd, µHLow, µHMed, and µHHigh, which had undetectable (Und), low, medium (Med), and high levels of pre-BCR expression, respectively (Fig. 1A and Table I).


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  		Table I. Characterization of µH clones isolated from early preB cells in fetal liver and adult bone marrowa

 
An analysis of cell lysates by sandwich ELISA demonstrated that the higher the level of surface pre-BCR expression the transfectants displayed, the larger the proportion of µH chains associated with the {lambda}5 SL chain (Fig. 1B). This correlation between the level of surface pre-BCR expression and the extent of µH chain-SL chain association was further confirmed by coimmunoprecipitation of the {lambda}5 and VpreB chains with µH chains from each 38B9 transfectant (Fig. 1C). Thus, the level of surface pre-BCR expression appeared to be determined primarily by the avidity of each µH chain for the SL chain.

Higher pre-BCR expression confers better survival and proliferation on 38B9 cells when cultured with an Abl inhibitor

Abelson murine leukemia virus-transformed cell lines such as 38B9 survive and proliferate in a tyrosine kinase v-Abl-dependent manner (32). Indeed, when 38B9 cells that were uninfected or infected with empty vector (mock) were cultured for 24 or 48 h in the presence of 0.1 µM STI571 (33), an inhibitor of Abl, more than half the cells showed apoptosis as defined by the presence of subdiploid DNA, compared with <2% of cells cultured in the absence of the reagent (Fig. 2, A and B, not all data shown). As the pre-BCR has been suggested to transduce survival signals (34), we examined whether the 38B9 transfectants expressing µH chains could survive in the presence of 0.1 µM STI571. The 38B9 cells that were infected with µHUnd and made no surface pre-BCRs were almost as sensitive as mock-infected 38B9 cells to the STI571-induced apoptosis (Fig. 2, A and B). In contrast, 38B9 cells infected with µHLow, µHMed, or µHHigh were all resistant to the STI571-induced apoptosis regardless of the level of pre-BCR expression (Fig. 2, A and B). These results suggested that the expression of pre-BCR rescued cells from apoptosis by transducing survival signals.

In the absence of STI571, all of the 38B9 transfectants expanded exponentially to a comparable extent during 3 days in culture, regardless of their levels of pre-BCR expression (Fig. 2C, left panel). In the presence of 0.1 µM STI571 during culture, however, the proliferation of each 38B9 transfectant was different (Fig. 2C, right panel). Consistent with the results of the cell survival assay, the µHUnd and mock transfectants did not proliferate and died during culture. µHLow transfectants proliferated transiently but soon began to die. In contrast, both the µHMed and µHHigh transfectants continued to proliferate even in the presence of STI571, albeit to lesser extent than in its absence. These results clearly indicated that the higher level of surface pre-BCR expression conferred on the cells a stronger proliferative activity as well as better survival.

Greater surface pre-BCR expression leads to stronger pre-BCR signaling

As expected, after 24 h in culture with 0.1 µM STI571 the tyrosine phosphorylation of v-Abl was almost completely abolished in all the transfectants, regardless of their levels of surface pre-BCR expression (Fig. 3). This observation was also true for STAT5 phosphorylation. Concomitant with this drastic reduction in v-Abl and STAT5 phosphorylation, the serine phosphorylation of Akt was greatly diminished in mock and µHUnd transfectants after they were cultured with STI571. In contrast, 38B9 cells expressing µHLow, µHMed, or µHHigh showed significant levels of Akt phosphorylation even in the presence of STI571, in accord with the results of the cell survival experiment. Tyrosine phosphorylation of Igbeta, a signal-transducing module of the pre-BCR, was detected in all of the 38B9 cells that had been infected with the different µH clones, but not in the mock transfectants, with or without STI571. A clear correlation was observed between the level of pre-BCR expression and the strength of Igbeta phosphorylation when the cells were cultured with STI571 (Fig. 3). This finding was also true for the tyrosine phosphorylation of signaling molecules downstream of the pre-BCR, such as the tyrosine kinase Syk, the adaptor molecule BLNK, and ERK, even though the extent of their phosphorylation in the presence of STI571 was much less than that observed in its absence. Taken together, these data indicate that higher levels of pre-BCR expression led to stronger signals for inducing the phosphorylation of critical molecules downstream of the pre-BCR.

Reconstitution of pre-BCR in bone-marrow pro-B cells with different µH clones

Our observations in the 38B9 transfectants prompted us to examine the functional outcome of different levels of pre-BCR expression in cells more closely reflecting the in vivo situation. In mice deficient in the membrane exon of the µH chain gene (µMT mice) (21), B cell development in bone marrow is arrested at the pro-B cell stage due to the lack of membrane-bound µH chains. Bone marrow cells enriched in the B220+ pro-B cells of µMT mice were infected with retroviral vectors encoding µHUnd, µHLow, µHMed, and µHHigh or with empty vector (mock). One day after the infection, the GFP+ infected cells were analyzed for their surface expression of pre-BCR. Different levels of surface pre-BCR expression were clearly observed on bone marrow pro-B cells, and the level depended on the µH clone that was introduced (Fig. 4A, top panels, and summarized in B), as in the 38B9 transfectants (Fig. 1A). pre-BCR expression was almost undetectable on the surface of bone marrow pro-B cells infected with µHUnd, whereas the highest expression was detected on those infected with µHHigh. The levels of cytoplasmic µH chain expression were almost comparable among the bone marrow pro-B cells infected with the different µH clones (Fig. 4A, bottom panels). In contrast to the stable expression of pre-BCR on the surface of the 38B9 transfectants, the pre-BCR expression on infected bone marrow pro-B cells was transient in vitro (Fig. 4B), as in the case of normal pre-B cells in the bone marrow. Although the maximal level of pre-BCR expression differed among the transfectants, the kinetics of the up- and down-regulation of pre-BCR expression was very similar among them, with the peak on day 1 postinfection. In all cases, the pre-BCR expression was undetectable on day 3.


Figure 4
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  		FIGURE 4. Reconstitution of pre-BCR in bone marrow pro-B cells with different µH clones. B220+ pro-B cells were enriched from the bone marrow cells of µMT mice and infected with an empty retroviral vector (mock) or with those encoding µHUnd, µHLow, µHMed, or µHHigh. A, One day after the infection, GFP+ (top row) and GFP– (middle row) cells were separately analyzed for their surface expression of CD19 and pre-BCR. In parallel, cytoplasmic expression of µH chains (bottom row) was analyzed for GFP+CD19+ cells (thick histograms) and GFP–CD19+ cells (thin histograms). B, The mean fluorescence intensity of pre-BCR expression at the indicated time points after the infection is plotted for each GFP+ transfectant: mock (x), µHUnd ({diamond}), µHLow {square}), µHMed ({triangleup}), and µHHigh ({circ}). The value on day 0 represents that obtained from uninfected cells. The data shown in A and B are representative of four repeated experiments.

 
Higher pre-BCR expression confers better proliferation on bone marrow pro-B cells

We next compared the proliferation potential of bone marrow pro-B cells expressing different µH clones in vitro (Fig. 5). Without the addition of IL-7, a growth factor for lymphocyte progenitors, the mock and µHUnd transfectants showed no significant proliferation during the culture period from day 1 to day 5 postinfection (Fig. 5A). In contrast, the µHLow, µHMed, and µHHigh transfectants proliferated even in the absence of exogenous IL-7, and the cell number increased 5, 11, and 15 times, respectively, during the first 2-day culture period (Fig. 5A). Thus, the extent of proliferation correlated well with the level of pre-BCR expression as observed in the 38B9 transfectants. To visualize cell division in each transfectant, the cells were labeled with the red fluorescent dye PKH26 1 day after the retroviral infection, and the PKH26 fluorescence intensity of the GFP+CD19+ transfectants was analyzed by flow cytometry after 2 days in culture without exogenous IL-7 (Fig. 5B). The µHUnd transfectants showed almost no cell division, as was the case for the mock transfectants. The majority of the µHLow-transfectants divided once, and the majority of the µHHigh transfectants divided twice during the 2-day culture period. The µHMed transfectants divided once or twice. Thus, the number of cell divisions correlated well with the level of pre-BCR expression. The number of cell divisions was smaller than expected given the increase in cell numbers shown in Fig. 5A. This could be due to a side effect of labeling the cells with PKH26.


Figure 5
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  		FIGURE 5. Higher level of pre-BCR expression confers better survival and proliferation on bone marrow pro-B cells. A, B220+ bone marrow pro-B cells of µMT mice were infected with retroviral vectors as shown in Fig. 4 and cultured without exogenous IL-7 at 2 x 105 cells/1 ml/well. The number of GFP+CD19+ cells was counted at the indicated time points postinfection. The data show the kinetics of relative cell number for each transfectant during the 5-day culture, where 1 presents the cell number of each transfectant 1 day after the infection; the absolute number of GFP+CD19+ cells was 2 x 104 in the mock transfectant, 1.2 x 104 in the µHUnd transfectant, and 1.0 x 104 in the µHLow, µHMed, and µHHigh transfectants. The symbols representing each transfectant are the same as in Fig. 4B. The data are representative of three repeated experiments. B, One day after infection the cells were labeled with the red dye PKH26 and cultured without exogenous IL-7 for 48 h. The left panels show the PKH26 histograms for the GFP+CD19+ cells after 48 h of culture. The right panels show the diagrams processed with the Proliferation Wizard module of ModFit software so that software deconvolution draws Gaussian distributions, allocating cells of different PKH26 intensities to different generations. Populations that underwent one (hatched histogram), two (gray histogram), or three (gray histogram) rounds of cell division were determined by defining mock transfectants as the parent (white histogram). C, The cells prepared as in A were cultured in the presence of 0, 0.01, or 10 ng/ml IL-7. The data show the relative cell number of GFP+CD19+ infected pro-B cells (upper panel) and GFP–CD19+ uninfected pro-B cells (lower panel) on day 3 postinfection, where 1 presents the cell number of each transfectant 1 day after the infection. The data are representative of three repeated experiments.

 
The addition of IL-7 (0.01 or 10 ng/ml) to the culture medium induced the proliferation in a dose-dependent manner in the mock and µHUnd transfectants that showed no proliferation in the absence of IL-7 (Fig. 5C, upper panel). The IL-7 addition also promoted the proliferation of the µHLow, µHMed, and µHHigh transfectants even though they could expand in the absence of IL-7. The extent of proliferation correlated with the level of pre-BCR expression in each transfectant, regardless of the absence or presence of IL-7 in culture (Fig. 5C, upper panel). In contrast, GFP– uninfected pro-B cells in the same culture with the GFP+ infected cells proliferated to a comparable extent in the presence of IL-7 no matter which retroviral vector was used (Fig. 5C, lower panel). Thus, the proliferation potential of pre-B cells appeared to be determined primarily by the level of pre-BCR expression and further enhanced by IL-7.

Greater pre-BCR expression confers better differentiation on bone marrow pro-B cells

We next compared the differentiation potential of bone marrow pro-B cells expressing different µH clones in vitro. For this purpose, a small amount of IL-7 (0.01 ng/ml) was added to the culture because almost all of the mock and µHUnd infected cells died during the culture without IL-7, as shown in Fig. 5A. The surface expression of c-kit and CD2 in the transfectants was analyzed first, because the c-kit expression is down-regulated at the transition from pro-B to pre-B cells in normal B cell development, whereas the CD2 expression is up-regulated (35, 36). On day 2 postinfection, the down-regulation of c-kit expression was apparent in the µHLow, µHMed, and µHHigh transfectants, with the µHHigh transfectants expressing the lowest levels of c-kit, whereas down-regulation was not obvious in the µHUnd transfectants or in the mock transfectants (Fig. 6A, top row). In the µHUnd transfectants, as well as in the mock transfectants, the up-regulation of the differentiation marker CD2 was not observed during the 4 days in culture (Fig. 6A, second row from top). In contrast, it was apparent in the µHLow, µHMed, and µHHigh transfectants 4 days after the infection, with the µHHigh transfectants expressing the highest levels of CD2.


Figure 6
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  		FIGURE 6. Higher level of pre-BCR expression confers better differentiation on bone marrow pro-B cells. A, B220+ bone marrow pro-B cells of µMT mice were infected with retroviral vectors as shown in Fig. 4 and cultured with 0.01 ng/ml IL-7 at 2 x 105 cells/1 ml/well. GFP+ cells were analyzed with flow cytometry for their expression of surface c-kit on day 2 postinfection, and that of surface CD2, cytoplasmic {kappa}L chain, and surface IgM on day 4. The number in each panel gives the percentage of stained cells. The data are representative of five repeated experiments. B, On day 2 postinfection the GFP+ cells were sorted to extract the total RNA, which was then subjected to RT-PCR to detect germline {kappa}L chain transcripts and beta-actin transcripts, as the control. C, The number of surface IgM+ B cells generated from 100 cells of each pro-B transfectant during the 3-day culture with 0.01 ng/ml IL-7 is shown and was calculated from the comb ination of the proliferation rate shown in Fig. 5C and the frequency of surface IgM+ cells shown in A.

 
A similar difference among the transfectants was observed for the production of {kappa}L chains (Fig. 6A, third row from the top). The frequency of {kappa}L chain-producing cells on day 4 was ~2, ~8, ~12, ~20, and ~40% in the mock, µHUnd, µHLow, µHMed, and µHHigh transfectants, respectively. This finding suggested that higher level of pre-BCR expression induced more rearrangements at the {kappa}L chain locus. Indeed, 2 days after the infection, the µHLow, µHMed, and µHHigh transfectants showed significant levels of germline {kappa}L transcripts in correlation with the level of pre-BCR expression (Fig. 6B).

Concomitant with the production of {kappa}L chains in the µH-transfectants, the expression of IgM on the cell surface was detected on day 4. The frequency of surface IgM+ B cells was <1, <1, ~1, ~4, and ~12% in the mock, µHUnd, µHLow, µHMed, and µHHigh transfectants, respectively, in correlation with the level of pre-BCR expression (Fig. 6A, bottom row). Interestingly, the frequency of surface IgM+ B cells among the cells expressing cytoplasmic {kappa}L chains differed among the µH transfectants: 8, 19, and 29%, respectively, in the µHLow, µHMed, and µHHigh transfectants. This result suggested that there was a qualitative difference among the µH clones in the ability to assemble with {kappa}L chains in correlation with their ability to pair with SL chains.

As a consequence of the different extents of proliferation and differentiation among the µH transfectants (Fig. 5 and 6A), the number of surface IgM+ B cells that differentiated from the pro-B cells differed greatly among them (Fig. 6C). During the 3-day culture period, which extended from day 1 to day 4 postinfection, ~8, ~90, and ~430 IgM+ B cells were produced from 100 pro-B cells infected with µHLow, µHMed, and µHHigh, respectively. In other words, 50 times as many as B cells were derived from the µHHigh transfectants than from the µHLow transfectants. Thus, the qualitative differences among the different µH chains expressed in the pro-B cells had an immense impact on the final outcome of B cell differentiation.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The combination of a variety of Ig H and L chains creates a huge repertoire of BCRs in the B cell population. It is well understood how a variety of H and L chains are generated in the DNA level during B cell development (1, 2). However, it remained largely unknown whether and how each component of the BCR is monitored for its quality and selected before assembly into the BCR. The findings in the present study cast new light on the role of the pre-BCR in this process as a key player in judging the quality of H chains and tuning the primary B cell repertoire before L chains are produced.

The pairing or nonpairing of µH chains with SL chains for the pre-BCR formation turned out not to be a discontinuous all-or-nothing phenomenon, but rather the µH chains showed a spectrum of fitness for the SL chain. The level of pre-BCR expression differed widely among the µH chain-expressing pro-B cells and correlated well with the avidity of each µH chain for the SL chain. The four µH clones analyzed extensively in the present study used the same VH7183 family and JH4 segment (Table I). It is notable that eight clones, including three of the four, use the same VH7183.1b and JH4 segments but carry different CDR3 sequences and differ in their levels of pre-BCR expression (Table I). This finding suggested the importance of the CDR3 region in determining the levels of pre-BCR expression, at least in case of VH7183.1b-bearing µH chains, extending the previous findings (14, 37, 38). The variability in pre-BCR-forming capacity among µH clones was also observed in those using the VHJ558 family (Table I) and, hence, appeared not restricted to those using the VH7183 family even though the average of pre-BCR-forming capacity might depend on the VH segment used. Although the surface pre-BCR expression is too low to detect on bone marrow pre-B cells in normal mice (39, 40), a wide range of surface pre-BCR expression levels was detected on the bone marrow cells of BLNK–/– and BLNK–/–CD19–/– mice, which have augmented pre-BCR expression (41, 42). Therefore, our observation in vitro is likely to reflect the in vivo situation.

The functional consequences of the different levels of pre-BCR expression among the cells were clarified in the present study. In the 38B9 transfectants cultured with STI571, a clear correlation was observed between the level of surface pre-BCR expression, the extent of the phosphorylation of signal transducers including Igbeta, and cell survival and proliferation. This observation indicated that the quality of µH chains and the resulting pre-BCR expression level determined the strength of the pre-BCR-mediated signals that promote cell survival and proliferation. A little phosphorylation of Igbeta was observed in the µHUnd transfectants (Fig. 3), whereas they displayed no apparent pre-BCR expression or proliferation in culture with STI571 (Figs. 1A and 2C). This discrepancy could be explained as follows. A small proportion of µHUnd chains were associated with SL chains (Fig. 1C) and, hence, a small amount of pre-BCR could be expressed on the cell surface even though its level was not high enough for detection by flow cytometry. Such a tiny amount of pre-BCR could transduce a signal to phosphorylate Igbeta to a certain extent, but the signal was too weak to drive cell survival and proliferation.

The relationship between the pre-BCR expression and the cellular proliferation was further extended to primary pro-B cells. The extent of their proliferation correlated with their levels of pre-BCR expression, regardless of the absence or presence of IL-7. Thus, the quality of µH chains produced by individual pre-B cells appears to primarily determine their proliferation potential, even though IL-7 further promoted their proliferation in a dose-dependent manner. Pre-B cells expressing µH chains with higher ability to form the pre-BCR would need lesser amounts of IL-7 than those with lower avidity to have the same extent of expansion. Thus, the IL-7 dependency varies among individual pre-B cells and depends on the quality of µH chains produced by them. This finding appears to reconcile the conflicting results in previous reports with regard to the IL-7 dependency of pre-B cell proliferation (17, 18, 19, 43).

The different proliferation potential among individual pre-B cells in correlation with the quality of their µH chains could account for the previous observation that individual bone marrow pro-B cells showed different numbers of cell divisions, from two to five, in vitro (17). A hypothetical model has been proposed to explain the different numbers of cell divisions by individual cells; as they divide, the pre-B cells dilute the SL chains and stop dividing when the level of pre-BCR expression drops below the threshold necessary for proliferation (15, 17). Therefore, the higher the initial level of pre-BCR, the longer pre-B cells express pre-BCR and, hence, the longer the cells expand. We found in the present study that the level of pre-BCR expression indeed varied among the pro-B transfectants and correlated with the strength of pre-BCR signaling as well as with their proliferation potential. Interestingly, the duration of the pre-BCR expression was very similar among the different transfectants of bone marrow pro-B cells, even though the peak expression level differed. Therefore, the strength of pre-BCR signaling, rather than the half-life of the surface pre-BCR, appeared to account for the difference in the proliferation potential of individual pre-B cells. It remains to be determined whether and how the microenvironment in bone marrow and fetal liver, including cytokines and adhesion molecules, modifies this potential in vivo.

The difference in the level of surface pre-BCR expression impacted not only cell proliferation but also differentiation. A higher level of pre-BCR expression appeared to confer better differentiation on bone marrow pro-B cells. Germline {kappa}L chain transcription became detectable as early as on day 2, whereas the pre-B cells still expressed pre-BCR and progressed in the cell cycle. This finding suggested that the pre-BCR delivered signals that not only drove proliferation but also opened the {kappa}L chain locus. However, it remains to be clarified whether each differentiation event was induced by pre-BCR signaling directly or indirectly. We cannot formally exclude the possibility that the apparent accelerated differentiation observed in cells with higher levels of pre-BCR expression was a consequence of the accelerated proliferation of differentiated pre-B cells. Nevertheless, the present study clearly demonstrated that the quality of µH chains produced at the pro-B cell stage had a great impact on the final outcome of B cell differentiation, namely the production of surface IgM+ B cells. The expression of µHHigh led to the production of 50 times as many surface IgM+ B cells as did µHLow. Interestingly, the frequency of surface IgM+ B cells among the cells producing cytoplasmic {kappa}L chain varied in the individual µH transfectants and correlated positively with the ability of their µH chains to form the pre-BCR. This finding suggests that µH chains with higher avidity for the invariant SL chains display higher capability to pair with a variety of conventional L chains. Therefore, it appears that µH chains with higher potential to assemble with L chains are positively selected and amplified through the assessment of their ability to pair with SL chains at the pre-BCR checkpoint in advance of association with L chains.

In conclusion, we demonstrated that the level of pre-BCR expression varied in individual pre-B cells to a great extent, depending on the ability of their µH chains to assemble with SL chains. The extent of survival, proliferation, and differentiation of individual pre-B cells was primarily determined by the ability of their µH chains through differential pre-BCR signaling. We proposed a mechanism whereby µH chains with higher potential to assemble with L chains are positively selected and amplified at the pre-BCR checkpoint before assembly into the BCR.


   Acknowledgments
 
We thank Novartis Pharmaceuticals for providing the STI571, H. Hayashi for his expert assistance with cell sorting, and A. Kakefuda for secretarial assistance in preparing the manuscript.


   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 by Grant-in-Aid 17047013 from the Japanese Ministry of Education, Culture, Sports, Science and Technology and Grant-in-Aid 2212131 from the Japanese Ministry of Health, Labor and Welfare. Back

2 Address correspondence and reprint requests to Dr. Hajime Karasuyama, Department of Immune Regulation, Tokyo Medical and Dental University Graduate School, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail address: karasuyama.mbch{at}tmd.ac.jp Back

3 Abbreviatons used in this paper: SL, surrogate light; BLNK, B cell linker; Med, medium; Und, undetectable. Back

Received for publication February 8, 2006. Accepted for publication June 6, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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