Affiliation to mature B cell repertoire and positive selection can be separated in two distinct processes

Soulef Hachemi-Rachedi1, Anne-Marie Drapier1, Pierre-André Cazenave1 and Pierre Sanchez1,2

1 Immunochimie Analytique, Institut Pasteur and
2 Immunobiologie, Université Denis Diderot, 75251 Paris, France

Correspondence to: P. Sanchez, Laboratoire d'Immunobiologie Case 7048, Université Denis Diderot (PARIS VII), 2 Place Jussieu, 75251 Paris Cedex 05, France


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Using an `oligoclonal' model, we have previously shown that mice transgenic for a µ chain (H3) and deficient for {kappa} chain expression display a mature B cell repertoire largely dominated by the H3/{lambda}1 pair, while the four H3/{lambda} available combinations can be observed in the immature B cell compartment. This led us to propose the existence of a positive selection process. To test this hypothesis, we have introduced the SJL {lambda} locus coding for a defective {lambda}1 chain ({lambda}1s) that creates a dysfunctional Ig receptor complex during B cell differentiation. Our results show that the {lambda}1s defect impairs the development of mature B cells when the H3-µ transgene insert is present in the hemizygous state. This suggests that the Gly -> Val substitution present in the C{lambda}1s chain at position 155 is sufficient to abrogate the selection of the H3/{lambda}1 pair. Unexpectedly, when the H3-µ transgene array is present in a homozygous state in {lambda}1s mice but not in `wild-type' {lambda}1 mice ({lambda}1+), a significant number of mature B cells expressing all H3/{lambda} combinations can be developed. These results indicate that the overriding H3/{lambda}1 dominance observed in {lambda}1+ mice is due to a positive selection process and not to a negative selection of other H3/{lambda} combinations. They also show that the export of B cells to the periphery can be controlled by the expression of the µ chain.

Keywords: B lymphocytes, cellular differentiation, repertoire development, transgenic/knockout


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
During B cell development, multiple Ig V(D)J gene segments are rearranged, and H and L chains are assembled consecutively to be expressed on the surface of B cells. The emergent B cell repertoire therefore displays a large diversity. However, certain H/L chain combinations can be disadvantaged due to incompatible pairing with each other or with different molecules responsible for folding, transport to the cell surface and signaling of the B cell receptor (BCR). For example, the expression of some VH genes is prevented by counter-selection due to the inability of encoded µ chains to form a pre-BCR with the surrogate light chain (13). Moreover, some B cells with nascent autoreactive BCR can be eliminated by negative selection or change their specificity by receptor editing (49). In addition, positive selection has been postulated to favor the entrance of certain newly formed B cells in the long-lived B cell pool (1013). Competition between cells can also act synergistically with the positive selection processes (1416). However, the ligands responsible for such positive selection are unknown and the real existence of these ligands is questioned by self-signaling BCR aggregation models (1720).

Studies on the formation of the B cell repertoire are limited by the huge diversity of generated BCR. To overcome this inconvenience, transgenic mice displaying an `oligoclonal' B cell repertoire have been developed (2123). Using such an experimental model, we have previously described a strong modification of the repertoire between emergent immature and peripheral mature B cell pools (22). More precisely, in the spleen of H3-µ transgenic mice (H3-Tg) crossed with {kappa} knockout mice ({kappa}–/–), B cells always express an H3-µ chain paired with a {lambda}1 chain, whereas newly formed immature B cells can expressed the transgenic heavy chain with one or another of the four available {lambda} subtypes [{lambda}1, {lambda}2(V2), {lambda}2(Vx) and {lambda}3] (24). We have postulated that a positive selection process is responsible for the H3/{lambda}1 dominance. However, we cannot exclude that the H3/{lambda}1 dominance is due to the negative selection of other combinations (i.e. H3/{lambda}2 or {lambda}3). To test this hypothesis, we have introduced the SJL {lambda} locus into the H3-Tg, {kappa} knockout mouse genome. This locus encodes a {lambda}1 chain ({lambda}1s) that causes a dysfunction of the {lambda}1-containing Ig receptor during B cell differentiation (2527). Our results show that (i) the nature of the light chain [i.e. {lambda}1s versus wild-type {lambda}1 ({lambda}1+)] determines whether or not the positive selection of H3/{lambda}1 B cells occurs in the periphery, (ii) the level of µ transgene expression controls the capacity of B cell replenishment in the periphery, (iii) the peripheral B cell repertoire comprises an abundant H3/{lambda}2 + {lambda}3 B cell population only in the absence of positive selection of H3/{lambda}1 B cells. Our results therefore demonstrate a versatile B cell positive selection independent of the B cell differentiation program. We propose a model in which the formation of the mature B cell repertoire is determined by positive selection, which is dispensable in `oligoclonal' mice when the B cell differentiation is effective and no H/L combination is available to be positively selected.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals and screening
Mice (8–20 weeks old) were housed in the animal facilities at the Institut Pasteur, Paris. Mice homozygous for the C{kappa} mutation were produced by mating of chimeras derived from 129/Sv ES cells with C57BL/6 ({lambda}1+/+ genotype) (24). Subsequently, the {lambda}1s locus was introduced by mating with the SJL strain ({lambda}1s/s genotype). The screening of {lambda}1+ versus {lambda}1s mice was done by Southern blot analysis using the presence or the absence of a KpnI restriction site in the C{lambda}1 segment (26,27). H3-Tg C57BL/6 mice were obtained by Honjo's group (28). The association of the H3-Tg chain with the L3 transgenic chain (a {kappa} light chain) displays an activity against mouse erythrocytes. However, mice expressing the H3-Tg µ chain only do not develop anemia (28). The H3 transgene was first introduced by crossing H3-Tg C57BL/6 mice with {lambda}1+/+{kappa}–/– mice and second by crossing H3-Tg {lambda}1+/+{kappa}–/– mice with {lambda}1s/s{kappa}–/– mice. The presence of the H3 transgene can be detected by FACS analysis to discriminate between the transgenic µa and endogenous µb chains or by Southern blot analysis using an XbaI–HindIII Cµ probe (29).

Antibodies and flow cytometry analyses
Cells were stained with the indicated mAb labeled with FITC (Sigma, St Louis, MO) or biotin (Boehringer Mannheim, Mannheim, Germany). Streptavidin coupled to phycoerythrin was obtained from Southern Biotechnology Associates (Birmingham, AL). Anti-CD24 was a gift from Dr A. Freitas. Anti-CD23 (B3B4) and anti-CD21 (7G6) were purchased from PharMingen (San Diego, CA). µ, {lambda}1 and {lambda}2 + {lambda}3 cells were defined by labeling with biotinylated goat anti-mouse IgM (Sigma), AF6-78, DS-1 (PharMingen), MS40-13 (30), 189B3 (31) antibodies and with FITC-labeled anti-CD45R/B220 (RA3-6B2) mAb (32). Analyses were done on a FACScan (Becton Dickinson) or on an Epics Elite-ESP flow cytometer (Coultronics, Margency, France).

In vitro B cell differentiation
Confluent S17 stromal cells in 24-well plates were {gamma}-irradiated at 2000 rad (33,34). Bone marrow cells (5x105) were then cultured in 1.5 ml in regular medium in the presence of 50–150 U rIL-7 in each well. After 4 days, cells were transferred to a new confluent S17 layer and grown in the absence of rIL-7 for 3 days. Cells were then cultured again under the same conditions for 3–4 days and subsequently analyzed by FACS.

Spleen B cells (3x106) were cultured in 1.5 ml in regular medium in the presence of lipopolysaccharide (LPS) (25 µg/ml) (L-2880; Sigma) over 9 days. Subsequently, supernatants were tested by ELISA using a sandwich test in which goat anti-mouse IgM were coated and endogenous or transgenic secreted IgM are revealed by specific biotinylated anti-allotype DS-1 or AF6-78. Finally, streptavidin coupled to horseradish peroxidase (Southern Biotechnology Associates) was used for the last step.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The {lambda}1s defect leads to a decrease in B cell development in {kappa}–/– mice
Among the four {lambda} chains encoded by the {lambda} locus [i.e. {lambda}1, {lambda}2(V2), {lambda}2(Vx) and {lambda}3], {lambda}1 is over-expressed in normal and {kappa} knockout mice (35). This dominant expression (~50–65%) is detectable in central and peripheral lymphoid tissues, except in the peritoneal cavity. We have shown that the preferential usage of {lambda}1 chains results from a higher frequency of V1–J1 rearrangements (36). In contrast, in {kappa}–/– mice with the SJL {lambda} locus, {lambda}1-expressing cells represent only 5–15% of cells in the periphery(27) (Fig. 1Go). The decrease in the {lambda}1 subpopulation can be first observed among immature B cells (B220loIgM+), in the bone marrow. The {lambda}1s phenotype probably results solely from a Gly -> Val substitution in the {lambda}1 constant region. Indeed, no other difference in the coding or regulatory sequences is found between SJL {lambda}1 ({lambda}1s) and the common {lambda}1 ({lambda}1+) locus and the higher frequency of V1J1 rearrangements is unchanged in {lambda}1s mice (27) (unpublished data). It has been proposed that the Gly -> Val substitution creates an Ig receptor complex which is dysfunctional during B cell differentiation (27). This could be due to an impairment in BCR signaling, although the precise mechanism responsible for this defect remains elusive. As can be seen on Fig. 1Go, the percentages of {lambda}1 cells, in {lambda}1s/s {kappa} knockout mice, decrease as B cells progress from the bone marrow to the spleen, suggesting a selection against {lambda}1 cells in the periphery. This result therefore reinforces the idea of an inefficient survival signaling via the {lambda}1s BCR. Moreover in in vitro culture conditions which allow the development of immature B cells only (22,37), the percentage of {lambda}1 cells (near to 30%) is highest, although it remains 2-fold lower than in the {lambda}1+/+ mice (Fig. 1Go).



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Fig. 1. Continuous {lambda}1s defect during B cell development. The percentage of {lambda}1 subtype among newly generated B cells in vitro (B220low), B cells in the bone marrow ex vivo (B220low and B220high) and spleen B cells (B220+) was determined by FACS analysis. Bars represent SD and data were obtained from five to 11 mice/group.

 
As 50–60% of immature B cells are of the {lambda}1 subtype in {lambda}1+/+ mice, the fact that there are 30% {lambda}1 immature cells in {lambda}1s/s mice should lead to a decrease in the number of newly formed B cells in the bone marrow in the absence of compensatory phenomena. To examine the efficiency of B cell development, we compared the ratio of IgM+B220lo/ total B220lo cells in ex vivo and in vitro different B cell differentiation conditions. As shown in Fig. 2Go, B cell development is impaired in {lambda}1s/s mice. Indeed, in ex vivo and in vitro conditions, the ratio passes from 9.8 and 16.8% in {lambda}1+/+ mice to 4.4 and 8.5% in the {lambda}1s/s mice respectively. However, the percentage of spleen B cells is similar in both mice, indicating a peripheral compensation for the defect in B cell lymphopoiesis by amplification of the {lambda}2 + {lambda}3 B cell compartment (see discussion).



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Fig. 2. Inhibition of immature B cell production in H3-Tg {kappa}–/–{lambda}1s/s mice. FACS analysis of B cell lineage ex vivo (spleen and bone marrow) and after in vitro differentiation of bone marrow cells. Numbers indicate the percentage of the cells in the light scatter gate corresponding to lymphocytes.

 
The {lambda}1s defect predominates over processes responsible for the H3/{lambda}1 selection
Using H3-µ transgenic mice obtained by Honjo's group (28), we have previously shown that H3-µ transgenic mice deficient for {kappa} chains (H3-Tg {kappa}–/– mice) express a quasi-monoclonal repertoire since ~80–90% of mature B cells express H3-µ in association with the {lambda}1 chain (22). In the immature B cell pool, however, all H3/{lambda} combinations are normally expressed. This led us to suggest that the H3/{lambda}1 combination is positively selected over other H3/{lambda} combinations by unknown ligand(s) during the immature to mature B cell transition. Introduction of the {lambda}s locus in H3-Tg {kappa}–/– mice allowed us to test the influence of {lambda}1s defect on this selective process. We therefore analyzed spleen B cells and estimated the proportion of {lambda}1 cells among the µ+ B cells. As expected, {kappa}–/–{lambda}1s/s and H3-Tg {kappa}–/–{lambda}1+/+ mice display very low (<15%) and very high (>75%) percentages of the {lambda}1 cells respectively (Fig. 3Go), compared to ~55% of {kappa}–/–{lambda}1+/+ mice (see Fig. 1Go). In contrast, H3-Tg {kappa}–/–{lambda}1s/s mice show a drastic decrease not only in {lambda}1 but also in total B cells (Fig. 3Go). These results indicate that (i) the {lambda}1s defect prevents the H3/{lambda}1 combination selection, (ii) there is no compensation with H3/{lambda}2 or H3/{lambda}3 combinations and (iii) the {lambda}1s defect leads to a quasi-absence of B cells in H3-Tg {kappa}–/–{lambda}1s/s mice. We conclude that the {lambda}1s defect is dominant over the selection of the H3/{lambda}1 combination, most probably because the {lambda}1s Ig receptor displays a deficient signaling function.



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Fig. 3. Deficiency of B cell development in H3-Tg {kappa}–/–{lambda}1s/s mice. The numbers indicate the percentage of cells in the light scatter gate corresponding to spleen lymphocytes.

 
The H3/{lambda}1 combination dominance is most probably due to positive rather than negative selection
As H3-Tg {kappa}–/–{lambda}1s/s mice have few spleen B cells, it is necessary to verify whether B lymphopoiesis is efficient and whether newly formed B cells can express all {lambda} chains. Spleen and bone marrow cells were therefore analyzed with anti-{lambda}1 and anti-{lambda}2 + {lambda}3 antibodies in conjunction with anti-B220 antibodies (Fig. 4Go, left in top). As already noted, these mice have very few spleen B cells (<5%). These cells express the different {lambda} subtypes and many of them express endogenous µ chains as observed using anti-µ allotype antibodies (see Fig. 6Go). In contrast, immature B cells are present in the bone marrow and all express the µ transgenic gene (not shown). Moreover, all {lambda} subtypes are expressed in the immature B cell compartment and no mature B cells in the bone marrow are detected. This suggests a decreased recruitment of B cells in the periphery rather than a blockade in early B cell maturation steps.



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Fig. 4. Absence of positive selection of the H3-µ/{lambda}1 combination in the H3-Tg {kappa}–/–{lambda}1s/s mice. The two upper and two lower profiles correspond to analyses of spleen and bone marrow respectively in each mouse.

 


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Fig. 6. Transgenic H3-µ chain expression in H3-Tg homozygous and hemizygous {lambda}1s mice. Splenic B cells are analyzed with anti-µa or anti-µb antibodies to control the transgenic or endogenous µ chain expression respectively. The lower profile corresponds to cells double-labeled with anti-µa and anti-B220 antibodies as indicated on the upper profiles. MFI = 5.45 for H3-Tg+/+{kappa}–/–{lambda}1s/s B cells and MFI = 4.21 for H3-Tg+/–{kappa}–/–{lambda}1s/s B cells.

 
Unexpectedly, during the breeding of H3-Tg {kappa}–/–{lambda}1s/s mice, we observed two phenotypes with respect to the number of spleen B cells. The first phenotype corresponds to that described above, i.e. very few spleen B cells, and the second one displays a significant number of spleen B cells (Fig. 4Go, top right). These two phenotypes are directly correlated with the number of H3-µ transgenic array copies as quantified by Southern blot analysis (Fig. 5Go). The results show that mice can receive one or two copies of inserts as suggested by calculated transgene/endogene ratios. Thus, the {lambda}1s/s mice with transgenic inserts to a hemizygous state (H3-Tg+/–) display very few B cells, whereas the mice with transgenic inserts to a homozygous state (H3-Tg+/+) have a significant percentage of splenic B cells (Fig. 4Go, top). Nearly all B cells of H3-Tg+/+{kappa}–/–{lambda}1s/s mice express the transgenic heavy chain contrary to H3-Tg+/–{kappa}–/–{lambda}1s/s mice (Fig. 6Go). Interestingly, the H3-Tg+/+ state (MFI = 5.45) favors a significantly higher expression of the transgenic chain on the cell surface than in H3-Tg+/– mice (MFI = 4.21) (Fig. 6Go). Unexpectedly, a large number of H3-Tg+/+{kappa}–/–{lambda}1s/s spleen B cells expresses the {lambda}1 chain (~40%). Finally, the percentage of {lambda}1 in the spleen reflects the percentage of immature B cells (IgM+B220lo) in the bone marrow, indicating that there is no positive selection of the H3/{lambda}1 combination in the periphery. It is worthy to note that ~60% of B cells express H3-µ in association with {lambda}2 or {lambda}3 chains, demonstrating that their quasi-absence in H3-Tg mice with the {lambda}1+ locus does not arise from a negative selection (see Fig. 4Go, bottom). These results show that the number of copies of the µ transgenic array plays an important role in B cell development in the {lambda}1s mice but does not restore the positive selection process of the H3/{lambda}1 combination. To control the mature status of peripheral B cells, we compared the expression of maturation markers in transgenic and non-transgenic {lambda}1s mice. As shown in Fig. 7Go(A), non-transgenic and H3-Tg+/+{lambda}1s mice display similar patterns of CD24, CD23 and CD21 expression, whereas H3-Tg+/–{lambda}1s mice show a more immature profile for CD23 and CD21. These results are in accordance with LPS proliferation assay (Fig. 7BGo), which shows that transgenic IgM are secreted after LPS stimulation in H3-Tg+/+{lambda}1s mice. Moreover, both {lambda}1 and {lambda}2 + 3 cells can be stimulated in these mice (data not shown).



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Fig. 5. Quantification of H3-µ transgene inserts. Tail DNA was digested by BamHI and membranes were hybridized with a Cµ probe. The ratios between c and d bands were calculated after scanning of membranes with a PhosphorImager (Molecular Dynamics, Bondoufle, France), and the hemi- versus homozygous state of H3-Tg inserts deduced from them are indicated.

 



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Fig. 7. Splenic B cell competence in H3-Tg+/+{kappa}–/–{lambda}1s/s mice. (A) Splenic B cells are double-labeled with anti-B220 and anti-CD24, anti-CD23 or anti-CD21 antibodies. The profiles only show cells positively stained with anti-B220. The numbers represent the percentages of B cells in the indicated windows. (B) Splenic cells (1.5x106/ml) were cultured in the presence or not of LPS (25 µg/ml). After 9 days, supernatants were harvested. ELISA data were obtained from two independent LPS stimulations with a 1/400 dilution of supernatant and correspond to transgenic (IgMa) or endogenous (IgMb) Ig secreted.

 
As double copies of the H3 transgene array lead to an increase in H3/{lambda}2 and H3/{lambda}3 positive cells in {lambda}1s mice, it was important to study the same H3 genotype in {lambda}1+ mice. As illustrated in Fig. 4Go (bottom), no significant difference can be detected between mice with one or two H3 transgene copies since ~60% of H3/{lambda}1 immature cells in the bone marrow and 85–90% of H3/{lambda}1 mature B cells in the spleen were found in both mice. Taken together these results demonstrate that (i) the conditions of H3 transgene expression play a role in the export of newly formed B cells to the periphery, (ii) the H3/{lambda}2 and/or H3/{lambda}3 combinations in H3-Tg {lambda}1+ mice are not eliminated by negative selection due to autoreactive specificities, and (iii) the {lambda}1s defect inhibits the positive selection process of the H3/{lambda}1 combination.

The µ transgenic chain increases B cell lymphopoiesis but does not restore completely the peripheral B cell pool in {lambda}1s mice
As shown in Fig. 4Go (top), B cell development seems normal in H3-Tg+/+{lambda}1s mice, whereas it is compromised in H3-Tg+/–{lambda}1s ones. On the other hand, we also showed that the {lambda}1s defect leads to a decrease of B cell lymphopoiesis in non-transgenic {lambda}1s mice (Fig. 2Go). We therefore assessed the influence of µ transgenic chain expression both on the generation of newly formed B cells in the bone marrow and on the capacity of these cells to migrate to the periphery. The data are summarized in Table 1Go in which we compare µ transgenic {lambda}1s or {lambda}1+ mice with non-transgenic {lambda}1s mice. As shown, both Tg+/– and Tg+/+ {lambda}1s mice have a significantly lower percentage of spleen B cells than non-transgenic {lambda}1s mice (7.7 and 20 versus 36% respectively). The behavior of Tg+/– and Tg+/+ {lambda}1+ mice is similar to the non-transgenic {lambda}1s mice (33 and 43 versus 36%) although Tg+/–{lambda}1+ mice recurrently display a lower B cell percentage than Tg+/+{lambda}1+ mice. The results are identical if we analyze the total number of B cells per spleen (data not shown). Therefore, this result shows that the homozygous state of the transgene array favors the filling of the peripheral B cell compartment in H3-Tg {lambda}1s mice. However, this filling remains lower than in the non-transgenic {lambda}1s animals.


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Table 1. Balance between B cell lymphopoiesis and the presence of splenic B cells in various {kappa} knockout mice
 
To analyze the influence of the µ transgenes on B lymphopoiesis, we estimated the ratio of immature B cells (IgM+B220lo) to total B220lo cells. As shown in Table 1Go, both Tg–/– and Tg+/– {lambda}1s mice display a similar B cell lymphopoiesis. Their ratios are significantly lower than in Tg+/+{lambda}1s mice, indicating that the homozygous state of the transgene enhances the production of newly formed B cells, which is similar to that found in H3-Tg {lambda}1+ mice (Table 1Go) and non-transgenic {lambda}1+ mice (not shown). Altogether, these data clearly show that µ transgene expression is able to modulate B cell development in the {lambda}1s mice in two ways. The first should determine the rate of immature B cells produced in the bone marrow. The second should determine the ability of new B cells to colonize the periphery. However, even if B cell lymphopoiesis is restored, it is not sufficient to have a complete mature B cell repertoire in H3-Tg+/+{lambda}1s mice.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The molecular and cellular mechanisms controlling the transition from newly formed B cells in the bone marrow to the mature B cell pool in the periphery are still largely unknown (38,39). Using H3-µ transgenic {kappa}–/– mice with the {lambda}1s defect, we bring some insights into the generation of immature B cells and the requirements for the establishment of a mature B cell repertoire. Two developmental steps can be distinguished in our model. The first is characterized by an absence of clonal selection and depends on mechanistic constraints. The second is based on selective processes that ensure the persistence of B cells in the periphery. We will discuss these two aspects sequentially.

The H3-Tg µ chain favors B cell lymphopoiesis, restores the {lambda}1/{lambda}2 + {lambda}3 ratio and controls the export of newly formed B cells in {lambda}1s mice
Non-µ transgenic {lambda}1s{kappa}–/– mice display a lower rate of immature B cell production than {lambda}1+{kappa}–/– mice (Fig. 2Go and Table 1Go). This observation can be interpreted as a consequence of the decrease in the {lambda}1 B cell number that can be produced in bone marrow (27) (Fig. 1Go). Indeed, we have previously reported that the dominant {lambda}1 cell expression in {lambda}1+{kappa}–/– mice (~60%) results from a higher probability to rearrange {lambda}1 over other {lambda} subloci (36). Thus, numerous cells could have productively rearranged {lambda}1s but because of the defective {lambda}1s chain, these cells die in situ before they express their surface Ig, and they consequently prevent, by competition, a compensatory development of bone marrow {lambda}2 + {lambda}3 cells. Moreover, these cells cannot be rescued by secondary rearrangements in the {lambda} locus since V{lambda}1s–J{lambda}1s rearrangements in spleen {lambda}2 + {lambda}3 cells are all in a non-productive configuration, as already shown for the {lambda}1+ mice (data not shown). The defect of {lambda}1s chain is likely due to a Gly -> Val substitution at position 155 since no other difference has been described between the {lambda}1+ and {lambda}1s loci (27,40). This substitution could act on the function of the Ig receptor, although we cannot exclude a ineffective association of the {lambda}1s chain with chaperones before its pairing with the µ chain (41), even if the defect can be modulated by the H3-Tg chain. However, in {lambda}1s mice, the µ transgenic chain effect on bone marrow B cell development is complex (see Fig. 4Go): (i) in the bone marrow, the presence of the H3 chain seems to restore the {lambda}1/{lambda}2 + {lambda}3 ratio to ~47%, regardless of the H3-Tg genotype, (ii) when the H3-Tg inserts are in double copies, B cell lymphopoiesis in {lambda}1s mice increases, regardless of the {lambda} subtype expressed, and (iii) the H3-Tg genotype in {lambda}1+ mice has no effect on either the {lambda}1/{lambda}2 + {lambda}3 ratio or B cell lymphopoiesis.

We propose the following hypotheses to explain these multiple effects of the H3-µ transgene. The expression of the µ transgenic chain is different from that of µ endogenous chain and favors the folding, the transport to the cell surface and/or the signaling of Ig{lambda}1s receptors. This enables immature B cells to have a {lambda}1/{lambda}2 + {lambda}3 ratio near 50%. However, when the transgene inserts are present in one copy only, the condition of transgenic µ expression is not sufficient to obtain a normal B cell lymphopoiesis. This would probably be due to the subsequent death of {lambda}1s cells because of a deficiency in Ig{lambda}1s signaling, even if these cells pursued their development until they express the Ig{lambda}1s on their surface. A defect in B cell lymphopoiesis would therefore be one of the causes of the quasi-absence of peripheral B cells in H3-Tg+/–{lambda}1s mice. In contrast, when the transgene inserts are present in double copies, the condition of transgenic µ expression overrides the {lambda}1s chain defect in B cell lymphopoiesis. This leads to two phenomena: (i) normal B cell lymphopoiesis for both {lambda}1 and {lambda}2 + {lambda}3 cells, and (ii) an increased ability of these cells to be exported to the periphery. The fact that the suppression of the {lambda}1s defect indirectly causes the production of {lambda}2 + {lambda}3 cells can be explained by the hypothesis that, now, the newly formed {lambda}1s cells, instead of dying in situ, leave the bone marrow allowing `free space' in which {lambda}2 + {lambda}3 cell development is possible.

More unexpected is the peripheral colonization by the newly formed cells when the transgenes are in double copies. To our knowledge, such a fine regulation is only reported by two very recent publications (42,43). Indeed, a slight but significant differential surface expression of transgenic µa chain can be observed between H3-Tg+/+ and H3-Tg+/– {lambda}1s mice (see Fig. 6Go). Moreover, we also found a more pronounced differential expression of intracellular µ chain and surface Ig density between H3-Tg+/+ and H3-Tg+/– {lambda}1+ mice (not shown). Watanabe et al. reported a similar result in H3-Tg {kappa}+/+ mice (42). A possible hypothesis to explain the colonization would be better survival signaling via Ig receptors in H3-Tg+/+{lambda}1s mice. An alternative but not opposite hypothesis could be that, as {lambda}1s mice display a diminished B cell lymphopoiesis, the average time of recombination processes increases, favoring a deletion of an H3 transgene array on the basis of recently reported data (44). In this case, cells with two copies of H3 transgenes should have an advantage to express an H3-Tg Ig receptor.

It is remarkable to note that colonization occurs for both {lambda}1s and {lambda}2 + {lambda}3 cells. Such a situation is not found in the H3-Tg {lambda}1+ mice in which {lambda}1 cells largely dominate {lambda}2 + {lambda}3 cells. As we propose below, the difference between {lambda}1s and {lambda}1+ mice must arise from a lack of positive selection in the {lambda}1s mice. However, {lambda}1 and {lambda}2 + {lambda}3 cells in the H3-Tg+/+{lambda}1s mice seem immunocompetent as tested by the presence of maturation markers or by in vitro LPS stimulation (Fig. 7Go), suggesting that positive selection is dispensable for bone marrow immature B cells to be exported to the periphery. Our results are in agreement with two very recent reports (42,43). Both papers favor the idea that BCR surface density positively regulates B-1 cell compartment. Our results extend their observations to B-2 cell compartment since our H3-Tg {lambda}1s mice display a splenic B-2 phenotype as tested by the absence of CD5 marker (data not shown).

The {lambda}1s defect reveals that the dominance of the H3/{lambda}1 combination results from a positive selection process in H3-Tg {lambda}1+ mice
Taken together, our analyses of the peripheral B cell repertoire can be divided into four phenotypes depending on two criteria: B cell number and the {lambda}1/{lambda}2 + {lambda}3 ratio. For each phenotype, we attempted to separate the parameters conditioning the establishment of a mature B cell repertoire, i.e. formation of immature B cells and recruitment to the periphery, on one hand, and a clonal positive selection, on the other hand, as illustrated in our model (Fig. 8Go).



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Fig. 8. Formation of the mature B cell repertoire in the H3-Tg {lambda}1s mice. This model is based on the idea that the probability of producing a {lambda}1+ cell from the B cell precursor pool is the same in {lambda}1s and {lambda}1+ mice as indicated in a potential repertoire from the pro-B cell pool.

 
The first phenotype can be seen in both H3-Tg+/– and H3-Tg+/+ {lambda}1+ `oligoclonal' mice (Fig. 8DGo). It is characterized by an almost normal number of spleen B cells and {lambda}1 dominance although the number of spleen B cells of Tg+/–{lambda}1+ is often smaller than that of the Tg+/+{lambda}1+ mice. These results confirm our previous data which have been interpreted as positive selection of the H3/{lambda}1 combination (22). Indeed, these two mouse strains have normal B cell lymphopoiesis and ~60% of {lambda}1 cells are present in the immature B cell pool as found in non-transgenic {lambda}1+ mice (Fig. 1Go). These results demonstrate that selection takes place at the transition between newly formed immature and mature B cells and the conditions of µ transgene expression (H3-Tg+/+ versus H3-Tg+/–) have no effect on this selection. Moreover, since H3/{lambda}2 and/or H3/{lambda}3 cells can be found in the periphery of H3-Tg+/+{lambda}1s mice, the quasi-absence of these cells in H3-Tg {lambda}1+ mice does not result from negative selection by clonal deletion. This fact therefore shows that the H3/{lambda}1 dominance is due to a clonal positive selection process.

The second phenotype is represented by H3-Tg+/–{lambda}1s/s mice which have very few B cells in the periphery and a dominance of {lambda}2 + {lambda}3 cells (Fig. 8BGo). Numerous peripheral B cells express an endogenous µ chain suggesting that no H3/{lambda} combination is positively recruited (Fig. 6Go). These results fit very well with our hypothesis of a positive selection of the H3/{lambda}1 combination. Indeed, bone marrow immature cells are not recruited to the periphery, the {lambda}1 B cells because of the {lambda}1s defect and the {lambda}2 + {lambda}3 cells due to the lack of positive selection of H3/{lambda}2 or H3/{lambda}3 combinations as observed in H3-Tg+{lambda}1+ mice.

The third and unexpected phenotype arises from H3-Tg+/+{lambda}1s/s mice which display numerous mature spleen B cells and a {lambda}1/{lambda}2 + {lambda}3 ratio near to 1 (Fig. 8CGo). As we have already mentioned, the presence of the H3 inserts to the homozygous state, enhances B cell lymphopoiesis, probably by compensation of the {lambda}1s defect. This better lymphopoiesis could favor, by itself, the recruitment of immature B cells to the periphery. However, non-transgenic {lambda}1s mice with a defective B lymphopoiesis display a normal mature B cell repertoire suggesting that other elements control the recruitment. The simplest hypothesis is to imagine that the modification of H3 expression has two effects, an increase in immature B cell number and an increase in Ig signaling, both of which permit the export of the cells to the periphery. However, it does not reach the normal size as shown in Table 1Go. Moreover, B cell recruitment does not favor the H3/{lambda}1 combination, since H3/{lambda}2 + H3/{lambda}3 expressing cells represent nearly half the B cells. These results therefore suggest that the H3-Tg+/+ genotype can suppress the {lambda}1s defect, with respect to production of {lambda}1s cells and their peripheral export, but remains ineffective for the suppression of the {lambda}1s defect, with respect to clonal positive selection. It is tempting to propose that there are two processes for peripheral recruitment. The first depends on efficient signaling via the Ig receptor and can be observed without positive selection. In this case, this process should allow the export of not only {lambda}1s cells but also {lambda}2 + {lambda}3 cells. This process could result from survival BCR signaling in a ligand-independent way, but it would not be sufficient to have a complete mature B cell repertoire due to lack of positive selection. The second process leads to clonal positive selection, which is probably ligand dependent. In this case, the mature repertoire tends to reach its normal size as seen in H3-Tg {lambda}1+ mice even if the lymphopoiesis is diminished as proposed below for the {lambda}1s mice.

The fourth phenotype corresponds to non-transgenic {lambda}1s mice which have a normal number of spleen B cells and a large {lambda}2 + {lambda}3 dominance (Fig. 8AGo). These mice display a deficient B cell lymphopoiesis due to their {lambda}1s defect (Table 1Go) but a normal mature repertoire size. This phenotype reflects a powerful compensation process due to selection of {lambda}2 + {lambda}3 clones. We propose, here, that this compensation is possible because numerous newly formed {lambda}2 + {lambda}3 cells can be positively selected in a ligand-dependent way and that this positive selection allows a normal size B cell repertoire in the periphery.

Numerous models of transgenic mice have been used to explore the establishment of the B cell repertoire. The concept of negative selection has largely been supported by the finding of B cell development arrest in `monoclonal' transgenic mice in the presence of self antigen (12). The concept of positive selection has been supported, indirectly, in a certain number of different transgenic models (16,22,45,46). However, the balance between negative and positive selections remains difficult to evaluate, notably because of possible competition between cells (14,15,47). Our oligoclonal transgenic model brings new light on this balance. First, the block in B cell development in certain transgenic mice could be interpreted only by an absence of Ig signaling and not by the existence of negative selection (48,49). Thus, the comparison between H3-Tg+/+{lambda}1s and H3-Tg+/–{lambda}1s is exemplary. The only difference between these two mice is the number of H3 inserts but the impact of this difference on the establishment of the B cell repertoire is drastic. This result strongly suggests that the conditions of µ chain expression determine the level of (auto?) Ig survival signaling. Secondly, the existence of a B cell subpopulation is totally dependent on the nature of the other B cells. Indeed, the H3/{lambda}2 and/or H3/{lambda}3 cells are, a priori, identical in both H3-Tg+/+{lambda}1s and H3-Tg+/+{lambda}1+ mice. However, these cells can develop in the former mice and almost disappear in the latter mice. Their presence is possible only in the absence of positive selection of H3/{lambda}1s cells. These results strongly suggest that, in physiological conditions (non-transgenic), only positively selected B cells can belong to the mature B cell repertoire.

Our understanding of the {lambda}1s phenotype remains obscure. Our results indirectly demonstrate that only the Gly -> Val substitution is responsible for the {lambda}1s defect since the latter can be overcome under adequate experimental conditions (i.e. in H3-Tg+/+{lambda}1s mice). According to our hypotheses, the {lambda}1s defect has several facets. It can be corrected for B cell lymphopoiesis and for export to the periphery. However, it remains effective to inhibit the positive selection process. From a structural viewpoint, the Gly -> Val substitution, which is exposed to solvent, fits with the idea that positive selection requires an interaction with an external ligand. However, the presence of few normal mature {lambda}1s cells in {lambda}1s mice remains an enigma since these cells do not differ from {lambda}1+ cells in their capacity to be stimulated or their VH-associated repertoire (our unpublished data).


    Acknowledgments
 
We thank Drs Michelle Goodhart, Ana Cumano, Dominique Rueff-Juy and Colette Kanellopoulos-Langevin for helpful discussions. We gratefully acknowledge Ms Anne-Marie Crain and Marie-Claude Gendron for expert assistance in FACS analyses. S. H.-R. was supported by a fellowship from the `Fondation pour la Recherche Médicale'. URA CNRS 1961 and the Université Pierre et Marie Curie support The Immunochimie Analytique unit. This work is also supported by the Fondation pour la Recherche Médicale and the Association pour la Recherche sur le Cancer


    Abbreviations
 
BCR B cell receptor
LPS lipopolysaccharide

    Notes
 
Transmitting editor: J. F. Kearney

Received 12 August 1999, accepted 24 November 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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