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Role of µ Heavy Chain in B Cell Development. I. Blocked B Cell Maturation But Complete Allelic Exclusion in the Absence of Ig/ß¹

Frank E. Cronin^*, Ming Jiang, Abul K. Abbas^* and Stephan A. Grupp^2,

^* Immunology Research Division, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; and Division of Pediatric Oncology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
There is good evidence for a signaling role played by Ig heavy chain in the developmental transition through the pre-B cell stage. We have previously described signal-capable or signal-incapable mutants of µ heavy chain in which a signaling defect is caused by failure to associate with the Ig/ß heterodimer. To further characterize the role of Ig heavy chain-mediated signaling in vivo, as well as in B cell development and allelic exclusion, we have created transgenic mice in which the B cells express these signal-capable and signal-incapable mutant µ chains. Failure of µ to signal via Ig/ß results in a block in B cell development in mice expressing the signal-incapable µ. A small number of B cells in these animals do escape the developmental block and are expressed in the spleen and the periphery as B220⁺ transgenic IgM⁺ cells. These cells respond to LPS by proliferating but show no response to T-independent-specific Ag. In contrast, B cells expressing the signal-capable B cell receptor show a strong signaling response to Ag-specific stimulus. There is no Ig seen in association with signal-deficient IgM. Thus, the B cell receptor complex is not assembled, and no signal can be delivered. Despite the block in developmental signaling, allelic exclusion is complete. There is no detectable coexpression of transgenic IgM and endogenous murine IgM, nor is there rearrangement of the endogenous heavy chain genes. This suggests that differing signaling mechanisms are responsible for the developmental transition and allelic exclusion and thus allows for separate examination of these signaling mechanisms.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The maturation of B lymphocytes and the responses of mature B cells to Ags are believed to be dependent on signals initiated by the B cell receptor (BCR)³ complex. The BCR complex includes the Ag receptor, membrane-bound Ig (mIg), and Ig (the product of the mb-1 gene) and Igß (the product of the B29 gene) (1, 2, 3). The Ig/ß heterodimer (4, 5) has structural and functional homologies to the CD3 complex (6, 7). Ig and Igß are noncovalently associated with mIg via polar interactions in the transmembrane region of the mIg molecule (8). Ig/ß are required for release of the BCR from calnexin (9) and subsequent expression of the BCR complex on the cell surface (10) and for Ag-induced cellular activation (8, 11). The heterodimer is not required for binding of the BCR to the cytoskeleton and internalization of mIg after cross-linking (12, 13), while its role in Ag presentation remains uncertain (14, 15, 16).

The analysis of BCR complex signaling in mature B cells has been paralleled by similar work in earlier B cell stages. The stages of B cell development have been well characterized, especially in the mouse. Recombination of the heavy chain gene begins at the CD43⁺ pro-B stage (17), while at the pre-B stage, CD43 expression is lost and µ protein is detected in the cytoplasm. A variety of studies have shown that µ heavy chain participates in an analogous signaling complex in pre-B cells, a complex that signals the successful, in-frame recombination and expression of the µ gene and, in turn, is critical for the developmental transition through the pre-B stage. This pre-B cell receptor complex consists of heavy chain and surrogate light chain (5 and VpreB) as well as other molecules such as the Ig/ß heterodimer (18, 19). The complex is involved in several key events in pre-B cell maturation, including developmental transition (20) as well as allelic exclusion (21), but the roles of the individual molecules as well as those the downstream signaling mechanisms involved remain unclear. The importance of µ and 5 surrogate light chain has been demonstrated by targeted deletion experiments. Deletion of 5 (22, 23) or the J_H or transmembrane domain of the µ gene (24, 25) results in a block in B cell maturation at the pre-B stage. This observation has now been extended to humans in some cases of non-X-linked agammaglobulinemia, where various deletions in the µ gene have been detected (26). The importance of µ chain-mediated signaling in the maturation of pre-B cells has been further highlighted by the identification of Btk as the target of mutations that cause the classic, X-linked form of agammaglobulinemia, in which maturation of B cells though the pre-B stage is blocked (27, 28). The same mutation in mice causes a less profound maturational abnormality, suggesting differences in signaling mechanisms between murine and human B cell development (29). The importance of the Ig/ß heterodimer in development has been directly demonstrated by disruption of the assembly or function of this portion of the pre-BCR complex (30, 31, 32).

To define the importance of an intact, signaling-competent Ig in B cell development, we have examined the ability of various µ mutants expressed as transgenes to support B cell maturation. In previous experiments, we have transfected into mature B cell lines a number of human µ constructs with nonconservative mutations in the transmembrane and cytoplasmic domains (14) to define the functions of the BCR complex. One such mutant, called YS:VV, in which two polar transmembrane residues, Tyr⁵⁸⁷ and Ser⁵⁸⁸, are replaced with valines, fails to associate with Ig/ß and fails to signal upon Ag binding or to efficiently target bound Ag for intracellular processing and presentation. This transmembrane mutant µ is expressed on the surface of transfectants as an integral membrane protein despite its failure to assemble the BCR complex as a result of differential processing and release from calnexin in the endoplasmic reticulum (9).

By expressing mutant µ molecules as transgenes, we sought to extend these studies of BCR complex-mediated signaling to the pre-B cell developmental transition. Based on the prior work of the Nussenzweig group (21, 30), we anticipated that this mutant µ would recapitulate the signaling failure in the context of the pre-B transition. In designing these experiments, we took advantage of the known specificity of the complete IgM (phosphorylcholine (PC)) available to us, with the intent of being able to study Ag-specific signaling through the transgenic receptor. Thus, we provided both the heavy and the light chain genes to allow for study of signaling via both the pre-BCR and the complete BCR in mature B cells. Our data show that disruption of the binding of µ protein to the Ig/ß heterodimer causes a block in B cell development. Despite this block in developmental signaling, allelic exclusion is complete. This suggests the existence of dual signaling pathways directing allelic exclusion and maturation through the pre-B cell stage.

	Materials and Methods

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Ig constructs, cell lines, and transgenic mice

The Ig constructs used in these studies have been described in detail previously (14). The wild-type (WT) µ consists of a rearranged V-D-J from the mouse plasmacytoma S107 and human Cµ regions. The first poly(A) addition site has been deleted, ensuring the expression of this protein solely in the membrane-bound form (33). The two mutants are Tyr⁵⁸⁷/Ser⁵⁸⁸ to Val/Val (YS:VV) and Tyr⁵⁸⁷ to Phe (Y:F). The functional analysis of these mutants transfected into A20 cell lines has been described elsewhere (14). The purified PvuI/ClaI fragment of the µ construct and the PvuI/BamHI fragment of the construct were comicroinjected into fertilized FVB mouse oocytes using standard techniques (34). Microinjected eggs were transferred into the oviducts of pseudopregnant Swiss Webster foster females. Potential founders were screened for the presence of the µ heavy chain and light chain DNA and protein by Southern analysis with a heavy chain probe, PCR utilizing a primer pair recognizing the rearranged S107 light chain, and Ab detection of human µ on the surface of lymphocytes (see below). RAG-2-deficient mice were the kind gift of Dr. F. W. Alt, Boston, MA. Progeny of WT, YS:VV, and Y:F transgenic founders were studied at 8 to 12 wk of age except where noted.

Abs and Ag

Affinity-purified rabbit anti-human µ (RAHµ) absorbed against mouse Ig and goat anti-mouse µ (GAMµ) adsorbed against human Ig (Jackson Immunoresearch, West Grove, PA) were use for cytometric analysis and cross-linking experiments. These Abs and normal rabbit IgG (Sigma, St. Louis, MO) were coupled with CNBr-activated Sepharose CL4B (Pharmacia, Piscataway, NJ) for use in preclearing or immunoprecipitation. Phycoerythrin (PE)- and FITC-labeled Abs recognizing murine B220, CD3, and CD43 were obtained from PharMingen (San Diego, CA). For the three-color analysis of bone marrow, PE-B220, FITC-CD43, and Cy5-RAHµ were utilized. Anti-Ig antiserum was the kind gift of Dr. J. Jongstra, Toronto Western Hospital, Toronto Ontario, Canada. PC-dextran was obtained from Dr. Jim Kenny, National Cancer Institute, Frederick, MD (35). AB1-2 hybridoma specific for the S107 Id was obtained from American Type Culture Collection, Manassas, VA. Ab was purified from ascites and biotinylated for labeling experiments.

B cell purification and culture

B cells for culture experiments were purified by panning on rabbit anti-mouse Ig- and goat anti-human µ-coated plates (both from Cappel, Malvern, PA) at 4°C as described (36). Typically, this protocol yields >95% pure B cells. In proliferation assays, B cells were cultured at 10⁵ cells per well in the presence of LPS (Sigma), dextran, or PC-dextran and pulsed with [³H]TdR for the final 6 h of a 72-h culture.

Western blotting was performed as previously described (9). Briefly, 10 to 25 x 10⁶ cell equivalents were lysed with 1% dodecyl maltoside (Anatrace, Maumee, OH) or 1% Triton X-100 (Sigma) in lysis buffer. The lysates were precleared with goat IgG-Sepharose, immunoprecipitated with Sepharose-coupled goat anti-human µ, washed, electrophoresed through polyacrylamide SDS gels, and blotted onto polyvinylidene difluoride membranes. The blots were then probed with either horseradish peroxidase-anti-human µ or anti-Ig. To examine the assembly of the BCR complex, the anti-µ immunoprecipitated proteins were divided into two aliquots and electrophoresed separately. One aliquot was probed for µ heavy chain and the other for Ig.

Analysis of intracellular Ca²⁺ flux

Cells (2 x 10⁶/ml) were loaded for 30 min at 37°C with 4 µg/ml Indo-1/AM (Molecular Probes, Junction City, OR) in DMSO. After washing, the cells were resuspended at 10⁶/ml in RPMI + 2% FCS on ice, shielded from light. Cell were warmed to 37°C and maintained at that temperature for the signaling assay. Cells (10⁶) were assayed for Ca²⁺ response before and after addition of cross-linking Ab or Ag. The ratio of violet:blue fluorescence (405:485 nm) emitted from Indo-1/AM was measured on an Epics V cytometer (Coulter, Hialeah, FL) as previously described (37). This assay depends on the shift in fluorescence emission of Indo-1/AM from blue to violet (485 to 405 nm) upon binding of Ca²⁺ and enabled us to examine Ca²⁺ flux on a cell-by-cell basis. Fluorescence was measured in arbitrary units (405/485 nm), and unstimulated cells were used to establish a baseline for each measurement. The number of responding cells was also expressed as a percentage of the total cell population.

Analysis of VDJ recombination

We used a modification of the procedure of Marshall et al. (38). Splenocytes were labeled with PE-anti-mouse IgM and FITC-anti-human IgM, followed by sorting on a Coulter Epics Elite cell sorter. Sort windows were drawn to collect PE-positive or FITC-positive cells. Then, 20,000 sorted cells were lysed and subjected to PCR using primers designed to recognize 80% of VDJ recombination events: a degenerate V_H consensus primer for the 5' end of the V_H framework I region (V_Hall; 5'-AGGTC/GA/CAA/G CTGCAGC/GAGTCA/TGG-3') and a primer recognizing a sequence just 3' to J_H4 (J_H4; 5'-AAAGACCTGCAGAGGCCATTCTTACC-3') (38). A Perkin-Elmer GeneAmp 2400 PCR machine (Norwalk, CT) was used for lysis and proteinase K inactivation as well as for the PCR reaction. After agarose gel electrophoresis, the PCR products were transferred via vacuum blotting to nylon membranes, followed by Southern analysis using a radiolabeled J_H4 probe (J_H4IN, which recognizes the J_H4 coding sequence: 5'-GAGGAGACGGTGGACTGAGGTTCCAATCCAATGAATACGAATTCCCTTCCCATG-3').

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Creation of Ig transgenic mice

Transgenic mice were created to study the role of µ-mediated signaling in B cell development. The hypothesis driving these experiments was that µ heavy chain participates in a signaling complex of molecules in the pre-B cell, and that disruption of this pre-BCR complex would disturb the developmental signal that allows the pre-B to B cell transition. To test this assumption, we coinjected into oocytes constructs directing the synthesis of the signal-competent WT and Y:F heavy chains, as well as the signal-impaired YS:VV heavy chain (Table I), each together with the S107 light chain. Founders were screened for expression of both the transgenic heavy and light chain. In each positive founder, heavy and light chain were expressed and, based on analysis of subsequent offspring (data not shown), were cointegrated into the same site in the genome. Initially, founders were screened by Southern analysis of genomic DNA obtained from tail biopsies for the presence of the transgenic heavy chain. Subsequently, we screened offspring utilizing a combination of PCR detection of the rearranged light chain sequence present in tail DNA combined with cytometric analysis of peripheral blood to detect B cells expressing the human IgM. It was also possible to confirm the expression of the S107 Id by staining with the mAb AB1-2, as well as to detect PC Ag binding directly using biotinylated PC-dextran (not shown).

As shown in Figure 1 and Table II, expression of the YS:VV Ig transgene induced a significant, although incomplete, block in B cell development. Figure 1, A and B, shows cytometric analysis of peripheral blood and spleen in animals expressing the WT IgM transgene, the signal-capable IgM mutant Y:F, and the signal-defective mutant YS:VV, as well as a nontransgenic littermate. There were significantly fewer B220⁺ cells in both the blood and spleens of YS:VV transgenics, which in turn resulted in reduced spleen size (Fig. 1C). Specifically, spleens from YS:VV animals had 50% or fewer of the normal number of total splenocytes and, as can be seen in Table II, <10% of the splenic B220⁺ µ⁺ B cells seen in WT or nontransgenic animals.

View larger version (23K): [in this window] [in a new window]   FIGURE 1. Analysis of peripheral and bone marrow cells from transgenic mice A, Peripheral white blood cells were obtained after lysis of RBC. Transgenic µ+ circulating B cells were detected by staining with PE-anti-B220 and FITC-RAHµ. B, Analysis of transgenic µ+ B cells in the spleens of WT, Y:F, and YS:VV transgenic mice, as well as a nontransgenic littermate. C, Comparison of typical spleen size and weight between spleens from WT and YS:VV transgenic mice. The deficiency in B cell number seen in the YS:VV transgenic mice accounts for the difference in spleen sizes. D, Analysis of pre-B cells in the bone marrow of transgenic mice. Bone marrow cells were stained with PE-anti-B220, FITC-anti-CD43, and Cy5-RAHµ. A gate was drawn around the B220+ population, and these B220+ cells were analyzed for CD43 vs µ. Percentages shown indicate the B220+ CD43-- IgM- pre-B cell population (lower left quadrant). E, Decrease in number of transgenic BCR+ B cells in the periphery of older animals. YS:VV transgenic mice were sacrificed at 5 mo (YS:VV 195) and 7 mo (YS:VV 162) of age, and peripheral blood and splenocytes were stained with anti-B220 and RAHµ.

Interestingly, the signaling failure responsible for the block in B cell development becomes more apparent over time. Figure 1E shows cytometric analysis of spleen and peripheral blood from older animals expressing the YS:VV transgene (YS:VV 195, 5 mo old, and YS:VV 162, 7 mo old). By this time, B cells expressing the YS:VV µ on the surface have almost completely disappeared from the periphery (Table II), with animal 195 having 1.5% and animal 162 0.7% YS:VV µ⁺ cells. A greater proportion of the splenic B cells were B220⁺ YS:VV µ^- (36% µ^- and 2% µ⁺ in animal 195; and 21% µ^-, 4% µ⁺ in animal 162). Thus, 85 to 95% of the splenic B220⁺ cells were transgenic µ^- in the older YS:VV animals. This compares with 5 to 20% in WT and Y:F transgenic splenocytes and 33 to 80% in younger YS:VV transgenics (range of eight experiments). These results are most easily explained by a relative growth advantage possessed by YS:VV µ^- B cells, a population that can preferentially expand over time over growth-impaired YS:VV µ⁺ B cells. This would further indicate lack of signaling through the impaired B cell receptor, as the few YS:VV B cells that escape the block are not expanding in the periphery. However, these data do not exclude the possibility that the life span of YS:VV µ⁺ B cells may also be shortened.

Analysis of allelic exclusion

In addition to driving the pre-B to B transition, the presence of rearranged µ heavy chain also indicates to the recombination machinery of the B cell that the alternate heavy chain locus should not be rearranged. This phenomenon of allelic exclusion ensures that no B cell emerges from the developmental process capable of recognizing more than one Ag. It was our expectation that a failure of developmental signaling would be accompanied by a failure to induce allelic exclusion and that those YS:VV IgM⁺ B cells that escaped the developmental block would do so as a result of rearrangement and expression of the endogenous heavy chain gene. Thus, the endogenous heavy chain would provide the developmental signal and not the YS:VV µ, and the development of the small number of YS:VV IgM⁺ B cells would be dependent on the rearrangement of one of the endogenous heavy chain genes. This proved not to be the case.

We initially looked for coexpression of mouse IgM with the transgenic human IgM on transgenic B cells. As seen in Figure 2A, regardless of the transgenic line tested, there was no appreciable coexpression of mouse IgM and human IgM on the surface of splenic B cells. This was true of peripheral blood as well (data not shown). Although we could not detect mouse IgM expression on the surface of YS:VV transgenic B cells, this did not rule out the possibility of endogenous gene rearrangement that did not result in surface expression of the murine BCR. To explore this further, we relied on molecular analysis of µ gene rearrangement. We used a modification of a PCR detection procedure for VDJ recombination that had been published previously (38), using a degenerate primer that recognizes most V_H exons and a primer that recognizes a sequence immediately 3' of the J_H4 exon, a primer pair that detects the majority of VDJ recombination events (38). We labeled cells with the non-cross-reactive PE- and FITC-conjugated Abs used in the studies depicted in Figure 2A. These cells were then sorted into PE (mouse IgM)- and FITC (human IgM)-positive fractions. Cells (20,000–40,000) were lysed and the DNA in the lysate subjected directly to PCR; the assay has a detection threshold of 10,000 or fewer templates (data not shown; and 39 . The PCR products were separated by gel electrophoresis and vacuum blotted onto nylon membranes. The Southern blot was then probed with the J_H4IN probe complementary to a sequence in the coding region of J_H4 to confirm the identity of the rearranged bands. The postsort cytometric analysis and the results of the PCR and Southern analysis are shown in Figure 2B. These data show no evidence for µ gene rearrangement in any of the human IgM⁺ B cells from any transgenic line, including mice expressing the signal-defective YS:VV transgene. This also provided evidence that, even in the face of defective developmental signaling through the YS:VV µ, the signal driving allelic exclusion seems to function efficiently.

View larger version (38K): [in this window] [in a new window]   FIGURE 2. Allelic exclusion of endogenous heavy chain gene rearrangement in transgenic B cells. A, No coexpression of human and murine IgM is seen on YS:VV B cells. Splenocytes were labeled with non-cross-reactive PE-anti-mouse µ (adsorbed against human Ig) and FITC-anti-human µ (adsorbed against mouse Ig). As seen in the analysis of nontransgenic splenocytes, there is no labeling of mouse IgM+ cells with the anti-human Ab. Even in the B cells of mice expressing the signal-deficient YS:VV IgM, there is no evidence of coexpression of mouse and human IgM on the cells. B, PCR analysis of endogenous heavy chain rearrangement in transgenic B cells. In these experiments, splenocytes were labeled with PE-GAMµ and FITC-RAHµ. The cells were then sorted into PE+ and FITC+ populations. A small number of the sorted cells were analyzed on the cytometer and 20,000 to 40,000 cells were lysed and the DNA subjected to PCR to detect VDJ rearrangement. Southern analysis using a JH oligoprobe was conducted to confirm the identity of the PCR products: VDJH2, VDJH3, and VDJH4 products are seen.

View larger version (30K): [in this window] [in a new window]   FIGURE 3. Limited B cell development in YS:VV transgenics does not depend on endogenous gene rearrangement: expression in the RAG-2-/- background. YS:VV transgenic mice were bred with RAG-2-/- and F2 offspring were chosen that were YS:VV+ and RAG-2-/-. Any µ+ B cells in the RAG-2-/- background must result from the expression of the YS:VV µ transgene, and these cells are demonstrated in the B220/RAHµ stain of splenocytes from these animals (A). No T cells are detected by staining with anti-CD3 in these mice (B), nor are they seen in the transgene-negative, RAG-2-/- littermate (C).

These data indicate that recombination of the endogenous µ gene and subsequent expression of endogenous µ protein did not provide the mechanism by which the small number of YS:VV IgM⁺ B cells were able to traverse the developmental block imposed by this signal-incapable transgenic Ig. Given this finding, it was important to exclude signaling through the BCR and the Ig/ß heterodimer. Although our previous work has shown that no assembly of the BCR complex or signaling is seen in mature B lymphoma cells transfected with YS:VV IgM, it may be that signaling and/or complex assembly is possible in this in vivo system.

To explore signaling through the impaired BCR complex in YS:VV transgenics, we examined both early and late consequences of receptor cross-linking. As shown in Figure 4A, B cells isolated by panning from WT transgenic mice demonstrated a proliferative response to specific, T cell-independent Ag (PC-dextran) as well as to LPS. The B cells from mice expressing the signal-capable Y:F transgene also responded to Ag, while YS:VV transgenic B cells showed no proliferative response to PC-Ag and responded only to LPS. This lack of signaling response was also seen with the early event of Ca²⁺ mobilization (Fig. 4B). YS:VV B cells treated with anti-IgM or PC-Ag showed no calcium response, while this response was intact in the case of WT or Y:F B cells, with a significant percentage of splenocytes showing an increase in Ca²⁺ flux after receptor cross-linking. The data in Figure 4 demonstrate a complete lack of detectable signaling through the YS:VV BCR. We also analyzed B cells from the Ig transgenics for assembly for the BCR complex. As can be seen in Figure 5, Ig is found in association with WT and Y:F IgM in dodecyl maltoside lysates of B cells of these transgenic mice but not in YS:VV B cells. Taken together, these data indicate that the BCR in B cells expressing a signal-deficient IgM transgene neither associates with nor is capable of signaling through the Ig/ß heterodimer.

View larger version (33K): [in this window] [in a new window]   FIGURE 4. Ag-specific signaling via signal-competent (WT, Y:F) and signal-incompetent (YS:VV) transgenic BCR. A, Proliferative response to PC-Ag in transgenic B cells. B cells were purified from transgenic animals and nontransgenic littermates by panning of splenocytes with RAHµ and GAMµ Abs at 4°C. Cells were collected and plated at 105 cells per well with the indicated stimulus: LPS, 10 µg/ml; dextran, 100 ng/ml; and PC-dextran, 3 ng/ml. Stimulation index (SI) of [3H]TdR incorporation at 72 h; mean of triplicate measurements. B, Analysis of Ca2+ flux in transgenic B cells after receptor cross-linking. Increase in 405:485 nm fluorescence ratio measured in Indo-1-labeled splenic B cells after addition of cross-linking Ab or Ag: PC-dextran, 25 ng/ml; GAMµ, 20 µg/ml (anti-IgM, nontransgenic); RAHµ, 50 µg/ml (anti-IgM, WT, and YS:VV transgenic B cells); PC-OVA, 25 µg/ml; and PC-rabbit IgG (PC-RGG), 10 µg/ml.

View larger version (72K): [in this window] [in a new window]   FIGURE 5. Assembly of the BCR complex with transgenic µ expressed on splenic B cells. Splenocytes were lysed in 1% dodecyl maltoside (DM) or 1% Triton X-100 lysis buffer and the BCR complex isolated by immunoprecipitation using RAHµ Abs. The detergent DM preserves the BCR complex associations, while Triton X-100 does not. Ig was detected by Western blotting with Abs recognizing Ig and was found in association with transgenic WT and Y:F µ, but not with the YS:VV µ. No murine µ protein was immunoprecipitated or detected by rabbit anti-mouse µ Abs.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The contribution of transgenic µ⁺ B cells to the mature circulating compartment in YS:VV animals is small to begin with and diminishes over time. This may be the result of preferential growth of B cells over the life of the animal that do not express the YS:VV µ protein and can thus rearrange their own Ag receptor genes. Similarly, investigators studying the more subtle phenotype of Btk inactivity in mice have suggested that this phenotype is caused by impaired expansion of B cell progenitors (42). When allowed to grow in a RAG-deficient background, these Btk^-/- cells can expand into peripheral compartments, a phenomenon that we observe here as well. This points to a possible signaling redundancy in the developmental system, a redundancy that may be more apparent in mice but may also be detected in humans as the phenotypic spectrum of Btk inactivation in man becomes more fully appreciated (43, 44). Our data suggest that if there is such redundancy, it may involve transducers in a pre-BCR complex other than Ig/ß.

The role of Ig/ß in B cell development has also been carefully studied by the Nussenzweig group, using a construct with a different VDJ region but an identical alteration in the transmembrane region (21). Despite the similarity in approaches, our conclusions differ in that we see the YS:VV µ protein as mediating allelic exclusion. One possible explanation for the difference would involve greater binding of surrogate light chain to our µ protein. Surrogate light chain-mediated signaling may act to screen for µ chains that are capable of appropriate assembly with light chain (45, 46). In this regard, we note that our signal-impaired YS:VV µ heavy chain assembles with 5 (S. R. Rheingold et al., manuscript in preparation). The developmentally ectopic expression of light chain in our transgenic system could also play a role, as the ability of any given light chain to substitute for surrogate light chain in the pre-BCR is unknown. Additionally, we note that our YS:VV IgM is capable of surface expression despite failure to assemble with Ig/ß, both in our transgenic system and in cell lines (9). Whether or not (or to what extent) surface expression of the pre-BCR is necessary for its signaling function is controversial. Finally, we paid particular attention to analyzing only those B cells that expressed the transgenic µ to focus on the role of YS:VV µ in delivering the signal for allelic exclusion. PCR analysis of unsorted bone marrow in our transgenics could easily have detected rearrangements in B cells expressing endogenous µ in the absence of transgenic µ and led us to a conclusion opposite to the one we reached: that YS:VV µ does mediate allelic exclusion.

Using a transgenic system to study the effect of a signaling-deficient Ig on B cell development, Torres et al. (32) demonstrated results more consistent with those seen here. They detected a block in B cell development, with the most significant depletion seen in the peripheral B cell compartment. The block in development, although profound, was not complete, and allelic exclusion was intact. These latter two findings were attributed to signaling via Igß, which is intact in these animals and which may assemble with the BCR in the presence of the signal-impaired Ig. This and other work (30) point to redundancy between Ig and Igß. In this view, each may provide partial or complete coupling to the downstream signaling machinery. This redundancy is brought into question by observations in Igß-deficient mice in which there is a complete block in B cell development (31). However, the block is at the pro-B stage, not at the pre-B cell stage during which µ is expressed and the pre-BCR complex can form, and points to an entirely separate and earlier role for Igß.

The transmembrane mutation (YS:VV) used in the studies reported here abrogates assembly with both Ig and Igß (8), suggesting that the Ig/ß heterodimer may not be the only µ-associated structure through which signaling via the pre-BCR takes place. Both our observations and those of Torres et al. are consistent with a model in which separate or parallel signaling mechanisms control the developmental process and allelic exclusion. Stringent controls avoiding B cell dual specificity are required, since the result of such dual specificity could either be autoimmunity or excessive deletion at the immature B cell stage. Given this result, it is not surprising that the mechanism controlling allelic exclusion might be separate from or, more likely, redundant with the developmental signal. Thus, allelic exclusion may be seen in the context of impaired development as we report here, while intact B cell development in the absence of allelic exclusion would not be observed.

The existence of separable signaling pathways raises the possibility that the pre-BCR may involve signaling transduction molecules other than Ig/ß. The role of the surrogate light chain has yet to be clearly delineated in pre-B cell development, but data from the studies of 5 knock-out mice (22, 23) show clearly that 5 is involved. Although µ chains, which do no bind surrogate light chains, are poorly represented in the B cell repertoire, this does not indicate the mechanistic link between 5, Ig/ß, and any downstream signaling pathways. Further support for a role for surrogate light chain in pre-BCR signaling comes from the Nussenzweig group, which has demonstrated a requirement for surrogate light chain or conventional light chain for pre-BCR signaling, even when Ig/ß are present (47).

In summary, our data add to the growing body of evidence implicating µ heavy chain in a pre-BCR complex that controls development and allelic exclusion. Further, we provide evidence that these two pathways may be distinct. The transgenic and knock-out systems that focus on µ heavy chain and the molecules of the pre-BCR complex offer the best opportunity to study the in vivo phenomenon of pre-B cell death or development.

	Acknowledgments

 
We acknowledge the valuable suggestions of Drs. Gillian Wu and Fay Young as well as the materials kindly provided by Drs. Jan Jongstra and Jim Kenny.

	Footnotes

 
¹ Supported by National Institutes of Health Grants AI22802 to A.K.A. and AI40111 and 5 P30 HD28815 to S.A.G., as well as by a Special Fellowship of the Leukemia Society of America (S.A.G.).

² Address correspondence and reprint requests to Stephan Grupp, Division of Pediatric Oncology, ARC 902, Children’s Hospital of Philadelphia, 34th and Civic Center Blvd., Philadelphia, PA 19104.

³ Abbreviations used in this paper: BCR, B cell Ag receptor; mIg, membrane-bound immunoglobulin; GAMµ, goat anti-mouse µ Ab; PC, phosphorylcholine; RAHµ, rabbit anti-human µ Ab; WT, wild-type mIgM; YS:VV, Tyr⁵⁸⁷/Ser⁵⁸⁸ to Val/Val transmembrane mutant; Y:F, Tyr⁵⁸⁷ to Phe transmembrane mutant; OE, phycoerythrin; Btk, Bruton’s tyrosine kinase.

Received for publication April 30, 1997. Accepted for publication March 6, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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