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The Journal of Immunology, 2001, 166: 3083-3089.
Copyright © 2001 by The American Association of Immunologists

Expression of a V Region-Less B Cell Receptor Confers a Tolerance-Like Phenotype on Transgenic B Cells1

Daniel Corcos2,*, Alf Grandien{dagger}, Aimé Vazquez{ddagger}, Olga Dunda*, Patrick Lorès* and Danielle Bucchini*

* Institut Cochin de Génétique Moléculaire, Institut National de la Santé et de la Recherche Médicale Unité 257, Paris, France; {dagger} Department of Immunology, Wenner-Gren Institute, University of Stockholm, Stockholm, Sweden; and {ddagger} Institut National de la Santé et de la Recherche Médicale Unité 131, Clamart, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neoplastic B cells from H chain disease patients express a truncated B cell receptor (BCR), comprising a membrane Ig that lacks part of its extracellular domain. It has been speculated that deletion of the Ag binding domain would confer a constitutive activity on the BCR, as it has been shown for oncogenic growth factor receptors. A V region-less BCR has constitutive activity, because in transgenic mice it causes inhibition of endogenous H chain gene rearrangements and relieves the requirement for surrogate L chain in pre-B cell development. However, it has been speculated that normal Ag receptors also display constitutive activity. Here we show that transgenic B cells expressing a membrane H chain disease protein on their surface are phenotypically and functionally similar to B cells developing in the presence of their cognate Ag and that cells with normal levels of mutant BCR are eliminated in spleen via a bcl-2 sensitive pathway while progressing toward the mature stage. In contrast, cells with lower levels of mutant receptors develop as mature B cells. These findings support the view that the truncated BCR has a constitutive activity that mimics ligand binding, in analogy to what has been shown for oncogenic growth factor receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The B cell receptor (BCR)3 is essential for the development and the survival of B cells (1, 2). In cancer cells from patients suffering from µ H chain disease, a B cell neoplasia, the µ H chain is truncated and lacks a functional variable domain (3, 4). Deletion of the variable domain of the human µ H chain, as found in several cases of µ H chain disease, allows surface expression of a truncated BCR in the absence of L chain (5, 6). This truncated BCR lacking an Ag binding domain has constitutive activity, because in transgenic mice the membrane {Delta}Vµ protein of human origin causes inhibition of endogenous H chain gene rearrangements and relieves the requirement for surrogate L chain in pre-B cell development (5, 7). The constitutive activity of this truncated BCR is reminiscent of that observed for oncogenic growth factor receptors (8). Some of these receptors can transduce signals in the absence of ligand as a consequence of loss of ligand binding domain (9). The activity of several constitutively activated receptors is mediated through self-oligomerization (10). Although there is evidence for self-aggregation of {Delta}Vµ-bearing receptors (7), it is not known whether this is required for the constitutive activity of these truncated BCR. Recently, biochemical and biological evidence has led to the suggestion that the normal BCR itself might possess constitutive activity (11, 12). Constitutive activity of unligated receptors has also been postulated to explain the effects of a transgenic truncated mouse H chain and of a truncated pre-TCR (13, 14), prompting us to determine whether the H chain disease protein containing BCR behaves as a normal BCR or whether the activity of the truncated BCR mimics that induced upon Ag binding. In mice transgenic for Ig genes, ligation of the BCR leads in most cases to B cell tolerance (15). This tolerance is achieved by a variety of means, including a block in B cell differentiation, cell death or reduced lifespan, anergy, or editing of the BCR due to secondary L chain gene rearrangement (16).

We have previously reported that, in {Delta}Vµ mice, there is a reduction in peripheral B cell numbers and very low levels of human protein are found in the serum (5). It has also been determined that {Delta}Vµ B cells have a short half-life (17). These features are similar to those observed in B cell tolerance models involving transgenic mice (15, 16), and therefore we investigated the possibility that the truncated BCR induces tolerance on B cells. We thus undertook a detailed characterization of peripheral B cells in {Delta}Vµ-transgenic mice. {Delta}Vµ mice were compared with hµSp6-transgenic mice, which express a complete H chain with the same human constant region and, unlike {Delta}Vµ mice, have high levels of human IgM detected in serum (~50 µg/ml). Here we show that {Delta}Vµ cells have a phenotype consistent with the idea that the mutant BCR has enhanced constitutive activity. {Delta}Vµ cells are functionally similar to tolerized B cells and are eliminated at the transitional B cell stage (18), cells with lower levels of receptors being spared. We also show that the truncated BCR allows the exit in periphery of cells with a very immature phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transgenic mice

{Delta}Vµ-6 and hµSp6-39 mice (5, 7) were backcrossed for at least 10 generations with BALB/c mice. Transgenic pups were identified either by the presence of human IgM in serum by ELISA or by DNA PCR of tail DNA using primers specific for the human µ promoter for {Delta}Vµ or primers specific for the Amp gene for hµSp6. Other {Delta}Vµ lines that were examined to ensure the reproducibility of the phenotype were {Delta}Vµ-12, {Delta}Vµ-13, and {Delta}Vµm-5 (5, 7), backcrossed for at least six generations with BALB/c mice. C57BL/6 (B6)-Sp6 mice (transgenic for mouse µ H and {kappa} chains (19) were provided by George Köhler (Max-Planck Institute of Immunology, Freiburg, Germany). BALB/c-{Delta}Vµ-6 mice and BALB/c-hµSp6-39 were bred to B6-Eµ-bcl-2-22-transgenic mice (20), and F1 mice were genotyped by ELISA for {Delta}Vµ and PCR on tail DNA for bcl-2. {Delta}Vµ mice were bred twice to recombination activating gene (RAG)-2 deficient (21) B6 mice and to µMT (1) B6 mice obtained from the Center de Developpement des Techniques Avancees pour l’experimentation animale (CDTA) in Orléans (France). Homozygous RAG-2-deficient mice and homozygous µMT mice were identified by the absence of mouse (m) IgM in ELISA, while {Delta}Vµ mice in the absence of other mutation still have mIgM in serum. The {Delta}Vµ protein could be detected in the serum by ELISA in the µMT background and in the RAG-2-/- background. Most of the experiments described here were conducted on mice raised in a pathogen-free housing (CDTA).

Flow cytometry

Single-cell suspensions were stained as described previously with the following conjugated Abs against human (h) IgM: FITC-conjugated monoclonal anti-hIgM (Nordic, Lausanne, Switzerland) and biotinylated anti-hIgM (MH15-1) from Janssen Pharmaceutica (Titusville, NJ); and against mouse markers: FITC-conjugated and biotinylated goat anti-mIgM (Caltag, South San Francisco, CA), FITC-conjugated goat anti-IgD (Nordic), FITC-conjugated (Harlan Sprague-Dawley, Indianaopolis, IN) and biotinylated (Caltag) goat anti-{kappa}, biotinylated and FITC-conjugated anti-CD23 (BD PharMingen, San Diego, CA), biotinylated and FITC-conjugated anti-B220 (Caltag), FITC anti-CD24 (heat-stable Ag; HSA) (BD PharMingen), biotinylated anti-CD22 (BD PharMingen), FITC-conjugated anti-CD21/CD35 (CR2/CR1) (BD PharMingen), FITC-conjugated anti-CD5 (BD PharMingen), FITC-conjugated anti-CD11b (Mac-1) (BD PharMingen), biotinylated anti-CD80 (B7-1) and biotinylated anti-CD86 (B7-2) (Southern Biotechnology Associates, Birmingham, AL), biotinylated anti-I-Ad (BD PharMingen). Biotinylated Abs were detected with PE-conjugated streptavidin (Caltag). Cells were analyzed with an Elite Cytometer (Coultronics; Beckman Coulter, Fullerton, CA). Dead cells were excluded by propidium iodide staining. Analyses were conducted on cells in the lymphocyte gate defined by forward and side scatter.

ELISA and in vitro culture assays

Human IgM serum concentrations were determined as in Ref. 5 . For in vitro assays, the following Abs were used: b7-6 (rat anti-mouse µ; Ref. 22), 20.5 (mouse anti-Sp6 Id; a gift of Pierre-André Cazenave, Institut Pasteur, Paris, France), alkaline peroxidase-labeled goat anti-mouse IgM (Southern Biotechnology Associates), and biotinylated goat anti-human µ (The Jackson Laboratory, Bar Harbor, ME). Secretion of IgM from LPS-stimulated B cells was quantified as in Ref. 23 . In the proliferation assays, [3H]thymidine (Amersham, Arlington Heights, IL) uptake was performed at day 3 of culture by adding 1 µCi per culture 4 or 8 h before harvest. Pulsed cultures were harvested on glass-fiber filter papers using a Skatron 96-well microtiter harvester (Flow Laboratories, McLean, VA). Incorporated radioactivity was measured in a scintillation counter.

DNA PCR assays

Semiquantitative DNA PCR assays were performed on 30 ng of DNA as described previously (24). The primers used were as follows: V{lambda}1, 2, 5'-CAGGCTGTTGTGACTCAGGAATCTG-3'; J{lambda}1, 5'-CTCACCTAGGACAGTCAGTTTGGTT-3', derived from published sequences (25, 26); 5' intronic RS, 5'-GGTAGCATCCCTTGCTCCGCGTGG-3'; 3' RS, 5'-GGGTTTCGTTTGACTGTTTGCTAC-3' (27). The following oligonucleotides were used as probes: for {lambda} rearrangement, 5'-GTCGTTGGTAACCCACAAGCC-3'; for RS rearrangement, 5'-GAGCTCAACTGCGAGT3'. DNA PCR assays were normalized using insulin gene PCR products (as in Ref. 24). Quantification was performed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of B cell populations in {Delta}Vµ mice on the BALB/c background

{Delta}Vµ- and hµSp6-transgenic proteins cause allelic exclusion, and almost all spleen B cells from {Delta}Vµ-6- and hµSp6-39-transgenic mice express neither mouse IgM nor IgD on their surface (5, 7). For this reason, B cells from these transgenic mice were identified by surface staining for developmentally regulated markers. According to the levels of truncated BCR, it is possible to distinguish two {Delta}Vµ cell populations: {Delta}Vµlow cells, which are HSAlow, CD22high, CD23+, CD21+, and B220high and thus have all the phenotypic characteristics of normal or hµSp6 mature B cells; and {Delta}Vµhigh cells, which are HSAhigh, CD22low, B220low, CD23-, and CD21low ( Figs. 1–3GoGoGo and data not shown). Weak staining for CD22 distinguishes these {Delta}Vµhigh cells from marginal zone and B1 B cells (28). These cells are unlikely to be B1 cells because they do not express CD5 or Mac1 and because the majority of peritoneal cells in {Delta}Vµ mice express endogenous IgM on their surface but not the transgenic receptor (not shown), suggesting that this receptor is incompatible with differentiation or survival of B1 cells. The levels of CD22, CD23, and B220 of these immature {Delta}Vµ spleen cells are lower than those of immature (transitional) spleen cells of nontransgenic and hµSp6-transgenic mice and are similar to those of immature B cells found in the bone marrow of normal mice ( Figs. 1–3GoGoGo and data not shown). {Delta}Vµhigh B cells, despite their immature phenotype, are found in numbers that are comparable to those of transitional B cells from hµSp6-transgenic mice or normal BALB/c mice (Fig. 2GoD). In contrast, mature spleen B cells of {Delta}Vµ mice are present in much more reduced numbers (Fig. 3Go). Because {Delta}Vµ immature spleen cells stain only very weakly for CD23, this marker was used in further studies to identify mature spleen B cells. BALB/c-hµSp6 mice show a modest reduction in mature B cell numbers, comparable to that of mice transgenic for normal mouse BCR (29). Expression of the {Delta}Vµ transgene reduces the number of mature CD23+ B cells in {Delta}Vµ+hµSp6 double-transgenic mice (Fig. 3Go), although these cells express the complete transgene because they stain for both hIgM and mouse {kappa} L chain (not shown), indicating that the phenotype of {Delta}Vµ mice is not simply the consequence of lack of function.



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  		FIGURE 1. Two B cell populations expressing different levels of BCR are present in {Delta}Vµ mice. Spleen cells from 3-mo-old BALB/c mice transgenic for {Delta}Vµ or hµSp6 were stained with anti-hIgM and anti-HSA or CD22 and analyzed by flow cytometry. Live cells within the lymphocyte gate are shown. Analyses of spleen cells from a nontransgenic BALB/c-{Delta}Vµ littermate after staining with anti-mIgM are also shown, but it must be noted that the main expressed BCR of normal mature B cells is IgD.

 


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  		FIGURE 2. Characterization of immature hIgMhigh B cells from {Delta}Vµ mice. A, Cells from a {Delta}Vµ mouse and a nontransgenic BALB/c littermate were stained with anti-B220 and either anti-hIgM or anti-mIgM. B, The intensity of B220 staining of {Delta}Vµhigh cells (shaded area) was compared with that of control immature B cells (B220low, mIgM+) from bone marrow (open area, solid line) and to that of mIgMhigh (mainly transitional) spleen cells (open area, thin line). C, B220 staining of mature {Delta}Vµlow spleen B cells from {Delta}Vµ mice (shaded area) was compared with that of mature bone marrow B cells (open area, solid line) and mature spleen B cells (open area, thin line) from control mice after normalization for cell number. D, Number of immature B cells in 1-mo-old (left) and adult (right) transgenic mice of the indicated genotype. Cl, Nontransgenic BALB/c. Immature B cells were defined as HSAhigh B220low. Bars indicate SEM of n mice.

 


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  		FIGURE 3. B cell development is impaired by the mutant BCR. A, Two-color flow cytometric analysis of spleen cells of 3-mo-old {Delta}Vµ and hµSp6 mice on the BALB/c background, after staining with anti-hIgM and anti-CD23. The numbers indicate the percentages of cells within the rectangles. B, Number of mature B cells in the spleen of mice transgenic for the indicated constructs (BALB/c background). h+{Delta}, hµSp6+{Delta}Vµ double transgenic. Mice were 2–4 mo old. Mature B cells were defined either as CD23+ cells or as B220high HSAlow. Bars indicate SEM of n mice.

 
Mature B cells of {Delta}Vµ mice display lower levels of human-derived BCR on their surface than mature B cells from hµSp6-transgenic mice, although immature B cells from these two different transgenic mice display similar levels of hIgM (Figs. 1Go and 3Go). The difference observed in surface mIgM levels between mature and immature B cells of nontransgenic mice is related to the presence of surface IgD, which is the major Ag receptor of mature B cells and therefore does not reflect a decrease in BCR levels (data not shown). Thus mature cells of {Delta}Vµ mice display a specific reduction in the surface expression of their BCR. The phenotype described here for the {Delta}Vµ-6 line was consistent in the four different {Delta}Vµ-transgenic lines (with different copy numbers of the transgene) (5, 7) that have been studied.

In summary, spleens of {Delta}Vµ mice comprise two populations of cells, both of which express the transgenic truncated receptor in the absence of endogenous H chain on their surface: immature B cells with a bone marrow-like phenotype, expressing high (normal, as compared with complete H chain-transgenic mice) levels of truncated protein; and mature B cells with reduced levels of truncated receptors. Mature {Delta}Vµ cells develop also, and perhaps in increased numbers, in mice deficient for endogenous BCR expression (µMT mice) (see below), indicating that maturation depends solely on the truncated receptor. These results suggest that in {Delta}Vµ mice, B cells with lower level of BCR expression are selected in the mature pool. However, perhaps as a consequence of its limited life span, this B cell population does not increase in older (1 year) animals (data not shown).

In addition, {Delta}Vµ mature B cells are enlarged in size as compared with normal mature or {Delta}Vµ immature B cells (Fig. 4Go), but they do not express activation markers as B7-1 or B7-2 and display normal levels of surface MHC class II (not shown). In contrast, hµSp6-transgenic cells are smaller than normal B cells (Fig. 4Go). The small size of hµSp6 B cells might be due to the absence of cognate ligand, in line with results showing that in Ig-transgenic mice the cells that are activated are those expressing endogenous Ig genes (23). The increase in size in the absence of other feature of activation suggests that mature {Delta}Vµ cells are abortively activated and is reminiscent of findings in another tolerance model (30).



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  		FIGURE 4. {Delta}Vµ mature B cells are larger than normal B cells. Spleen cells of age-matched (3 mo old) BALB/c-transgenic and control mice raised in pathogen-free conditions were stained with anti-HSA and anti-B220 and analyzed by two-color flow cytometry A, Mature spleen B220high HSAlow were electronically gated and their size was determined by forward scatter (shaded areas) and compared in B to that of immature (B220low HSA high) B cells of the same mouse (open areas, dotted lines). Note that immature B cells were of the same size in all mice. In C, spleen mature (B220high HSAlow) B cells of four 3-mo-old, pathogen-free, BALB/c mice of each indicated genotype were analyzed by forward scatter. The percentages of cells over a fixed arbitrary value are indicated.

 
{Delta}Vµ B cells are anergic

A prominent feature of tolerized B cells is anergy. In attempts to derive hybridomas from BALB/c-{Delta}Vµ mice, we found that {Delta}Vµ cells, unlike hµSp6 cells (data not shown), did not proliferate in response to the polyclonal activator of B cells, LPS. To determine whether expression of the truncated BCR inhibits LPS responses in cells expressing a normal BCR, we studied proliferative and secretory responses of splenocytes from double-transgenic mice expressing both a normal mouse BCR and the {Delta}Vµ protein. We used offspring from BALB/c-{Delta}Vµ mice and B6-Sp6 mice (19). Although cells from double-transgenic animals coexpress both types of receptors on their surface (7), expression of the {Delta}Vµ molecule resulted in strong inhibition of [3H]thymidine incorporation and of mIgM secretion (Fig. 5Go). Expression of the normal H chain nonetheless allowed a weak proliferative response and secretion of human {Delta}Vµ, indicating that it counteracts the effect of membrane {Delta}Vµ, a finding reminiscent of the effect of the human µ chain on the generation of CD23+ cells (Fig. 3Go). {Delta}Vµ cells were also poorly responsive to Staphylococcus aureus Cowan I, unlike normal or hµSp6-transgenic cells (not shown).



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  		FIGURE 5. Expression of the {Delta}Vµ protein inhibits B cell responses to LPS. Splenocytes from (B6-Sp6 x BALB/c-{Delta}Vµ)F1 were cultured at 2 x 105 per ml in 0.2-ml cultures. The starting populations contained: Cl (nontransgenic), 48%; mµSp6, 50%; {Delta}Vµ, 24%; {Delta}+mµ (double transgenic), 37% B220+ cells, as determined by flow cytometry. A, Proliferation measured by [3H]thymidine incorporation after a 4-h pulse at day 3 of culture with LPS as previously described (23 ). Bars indicate SEM of triplicate cultures. B, Ig content in the culture determined by ELISA at day 6 of culture with LPS, using mIgM or hIgM as standard. *, Not detectable.

 
Evidence for secondary L chain gene rearrangement in {Delta}Vµ cells

Another consequence of signaling through BCR is secondary L chain gene rearrangement (16). It has been shown that while an autoreactive BCR will induce L chain gene rearrangement, a nonautoreactive BCR will not (31). Although editing in {Delta}Vµ mice cannot be manifested by a change in L chain, because {Delta}Vµ receptors do not contain L chains, it is possible to evaluate the nature and the extent of L chain gene rearrangement by semiquantitative methods (24). B cells of {Delta}Vµ mice have a high level of {kappa} L chain gene rearrangement (7), and intronic {kappa}-RS rearrangement (a rearrangement that deletes the C{kappa} locus and is present in a high fractions of {lambda}-expressing B cells) is increased in {Delta}Vµhigh spleen cells as compared with total spleen B cells (95% {kappa}-expressing cells and 5% {lambda}-expressing cells) of normal BALB/c mice, indicating secondary rearrangements on the {kappa} locus (Fig. 6Go). A high level of RS rearrangement is also found in {Delta}Vµ bone marrow cells (not shown), indicating that this rearrangement occurs early in B cell development. We do not know why {lambda} gene rearrangement is not enhanced in {Delta}Vµ cells, but the conservation of the {kappa}/{lambda} rearrangement ratio (7) is reminiscent of the effect of an activated ras oncogene on B cell development (32). Moreover, editing in the absence of {lambda} chain expression has previously been observed (33).



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  		FIGURE 6. Increase in RS recombination in {Delta}Vµ cells. {lambda} (V{lambda}1-J{lambda}1) and intronic {kappa}-RS (I{kappa}-RS) rearrangements were analyzed as described (24 ). A, Representative autoradiographs of rearrangement products after amplification of DNA from: tail (T); {lambda}- and {kappa}-expressing cells from spleen of control (Cl) BALB/c mice; mIgM+CD23lowcells (I) and CD23+ mature (M) B cells from control mice; BALB/c {Delta}Vµhigh cells ({Delta}I); and hIgM+ spleen cells from hµSp6 mice (H). Loaded volumes were normalized after a first electrophoresis and ethydium bromide staining of insulin gene PCR products (5 7 ). B, Quantification of intronic {kappa}-RS rearrangement in {Delta}Vµhigh cells. DNA PCR assays were normalized by hybridization of insulin gene PCR products (24 ). The value for {Delta}Vµhigh cells represents the mean from seven separate assays where total spleen B cells (IgM+) are the 100% value. Bars indicate SEM.

 
Rescue of {Delta}Vµhigh cells by a bcl-2 transgene

To investigate the mechanisms leading to B cell depletion and the short half-life of {Delta}Vµ spleen cells, we crossed BALB/c-{Delta}Vµ mice to B6 mice transgenic for bcl-2 (2028). {Delta}Vµ mice on the (BALB/c x B6)F1 background display the same two B cell populations as in the BALB/c background and significantly less CD23+ B cells than (BALB/c x B6)F1-hµSp6 mice (Fig. 7Go) but their mature B cell number is not as dramatically reduced as in the BALB/c background. Enforced expression of the anti-apoptotic gene bcl-2 in the B cell lineage rescues the development of a mature CD23+ B cell population of {Delta}Vµhigh cells (Fig. 7GoA), and mature B cell numbers in the spleen of {Delta}Vµ-bcl-2 double-transgenic mice are equivalent to those found in hµSp6-bcl-2 mice (Fig. 7GoB), indicating that, in mice that do not overexpress bcl-2, cells expressing the truncated BCR at high levels are eliminated by apoptosis.



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  		FIGURE 7. Mature B cells expressing high levels of mutant receptor develop when bcl-2 is overexpressed. A, Representative flow cytometric analysis of spleens from four littermates (2 mo old) born from a cross between BALB/c-{Delta}Vµ and B6-Bcl-2. The percentages of cells within the rectangles are indicated. B, Number of mature B cells defined as CD23+ cells in the spleen of 2- to 3-mo-old (BALB/c x B6)F1 animals transgenic for the indicated constructs. SEM are indicated.

 
We conclude that expression of {Delta}Vµ at a level comparable to that of normal transgenic IgM leads to cell death at the transitional or mature B cell stage and that cells with a reduced level of {Delta}Vµ can reach the mature B cell stage in the absence of endogenous BCR. Levels of hIgM are low (<10 µg/ml) in {Delta}Vµ-bcl-2 mice, suggesting that the functional defect of {Delta}Vµ cells remains when bcl-2 is overexpressed.

Elimination of {Delta}Vµhigh cells does not require T cells

To determine whether T cells are involved in the elimination of {Delta}Vµhigh cells, we backcrossed BALB/c-{Delta}Vµ mice to B6-RAG-2-deficient mice (21). F2 mice of different genotypes were obtained, and their spleens were analyzed by flow cytometry (Fig. 8Go). Immature, CD23- {Delta}Vµhigh B cells were present in normal or increased numbers, but the numbers of mature CD23+ {Delta}Vµlow cells were reduced. This reduction in mature B cell numbers is not observed in mice lacking endogenous BCR expression (1) on the same genetic background (Fig. 8GoB) and therefore suggests that T cell derived factors contribute to the survival of transgenic mature B cells. In any event, we can conclude that elimination of {Delta}Vµhigh cells does not require T cells.



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  		FIGURE 8. Development of {Delta}Vµ-expressing B cells in the absence of T cells. A, Representative flow cytometric analysis of spleens from two littermates (2 mo old) born from a cross between (BALB/c x B6)F1 {Delta}Vµ+/- RAG-2+/- (double heterozygous) and B6-RAG-2-/- mice. RAG-2-/- mice were identified by absence of mIgM in serum, which was confirmed by absence of CD4+ spleen cells. Rectangles indicate the percentages of CD23- hIgMhigh and CD23+ hIgMlow cells. B, Number of {Delta}Vµ CD23- and CD23+ B cells in the spleen of 2- to 3-mo-old animals in µMT or Rag-2-deficient background. Cl represents {Delta}Vµ cells in {Delta}Vµ+/- µMT+/- or {Delta}Vµ+/- RAG-2+/- animals. SEM are indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Altogether, our results indicate the following. First, expression of the truncated BCR at normal levels is responsible for two major effects, export to the periphery of cells with an immature phenotype and apoptosis in the periphery at the transition from the immature to the mature stage. The possibility that the export of cells with an immature phenotype to the periphery is related to homeostatic compensation seems to be excluded because one {Delta}Vµ mouse line ({Delta}Vµm-1) exhibited both a large number of mature B cells expressing endogenous BCR and immature bone marrow-like spleen B cells expressing the transgene (5). Second, the truncated receptor when expressed at low levels allows some survival of mature B cells. We favor the idea that, in the absence of the bcl-2 transgene, {Delta}Vµlow cells are preferentially selected into the mature B cell pool as a function of their lower levels of constitutive receptor. If selection is responsible for the low levels of {Delta}Vµ receptors in mature B cells, the molecular basis for the reduction in cell surface expression is not clear, because {Delta}Vµ homozygous mice do not show any increase in receptor numbers nor reduction in their mature B cell pool. An alternative interpretation for the low levels of truncated BCR on mature B cells would be down-regulation of the receptor. However, it is difficult to imagine why this down-regulation occurs only in mature and not in immature B cells and why it does not occur in bcl-2-transgenic mice.

Interestingly, we found that, unlike mature B cells, immature CD23- B cells tolerate a high level of constitutive, self-aggregating BCRs, suggesting that, in vivo, CD23- B cells do not die upon aggregation of their BCR. These results are in agreement with those of Carsetti et al. (18), who found that immature B cells, unlike transitional B cells, are resistant to BCR cross-linking. It has also been proposed that negative selection by self-Ags occurs mainly in the peripheral lymphoid system at the transition between the immature and the mature B cell stage (34, 35).

The {Delta}Vµ model presents two distinctive features as compared with other negative selection models. First, the {Delta}Vµ receptor is present at high levels on the B cell surface of immature B cells, or of mature B cells in bcl2-transgenic mice, instead of being down-regulated as is usually observed for anergic B cells. The absence of down-regulation of the truncated BCR might be important for enhanced constitutive activity. In support of this view, it has been suggested that the mitogenic effects of an oncogenic truncated mutant of the epidermal growth factor receptor are due to low constitutive activation, amplified by failure to attenuate signaling (36). Interestingly, it has been proposed that a normal BCR might possess constitutive activity (11, 12), and therefore this constitutive activity might be exacerbated by the absence of feedback. The mechanism leading to the absence of down-regulation is unknown. A highly speculative scenario is that it might involve the chaperone H chain binding protein (BiP), because BiP retains normal µ (37) but not {Delta}Vµ H chains inside the cell in the absence of L chains. Indeed, it has been shown that down-regulation of membrane IgM in tolerant B cells involves a postsynthetic step between the endoplasmic reticulum and the Golgi apparatus (38), while it is in the endoplasmic reticulum that the BiP chaperone retains µ chains that do not associate L chains.

The second feature, export to the periphery with an immature phenotype, might also be related to the previous mechanism. In the case of Ag-induced tolerance, down- regulation of the BCR would lead to a state akin to that induced by the ablation of the BCR with a rapid cell death in situ or the absence of export from the bone marrow, while lack of down-regulation of the constitutive BCR would allow the export to the periphery. The immature phenotype of splenic {Delta}Vµhigh cells might be the consequence of two processes, not necessarily exclusive: a block in B cell maturation, as has been shown after strong cross-linking of BCRs by self-Ags on the surface of immature B cells (39); or a premature exit from the bone marrow. This last possibility has been shown for B cells expressing an activated ras gene, but these cells seem to have a more mature phenotype (40).

We found that, when bcl-2 is overexpressed, mature {Delta}Vµ cells develop in the same way as mature hµSp6-transgenic cells. B cell numbers do not reach the levels observed in bcl-2 single-transgenic mice, but this difference is probably related to the effects of Ig transgenes early in development (7), and therefore we think that bcl-2 completely prevents {Delta}Vµ-induced deletion. In the membrane-bound lysozyme model, immature B cells with an anti-lysozyme BCR were rescued by bcl-2, but not mature B cells. This might be due to a different balance between anti-apoptotic activity and signals delivered upon strong BCR aggregation by membrane-bound lysozyme. It is interesting to note that expression of bcl-XL can enhance the survival of mature B cells (41) and that bcl-2 can rescue mature anti-H-2 autoreactive B cells in an Ag dose-dependent manner (42, 43). A comparison might also be made between {Delta}Vµ-induced cell death and that induced by BCR ablation (2). In the case of BCR ablation, cell size decreases while it increases in the mature (Fig. 4Go) and the very rare transitional (data not shown) spleen cells expressing {Delta}Vµ. Moreover, bcl-2 is not able to fully rescue cells from death induced by BCR ablation (2).

The {Delta}Vµ model seems to be suitable for the study of genes implicated in B cell development, because there is no effect related to the repertoire, either in B or T cells, and the {Delta}Vµ transgene can be easily introduced on the genetic background that is to be studied. We found a significant difference in mature B cell numbers between the BALB/c and the (BALB/c x B6)F1 backgrounds, and it will be interesting to study {Delta}Vµ cells in genetic models where B cell tolerance is compromised.

The finding of a decreased CD23+ mature B cell population in {Delta}Vµ-RAG-2-deficient mice, but not in {Delta}Vµ-µMT mice, suggests a role for T cells in the survival of mature {Delta}Vµ cells. Because nontransgenic B cells or B cells expressing a transgenic anti-H-2K BCR develop normally in the absence of cognate ligand in a RAG-1-deficient background, it is unlikely that all normal mature B cells display a similar T cell dependence (44). Interestingly, it has been shown that T cells support the survival of autoantigen binding B cells (45). Because {Delta}Vµ B cells are unable to bind an Ag, our results would mean that the role played by T cells might be independent of specific cognate interactions.

The dependence of {Delta}Vµ cells on nonspecific factors is reminiscent of the situation observed in the most common form of H chain disease, namely {alpha}-chain disease, which can be treated by antibiotics (46). It is also of interest to note that {Delta}Vµ mature B cells are eliminated by competition with normal B cells (17), as are autoreactive B cells (47), raising the possibility that, in the latter case, competition occurs for T cell-derived factors.

Finally, while the truncated Ag receptor behaves like an activated receptor, as predicted by our pathogenic model, {Delta}Vµ-transgenic mice do not suffer from lymphoproliferative disorders. One of the reason for this is that the genetic event leading to the production of a H chain disease protein probably occurs at the mature B cell stage (48), whereas in transgenic mice the {Delta}Vµ protein is expressed at an early stage, which is more likely to induce tolerance (49). In addition, in contrast to numerous in vitro findings showing that mature spleen B cells can be stimulated to proliferate after stimulation with anti-IgM, cross-linking of BCR on mature B cells in vivo induces cell death (18, 43, 50), and the factors that determine, in the absence of T cells, the outcome of the responses induced by BCR ligation, namely tolerance, proliferation, or differentiation to the B1 cell subset (51), remain to be elucidated. In the case of H chain diseases, a second signal, responsible for proliferation and H chain secretion, might be provided by a yet unknown cooperating genetic event.


   Acknowledgments
 
We thank J. Jami for support during this work, A. Harris and G. Köhler for providing bcl-2-22-transgenic mice and B6-Sp6-transgenic mice, M. Lemée and J.-P. Reignault from the CDTA for animal care, I. Bouchaert for help with flow cytometry, and S. Elsevier, C. Fournier, and M. Goodhardt for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by grants from the Comité de Paris de la Ligue Nationale contre le Cancer. Back

2 Address correspondence and reprint requests to Dr. Daniel Corcos, Institut National de la Santé et de la Recherche Médicale Unité 477, Hôpital Cochin, 27 rue du Faubourg St-Jacques, 75014 Paris, France. Back

3 Abbreviations used in this paper: BCR, B cell receptor; RAG, recombination activating gene; HSA, heat-stable Ag; B6, C57BL/6; BiP, heavy chain binding protein; m, mouse; h, human. Back

Received for publication November 1, 2000. Accepted for publication December 22, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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