The level of expression of µ heavy chain modifies the composition of peripheral B cell subpopulations

Pierre Sanchez, Anne-Marie Crain-Denoyelle, Philippe Daras, Marie-Claude Gendron1 and Colette Kanellopoulos-Langevin1

Laboratoire d'Immunobiologie, Case 7048, Université Denis-Diderot (Paris 7), 2 Place Jussieu, 75251 Paris Cedex 05, France
1 Institut Jacques Monod, 75005 Paris, France

Correspondence to: P. Sanchez


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The B cell receptor (BCR) has a decisive role in transducing signals required for the development of B cells and their survival in the periphery. However, the processes that initiate these signals remain unclear and concepts of constitutive and ligand-dependent signaling have been proposed. Using a µ-transgenic mouse model, we have analyzed the impact of high surface IgM expression on the composition of the splenic B cell population. {kappa}-deficient mice homozygous for the H3-µ transgene have B cells with a higher BCR surface density than H3 heterozygous mice. This higher BCR expression is associated with an increase in the percentage and the total number of splenic B cells. In addition, an important proportion of CD23CD21+ marginal zone (MZ) B cells can be observed in H3 homozygous mice. However, these modifications operate in the absence of impairment of the positive selection process of the H3-µ/{lambda}1 combination over the H3-µ/{lambda}2 + 3 ones. These results suggest that (i) a constitutive BCR signaling directly correlated with BCR surface density is responsible for the efficient B cell colonization of the periphery with an accumulation of B cells in the MZ and (ii) a ligand-dependent BCR signal is responsible for the clonotype composition of the mature B cell repertoire.

Keywords: B lymphocytes, B cell repertoire, {kappa} knockout, {lambda} chains, µ transgenic mice


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The heavy chain of Ig plays a key role in B cell development (for review, see 1). Linked to a surrogate light chain or a conventional light chain and associated with the Ig{alpha} and Igß chains, it forms the pre-B or B cell receptor (pre-BCR/BCR) respectively. These receptors can transduce signals by inducing phosphorylation of Ig{alpha}/Igß immunoreceptor tyrosine-based activation motifs (2). The pre-BCR is supposed to control the pre-B to immature B cell transition, whereas the BCR should drive transition from the immature to mature B cell stage. However, a crucial question which remains without conclusive answer to date is whether the pre-B/BCR constitutively transduces a basal signaling for cellular survival or whether the pre-B/BCR signaling depends on yet unknown ligands external to the cell (3,4). Different experimental models have shown that the absence of any element of the pre-B/BCR blocks B cell development but they did not address the mechanisms of pre-B/BCR signaling (510). On the other hand, studies comparing B cell repertoires among emergent and mature B cells suggest that an external signal could induce a clonal positive B cell selection (1114). These results, however, do not discriminate between signaling by self-ligand and signaling by environmental non-self-antigens. In some cases, a correlation has been shown between positive selection and the presence of a self-antigen (1517).

To distinguish between constitutive and external survival signaling, it can be postulated that the number of pre-B or BCR on the surface of the cell differently influences the two processes. In the constitutive signaling process, the density of receptors could be directly correlated with the intensity of the signal. On the other hand, in the external signaling process, the intensity of the signals depends on a supplementary constraint that is the number of ligand–receptor interactions in the micro-environment during cell development. To address this question, we analyzed the influence of the expression level of the Ig heavy chain on B cell development in our `oligoclonal' mouse model (18). In this model, we have previously shown that H3-µ-transgenic {kappa}-knockout mice displayed a peripheral B cell repertoire largely dominated by H3-µ/{lambda}1 B cells, while emergent immature B cells can express the transgenic µ chain in association with one or another of the four available {lambda} subtypes [{lambda}1, {lambda}2(V2), {lambda}2(Vx) and {lambda}3]. We have postulated that a positive selection of H3/{lambda}1 cells is responsible for their dominant presence in the periphery. We have also shown that this selection is hampered if the {lambda}1 chain is substituted by the SJL {lambda}1 chain ({lambda}1s) that leads to a dysfunction of the {lambda}1s-BCR (19,20). Indeed, B cell development is stopped at an immature B cell stage in {kappa}–/–, {lambda}1s/s mice heterozygous for H3-transgenic inserts. However, in H3 homozygous mice, B cell development can be observed in the absence of H3/{lambda}1 positive selection. This suggests that the expression level of the transgenic µ chain also controls the immature/mature B cell transition. In the present study, we analyzed the influence of the quantitative expression of the µ chain in B cell development in conjunction with the clonal positive selection process. We show that a high level of H3 transgene chain expression favors the colonization of B cells in the periphery but does not modify the positive selection process. These results suggest that the mature B cell repertoire is generated upon: (i) an constitutive signaling process directly dependent on BCR surface density that ensures survival in the periphery and (ii) a ligand-dependent signaling process which induces clonal positive selection responsible for modification of the emergent B cell repertoire.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
Adult mice were housed in the animal facilities according to Institutional Guidelines at the Institut Jacques Monod, Paris. Mice homozygous for the C{kappa} mutation ({kappa}–/–) were derived from chimeras from 129/Sv ES cells and crossed several times with C57BL/6 (21). H3-transgenic {kappa}–/– mice were derived from H3-transgenic C57BL/6 mice obtained by Honjo's group (22). The association of the H3-µ chain with the L3-transgenic {kappa} chain displays an activity against mouse erythrocytes. However, mice expressing the H3-transgenic µ chain only do not develop anemia (22). To exclude endogenous IgM expression, we also introduced the µMT mutation obtained by Rajewsky's group (5). All transgenic mice used in this study therefore are H3-Tg,{kappa}–/–,µMT/µMT with a genetic background several times backcrossed with C57BL/6. The transgenic and endogenous status of µ genes were checked by Southern blot analysis using an XbaI–HindIII Cµ probe as shown in Fig. 1Go (23).



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Fig. 1. Hetero- versus homozygous status of H3-transgenic mice. Tail DNA was digested by BamHI and hybridization was done with a Cµ probe. The band ratios were calculated after scanning membranes with a Phosphorimager (Molecular Dynamics, Bondoufle, France).

 
Antibodies and flow cytometry analyses
Two- or three-color analyses were done on an Epics Elite ESP flow cytometer (Coultronics, Margency, France). The antibodies were coupled to FITC, phycoerythrin or biotin depending on the combination used. For two-color analyses, streptavidin coupled to phycoerythrin (Southern Biotechnology Associates, Birmingham, AL) was used to reveal biotinylated antibodies. For three-color analyses, streptavidin coupled to Quantum Red (Sigma, St Louis, MO) was used. Anti-CD24 was a gift from Dr A. Freitas. Anti-CD23 (B3B4), anti-CD21 (7G6), anti-µb (AF6-78) and anti-µa (DS-1) were purchased from PharMingen (San Diego, CA). Goat anti-mouse IgM was purchased from Sigma. Other antibodies were purified and coupled by ourselves: anti-CD45R/B220 (RA3-6B2) (24), anti-{lambda}1 (MS40-13) (25) and anti-{lambda}2 + {lambda}3 (189B3) (26).

Four main {lambda} chains can be produced by the {lambda} locus depending on the rearrangements: {lambda}1 (V1J1C1), {lambda}2(V2) (V2J2C2), {lambda}2(Vx) (VxJ2C2) and {lambda}3 (V1J3C3) (27). MS40-13 mAb only recognizes {lambda}1 chain, whereas 189B3 does not discriminate between {lambda}2(V2), {lambda}2(Vx) and {lambda}3 chains, and so is called anti-{lambda}2 + {lambda}3.

For cytoplasmic staining, cells were first stained with biotinylated goat anti-mouse IgM and phycoerythrin–anti-CD45R/B220 antibodies, and subsequently stained with streptavidin coupled to Quantum Red. Then, cells were fixed with paraformaldehyde in a buffered saline solution and subsequently permeabilized with saponin in FCS-buffered saline solution, as recommended by BioSource International (Camarillo, CA). After staining with FITC–anti-mouse IgM, cells were analyzed by FACS. Cytoplasmic versus surface staining was also checked by fluorescence microscopy.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The level of µ-transgenic surface expression conditions the colonization of B cells in the periphery
Southern analyses with a Cµ probe were carried out to estimate the number of copies of the H3-µ transgene in transgenic mice. As shown in Fig. 1Go, it can be estimated that two to three copies per haploid genome are inserted. In addition, two transgenic genotypes can be clearly distinguished by comparing the ratios of intensities between transgenic and endogenous Cµ hybridized bands. Indeed, ratios between transgenic bands remain constant (mean c/b = 3.1) while ratios between transgenic and endogenous bands discriminate two groups defining two transgenic genotypes (H3+/+ and H3+/– for mean c/d = 2.4 and 1.1 respectively). We therefore tested the influence of the transgenic genotype on the surface density of transgenic BCR by flow cytometry analysis. As our mice are µMT, we can detect directly the presence of the µ transgene with goat antibodies to mouse µ chain (see Methods). As shown in Fig. 2Go, H3+/+ homozygous mice are characterized by a small increase in the percentage of B cells with respect to their heterozygous counterparts (44 versus 35%) and a higher surface density of Ig receptor (middle profile). A staining of permeabilized cells with anti-µ antibodies also shows higher intracytoplasmic production of the transgenic chain. As heterozygous mice displayed a weaker percentage of B cells, we checked that the number of spleen B cells decreased as well. Results shown in Table 1Go confirm that heterozygous mice have both the percentage and a number of splenic B cells significantly diminished, indicating that the higher expression of transgenic chain increases the absolute number of B cells in the periphery.



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Fig. 2. High µ chain expression in homozygous H3-transgenic mice. Spleen cells were analyzed by three-color labeling. First, surface staining was carried out with anti-µ and anti-B220 antibodies, and the cells were subsequently permeabilized for cytoplasmic staining with anti-µ antibodies. The middle profile represents the fluorescence intensity from cells in the gates defined on the upper profiles.

 

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Table 1. High level of µ chain expression is correlated with a higher number of splenic B cells
 
The high expression of H3-µ transgene does not seem to modify the efficiency of B cell differentiation in the bone marrow
The fact that the enhanced expression of µ chain increases the number of peripheral B cells could result from an increment in the entrance of newly formed B cells in the periphery. We have previously shown that the ratio of immature B cells (IgM+B220lo) to total B220lo cells is unchanged between H3+/+ and H3+/– non-µMT mice (19). We reinvestigated this in H3-transgenic µMT by including cytoplasmic anti-µ labeling to see whether higher µ expression could be detected early in B cell development. Figure 3Go shows that both cytoplasmic c) and surface (µs) expressions of µ chain are higher at different developmental B cell stages in H3+/+ mice compared to H3+/– mice. The ratio IgM+B220lo/total B220lo is similar in both mice (12.5% ± 2.5 for H3+/+ and 15.2% ± 6.0 for H3+/–, P > 0.5). These results show that the increased expression of µ chain has no detectable effect in the pro/pre-B -> immature B cell transition in these transgenic mice, confirming previous data (19). The pro-B/pre-B ratios (0.11 ± 0.01 for H3+/+ and 0.20 ± 0.14 for H3+/–, P > 0.5) as estimated by the B220+µcµs/B220+µc+µs ratio from flow cytometry analyses, is also compatible with an absence of changes in the pro-B -> pre-B transition. Altogether, these results suggest that an increased expression of the µ chain does not favor a more efficient B cell differentiation.



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Fig. 3. Higher expression of the µ-transgenic chain during the first steps of B cell development in H3+/+ mice. Bone marrow cells were treated as in the legend of Fig. 2Go. B220+ cells were gated for cytoplasmic and surface anti-µ staining as shown in the left profiles. The three lower histograms correspond to cytoplasmic staining of the cells gated as shown in the upper profile, i.e. B220loIgM (pre-B), B220loIgM+ (B220lo B cells) and B220hiIgM+ (B220hi B cells). The numbers represent the percentage of the analyzed cells in the gates or quadrants indicated.

 
Positive selection of the H3/{lambda}1 combination is characterized by the mature follicular profile of B cells, independently of the level of transgenic µ chain expression
Both H3-transgenic mice display a B cell repertoire strongly biased for the expression of H3/{lambda}1 combination. In the experiment shown in Fig. 4Go, 84% (37 of 44) and 96% (26 of 27) of splenic B cells express {lambda}1 chains in H3+/+ and H3+/– mice respectively. In non-transgenic mice, we recurrently found a {lambda}1 percentage of ~60% (18,19,21,28). This result favors the selection of H3/{lambda}1 cells regardless of the level of expression of the µ-transgenic chain. On the other hand, it is interesting to note that the percentage of {lambda}1 B cells is slightly higher in H3+/– than in H3+/+ mice (95.3 ± 0.3 and 85.6 ± 2.8% respectively, with P < 0.001). These results suggest that, as the number of B cells increases in H3+/+ mice, a part of this increment could be due to an accumulation of {lambda}2 + {lambda}3 cells in H3+/+ mice. Since we have shown that transgenic µ chains are expressed at higher density by B cells of H3+/+ mice, it was important to know whether this phenotype was common for both the selected H3/{lambda}1 and the non-selected H3/{lambda}2 + {lambda}3 combinations. The comparison of fluorescence intensity clearly shows that the homozygous status of the H3 transgene allows an higher Ig surface expression regardless of the nature of the light chain (Fig. 3Go, lower). Altogether, these results suggest that the small increase in the percentage of {lambda}2 + {lambda}3 cells could be due to a higher BCR density which leads to an increased efficiency of the survival signals to non-selected B cells.



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Fig. 4. Higher expression of the µ-transgenic chain regardless of the associated {lambda} chain in H3+/+ mice. Spleen cells were analyzed by two-color immunofluorescence. The lower histograms correspond to cells stained double positive in the gates indicated in the upper profiles. The numbers indicate the percentages of analyzed cells in the gates indicated.

 
Expression of CD23 and CD24 markers can discriminate between transitional or mature splenic B cell subpopulations (2931). We analyzed the presence of these markers among positively selected {lambda}1+ or non-positively selected {lambda}2 + {lambda}3+ B cells. The results shown in Fig. 5Go and Table 2Go demonstrate that cells expressing {lambda}1 chains display a more mature phenotype than cells expressing {lambda}2 or {lambda}3 chains, in both H3+/+ and H3+/– mice. Indeed, a higher percentage of H3/{lambda}1 cells expresses the CD23 marker compared to H3/{lambda}2 + {lambda}3 cells (~15% more). This finding support that the clonal positive selection of H3/{lambda}1-expressing B cells is an active phenomenon which leads to an increase in the fraction of cells with a follicular CD23+ mature phenotype. More unexpectedly, heterozygous transgenic mice display a percentage of mature B cells slightly higher than homozygous mice regardless of the {lambda} light chain (51 versus 45% for {lambda}1 cells and 36 versus 30% for {lambda}2 + {lambda}3, as shown in Fig. 5Go). This difference is also observed when total B cells (B220+) are analyzed (65 versus 52% in Table 2Go). These latter results suggest a dual effect of the BCR. In cells with a higher Ig surface density, survival signals are more efficiently transduced and lead to an increase of the percentage of {lambda}2 + {lambda}3 cells in the absence of positive selection of follicular mature cells. In cells with a lower BCR density, the level of survival signaling is not sufficient to rescue these cells, which results in the relative increase in selected {lambda}1 follicular mature cells. Thus, our data clearly indicate that the clonal positive selection favoring the H3/{lambda}1 combination and the high expression of surface Ig act by two distinct processes on the formation of the B cell repertoire.



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Fig. 5. H3/{lambda}1 cells displayed a more mature B cell phenotype than H3/{lambda}2 + {lambda}3 cells. Spleen cells were analyzed by three-color labeling. For the analysis with anti-CD23/anti-CD24 staining (second and fourth horizontal profiles), positive {lambda}1 and {lambda}2 + {lambda}3 cells were gated as indicated on the first and third horizontal profiles respectively. For the {lambda} profiles, numbers are the percentages of {lambda}+ cells in the light scatter gate corresponding to lymphocytes. For the CD23/CD24 profiles, the numbers are the percentages of CD23+ or CD23 cells in the {lambda}+ gates defined in {lambda} profiles. Non-Tg mice are related {kappa}–/– mice.

 

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Table 2. Influence of surface Ig expression on the follicular splenic B cell subsets
 
High surface Ig density on the B cell favors the emergence of cells with the marginal zone (MZ) phenotype for both selected and non-selected B cells
Splenic CD23 B cells comprise transitional and MZ B cells which can be distinguished through the CD21 marker (3236). We analyzed CD21 expression among the positively selected H3/{lambda}1-expressing B cells. The results shown in Fig. 6Go compare the expression of the CD21 marker in two major splenic B cell subsets, i.e. mature follicular B cells (CD23+) and Ig{lambda}1hiCD23 B cells. All CD23+ cells display a similar pattern of CD21 expression although this marker is slightly more expressed by B cells of H3+/+ mice. In contrast, the CD23 B cells are represented by quite different subpopulations. In H3 homozygous mice, many CD23 cells also express high levels of CD21, defining MZ B cells, whereas in H3-heterozygous mice, the majority of these cells are CD21. These results show that the higher Ig surface density modifies the ratio of follicular/marginal B cells in favor of MZ B cells. Moreover, the difference between the CD23 cells in H3+/+ and H3+/– mice was also observed among H3/{lambda}2 + {lambda}3 cells (not shown). This latter result suggests that the relative increase in the size of the MZ B cell compartment is dependent on surface Ig density and is independent of the clonal positive selection, as the expression of H3/{lambda}1 combination is always largely dominant in this compartment.



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Fig. 6. Surface Ig{lambda}1hi CD23 cell subsets define different B cell populations in H3+/+ and H3+/– mice. Spleen cells were analyzed by three-color labeling. Only {lambda}1+ cells were analyzed. Right profiles show the CD21 fluorescence intensities for the cells present in the gates defined in the left profiles.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The BCR plays a central role in the recruitment of immature B cells into the mature B cell pool. Its presence on the cell surface mediates signaling via key proteins such as CD79{alpha} and Syk (37,38). Indeed, a dysfunction of these molecules blocks the transition from immature to mature B cells. However, the nature of this BCR signaling remains to be elucidated and at least two crucial questions must be answered (3,4,39). The first question raises the problem of the intensity of such signaling since a balance must be found between positively and negatively selecting signals. The second question is whether maturation signals are generated through ligand-independent or ligand-dependent mechanisms? Some experimental models strongly suggest that signaling can be generated in a cell autonomous fashion independently of exogenous ligands (40,41). These models show that a B cell differentiation can be obtained without BCR, suggesting that ligand-dependent signaling is dispensable. On the other hand, many analyses reported modifications of the B cell repertoire between immature and mature B cell pools (1114,42). These findings also suggest that a ligand-dependent signaling must be responsible for a positive clonal selection. However, the nature of ligands responsible for such signaling remains unknown. It could be self- and/or non-self-antigens even if in certain models self-antigens are privileged (15,17,43). From all these observations, it can be proposed that a further maturation of immature B cells is warranted by a minimal signaling mediated by the BCR. This minimal signaling could be a sum of constitutive and ligand-dependent signals allowing both to check the quality of BCR to transduce subsequent activation signals and to choose immature B cell clones conferring an immunological benefit to the mature B cell pool. The results reported in the present paper support this proposition in suggesting that constitutive and ligand-dependent parts of the maturation signaling can be distinguished.

Using an oligoclonal mouse model, we have shown previously that the peripheral B cell repertoire of H3-µ-transgenic {kappa}-knockout mice is composed of >80% of B cells expressing the H3/{lambda}1 combination (18). By contrast, only ~60% of immature B cells display this combination, as analyzed either ex vivo in the bone marrow or after in vitro differentiation of bone marrow cell precursors (18,19). Both negative and positive selection processes could formally give such results. However, the hypothesis of a clonal positive selection is strengthened in the present paper. First, B cells expressing the H3/{lambda}1 combination also express, in greater number, CD24lo and CD23+ maturation markers (Fig. 5Go), characteristic of circulating and follicular immunocompetent B cells. Second, H3/{lambda}1 dominance is found in both H3+/+ and H3+/– mice. Thus, the decrease of H3/{lambda}2 + {lambda}3 B cells cannot be explained by a negative selection due to a self-antigen since a higher expression of an autoreactive BCR should favor the deletion process. On the contrary, our results show that the fraction of H3/{lambda}2 + {lambda}3 cells is more important when the BCR density increases, suggesting that this higher percentage is due to better ligand-independent signaling. Therefore, we conclude that a positive clonal selection process is responsible for the H3/{lambda}1 dominance. Very recently, several reports seem to give credit to the idea that the positive selection is restricted to B-1 subsets (17,4347). Our observations extend the positive selection process to the B-2 subset and are consistent with those of Kearney and collaborators (34,48). However, in our transgenic model, we associate the positive selection process to CD23+ cells even if the selected H3/{lambda}1 cells are also predominant in the MZ.

Although positive selection for the H3/{lambda}1 B cells is observed in both H3+/+ and H3+/– mice, some modifications in the B cell repertoire occur induced by the modulation of the BCR surface density. First, the higher expression of BCR leads to an increase in the number of splenic B cells (Table 1Go). Second, this increase is correlated with a higher relative proportion of CD23 cells expressing {lambda}1 or {lambda}2 + {lambda}3 chains even if {lambda}1 cells are always largely dominant (Fig. 5Go). Third, in H3+/– mice, the CD23 population mainly consists of IgMhiCD24hiCD21 cells, while in H3+/+ mice, IgMhiCD24hiCD21++ cells are predominant (Fig. 6Go). These phenotypes have been recently referred to as immature or transitional (T1) cells and MZ cells respectively (36).

The fact that a high BCR surface density favors B cell colonization in the periphery with no alteration of the positive selection process of the H3/{lambda}1 combination has important implications for the inducing signals mediated by the BCR. These signals can be separated in two types depending on their effects on the B cell repertoire. The first type should correspond to auto-signaling, i.e. constitutive signals. In this case, each B cell receives this signal independently of the BCR specificity. The second type should correspond to preferential signaling for certain B cells depending on the BCR specificity and therefore would be ligand-dependent signaling. Obviously, a B cell cannot discriminate between these two types, and receives a signaling that arises from the sum of constitutive and ligand-dependent signals. Thus, in the present model, the selective process prevails on the constitutive signaling. This selection seems to take place early in B cell development since the overexpression of the H3/{lambda}1 combination begins in the CD23 B cell compartment which is rich in transitional cells in H3+/– mice. Unfortunately, the proportion of 493+ cells among this population cannot be clearly determined in our hands to ascertain whether the selection is central or peripheral (49). However, this selection is dispensable for a given B cell if the BCR auto-signaling is sufficient and other `sister' B cells have no selective advantages (19,50,51). The notion that a high BCR surface density can furnish sufficient auto-signaling for the survival of mature B cells is also reminiscent of the observations made in CD45–/– or CD19–/– mice which present deficient B cell development (reviewed in 39). These animals display peripheral B cells with an unusually high level of surface IgM which can compensate for other defective constitutive signals.

In conclusion, our data suggest that the impact of constitutive and ligand-dependent signalings on the composition of the splenic B cell population can be distinguished. In the present experimental model, we observed a predominant effect of ligand-dependent signaling on the B cell repertoire. This predominance could be due to the fact that the requirement for signaling is modulated during the different steps of B cell development. Thus, checkpoints in the development would be preferentially dependent on ligand-dependent signaling while the survival of the cell in a steady state of development would be preferentially dependent on constitutive signaling. A higher surface density of the BCR could increase constitutive and ligand-dependent signalings. Its effect will be variably observed on the peripheral B cell subpopulations, depending on the moment and the site where these signals are received by the B cell during its development. However, the cumulative intensity must remain below the threshold inducing negative selection. A very recent report suggests such a mechanism, by which autoreactive B cells escape negative selection by dilution of surface autoreactive BCR density (52).


    Acknowledgments
 
We are indebted to Antonio Freitas for his continuous support. We thank Drs Ana Cumano, Antonio Freitas, Fabio Grassi and Dominique Rueff-Juy for helpful discussions. We gratefully acknowledge Sébastien Paturance and his staff for excellent animal care. This work is supported by the Université Denis-Diderot, the Association pour la Recherche sur le Cancer and the Fondation pour la Recherche Médicale.


    Abbreviations
 
BCR B cell receptor
MZ marginal zone

    Notes
 
Transmitting editor: J. F. Kearney

Received 21 April 2000, accepted 6 July 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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