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Expression levels of B cell surface immunoglobulin regulate efficiency of allelic exclusion and size of autoreactive B-1 cell compartment.

Surface-expressed immunoglobulin (Ig) has been shown to have a critical role in allelic exclusion of Ig heavy (H) and light (L) chains. Although various degrees of suppression of endogenous Ig expression are observed in Ig transgenic (Tg) mice, it was not clear whether this difference is due to different onsets of Tg expression or to different levels of Tg expression, which are obviously affected by integration sites of the transgene. In this study we generated antierythrocyte antibody Tg mice that carry tandem joined H and L chain transgenes (H+L) and confirmed that homozygosity of the transgene loci enhances the level of transgene expression as compared with heterozygosity. Suppression of endogenous H and L chain gene expression was stronger in homozygous than in heterozygous Tg mice. Similar results were obtained in control Tg mice carrying the H chain only. These results suggest that there is a threshold of the B cell receptor expression level that induces allelic exclusion. In addition, despite the same B cell receptor specificity, the size of Tg autoreactive B-1 cell compartment in the peritoneal cavity is larger in homozygous than in heterozygous mice, although the number of the Tg B-2 cell subset decreased in the spleen and bone marrow of homozygous Tg mice as compared with heterozygous Tg mice. By contrast, homozygosity of the H chain alone Tg line, which does not recognize self-antigens, did not increase the size of the peritoneal B-1 subset. These results suggest that the size of the B-1 cell subset in the Tg mice may depend on strength of signals through B cell receptors triggered by self-antigens.

. This phenomenon is called H chain allelic exclusion. Surface expression of the chain has a critical role in allelic exclusion of the H chain locus because targeted disruption of the chain membrane exon results in loss of H chain allelic exclusion (2). The essential role of the cell surface chain expression in allelic exclusion is also supported by studies using Ig transgenic (Tg) 1 mice (3). Mice expressing the membrane-form chain transgene inhibit expression of the endogenous chain, whereas Tg mice with the secreted form chain do not show such inhibition (4)(5)(6). In addition, expression of Ig L chain transgenes also exerts suppression on the rearrangement and expression of the endogenous L chain Ig locus (6,7). However, the suppression of endogenous H and L chain gene expression in Ig Tg mice are generally not complete, and efficiencies of the suppression are variable among different lines of Tg mice (8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Variable integration sites of transgenes may influence the onset and level of transgene expression, which may affect the efficiency of allelic exclusion. Since the V(D)J recombination event in each B cell is all or none, it has not been clear whether allelic exclusion can be induced by a small number of surface chain or if it requires a relatively higher number of surface chain (a threshold). To examine these possibilities, quantitative comparison between the levels of transgene expression and suppression of endogenous H and L chain gene expression should be carried out using Tg lines with the same integration site to avoid the difference due to the developmental onset of transgene expression.

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Quantitative Effects of Surface Ig Levels on Allelic Exclusion We have generated Tg mice that produce an autoantibody (4C8) to RBCs, and have analyzed the mechanisms of B cell tolerance and B-1 cell activation (18)(19)(20)(21)(22)(23)(24). Since autoreactive B cells are eliminated at the immature stage in the bone marrow, the number of self-reactive B cells is markedly decreased in the bone marrow, peripheral blood, spleen, and lymph nodes in the Tg mice. In contrast, the peritoneal cavities of the Tg mice contain a normal number of autoreactive B-1 cells that can be eliminated by interaction with RBCs (18,19). B-1 cells show distinct surface antigen expression and anatomical localization from conventional B (B-2) cells (25)(26)(27)(28). Furthermore, the V H gene usage, N region diversity, and antigen specificity of B-1 cells are also unique (29)(30)(31)(32)(33)(34). B-1 cells are thought to be generated from fetal liver cells, and two independent studies have demonstrated that adult bone marrow cells do not give rise to CD5 ϩ B-1 cells after transfer to irradiated hosts (35,36). In contrast, Wortis and colleagues (37)(38)(39) have shown that adult bone marrow contains precursors for CD5 ϩ cells and that CD5 expression in splenic B cells is induced by surface IgM cross-linking, suggesting that B-1 cells are generated from common precursors to conventional B cells. Arnold, Clarke, and colleagues (40,41) have reported that B-1 cells differentiate from B-0 cells after expression of specific antigen receptors. Thus, it is not yet clear whether B-1 cells constitute a B cell lineage distinct from conventional B cells. In addition, even if some B-1 cells are derived from activation of B-2 cells, it is not clear whether a certain threshold level of B cell receptor (BCR) expression is required for B-1 cell differentiation or if the receptor specificity alone plays a major role.
To answer these questions we generated new lines of anti-RBC antibody Tg mice that carried tandem joined H and L chain (H ϩ L) transgenes, and we compared the levels of allelic exclusion and the size of B-1 cell compartment between homozygotes and heterozygotes that differ only in the level of the surface Ig expression. Suppression of endogenous H and L chain gene expression was more prominent in homozygotes than in heterozygotes, suggesting that allelic exclusion depends on a certain level of surface Ig expression. In addition, the size of the Tg B-1 cell compartment in the peritoneal cavity is larger in homozygous than in heterozygous H ϩ L mice, suggesting that the increase in the B-1 cell compartment in the Tg mice may be due to augmentation of signals through surface Ig by the self-antigen (RBC).

Materials and Methods
Tg Mice. Tg mouse lines carrying either H or L chain gene for the anti-RBC mAb (4C8 mAb) have been established previously (18). Double Tg (H ϫ L) mice with H and L chain transgenes were obtained by mating H and L chain Tg mice. In this study we generated new lines of 4C8 mAb Tg mice that carried tandem joined H and L chain transgenes. To construct tandem joined transgenes, a 5.6-kb BamHI fragment of pMO-4C8 was first subcloned in the BamHI site of pSP73 Vector (Promega Corp.). Next, a 15.5-kb XhoI fragment of pMO-4C8 was sub-cloned between the SalI and XhoI sites. The pSP73 plasmid containing both H and L chain genes was designated as pSP73-4C8. Tandem joined H and L chain Tg (H ϩ L) mice were generated by injecting a 22.0-kb PvuI-XhoI fragment of pSP73-4C8 (see Fig. 1 A). Heterozygous mice were mated to get homozygous mice in two representative lines, H ϩ L5, with 4-12 copies, and H ϩ L6, with 1-3 copies of the transgene. The presence of the transgenes and homozygosity of the Tg loci were screened by PCR and Southern blot analysis. Tg mice were maintained under conventional conditions in our animal facility.
Flow Cytometric Analysis. Single cell suspension from bone marrow was prepared by flashing femur bones with a staining buffer (PBS containing 2% FCS and 0.05% sodium azide), gently pipetted, and washed with the staining buffer. Single cell suspensions from bone marrow, spleen, and peritoneal cavity were prepared with the staining buffer and pretreated with 10% heat-inactivated normal rat serum. After 15 min of incubation, FITC-and/or biotinconjugated mAbs at appropriate dilutions in the staining buffer were added directly. After 30 min of incubation, PE-conjugated mAbs and/or FITC-or PE-conjugated SA was added. Before and after each step of incubation, cells were washed with the staining buffer. Stained cells were then applied to FACSCalibur ® (Becton Dickinson). After excluding dead cells by propidium iodide staining, cells present in the lymphocyte gate defined by forward and side light scatters were analyzed.

Surface Expression of the Transgene Is Higher in Homozygous than in Heterozygous Mice.
To assess whether the levels of transgene expression are higher in homozygous than in heterozygous mice, we analyzed the bone marrow cells of H ϩ L5 and H ϩ L6 mice that carried tandem joined H and L chain transgenes cording for the 4C8 anti-RBC antibody ( Fig. 1 A). In H ϫ L mice, B220 ϩ IgM ϩ bone marrow cells almost completely disappeared (18). In contrast, both lines of H ϩ L mice had significant numbers of B220 ϩ IgM ϩ cells in bone marrow ( Fig. 1 B). The majority of these cells were stained both with antiallotypic mAb for IgM a (Tg) and with antiidiotypic mAb (S54) for the anti-RBC anti-body. We compared the fluorescence intensity of Id ϩ bone marrow cells in homozygous mice to that in heterozygous mice ( Fig. 1 C). The mean fluorescence intensities (MFI) of Id ϩ cells in heterozygous and homozygous H ϩ L5 mice are 481.70 and 717.42, respectively. Similarly, the MFI of Id ϩ cells in heterozygous and homozygous H ϩ L6 mice are 997.76 and 1670.11, respectively. These results indicate that in both H ϩ L5 and H ϩ L6 lines the levels of transgene expression are clearly higher in homozygous than in heterozygous mice. We also examined surface Ig expression on the bone marrow cells of H3 Tg mice that had only H chain transgene. Fluorescence intensity of antiallotypic mAb for IgM a (Tg) of bone marrow cells was also stronger in homozygous (MFI ϭ 908.77) than in heterozygous (MFI ϭ 319.25) mice. In a parallel experiment, anti-IgM a mAb MFI of homozygous H ϩ L5 and H ϩ L6 were 173.07 and 250.72, respectively, indicating that homozygous H ϩ L B cells had not saturated the capacity of surface Ig expression ( Fig. 1 D). Taken together, homozygosity of Tg Ig loci enhances the level of transgene expression on B cell surface.

Inhibition of Endogenous H Chain Expression Is Stronger in Homozygous than in Heterozygous Mice.
To test whether inhibition of endogenous H chain expression is stronger in homozygous than in heterozygous mice, we analyzed lymphocytes in bone marrow and spleen of H ϩ L5, H ϩ L6, and H3 mice by flow cytometry. We stained bone marrow and spleen cells with antiallotypic mAbs (which can clearly distinguish IgM of the control mice; Fig. 2, A and B) for IgM a (Tg) and IgM b (endogenous). In all heterozygous Tg mice, the numbers of IgM a ϩ IgM b ϩ cells (allelic inclusion) were small but significant, whereas these cells almost completely disappeared in all homozygous Tg mice. This conclusion is further confirmed by the finding that both bone marrow and spleen B cells with only endogenous H chains (IgM a Ϫ IgM b ϩ ) decreased in homozygous Tg mice as compared with heterozygous Tg mice (Fig. 2, A and B). In all lines of Tg mice, total numbers of endogenous H chain- expressing (IgM bϩ ) spleen cells were lower in homozygous than in heterozygous mice (Fig. 2 C). These results indicate that inhibition of endogenous H chain expression (allelic exclusion) is stronger in homozygous than in heterozygous mice. Taken together, higher levels of Ig transgene expression appear to cause stronger inhibition of endogenous H chain expression, suggesting that a certain level of surface Ig expression is required for allelic exclusion of the H chain locus.
Inhibition of Endogenous L Chain Expression Is Stronger in Homozygous Mice than in Heterozygous Mice. Next, we asked whether homozygosity of Tg loci also reduces endogenous L chain expression. Since the allotypic specificity for the Tg L chain is not known, we estimated the degree of endogenous L chain expression by difference in expression of Tg H chain (IgM a ) and Id (the combined epitope of the H and L chains of the 4C8 anti-RBC mAb) that is recognized by the S54 mAb. In the HϩL5 line, two populations of IgM aϩ cells were observed: one stained with both anti-IgM a and anti-Id mAbs diagonally, and the other stained more weakly with anti-Id mAb than with anti-IgM a mAb (Fig. 3,  A, B, and D, tops). As a control, IgM aϩ cells from H3 mice did not contain the population stained with the S54 mAb (Fig. 3, B and D, bottoms). We presume that the cells that stained diagonally with both mAbs express the Tg H and L chains equally, and that the IgM aϩ Id low cells express the Tg H chain with both endogenous and Tg L chains. In support of this assumption, IgM aϩ bone marrow cells in heterozygous HϩL6 mice, which consisted of almost exclusively IgM aϩ Id ϩ cells and only few IgM aϩ Id low cells, contained Ͻ1.2% ϩ cells, which represent a fraction of B cells that express endogenous L chains (Fig. 3 A). In contrast, IgM aϩ bone marrow cells in heterozygous HϩL5 mice, which consisted of ‫%82ف‬ IgM aϩ Id ϩ and ‫%27ف‬ IgM aϩ Id low cells, contained Ͼ5.5% ϩ cells (Fig. 3 A). Furthermore, IgM aϩ spleen cells in heterozygous HϩL5 mice, which consisted of ‫%4ف‬ IgM aϩ Id ϩ and ‫%69ف‬ IgM aϩ Id low cells, contained Ͼ14% ϩ cells (Fig. 3 A). The total number of cells expressing endogenous L chains (IgM aϩ Id low ) in bone marrow (Fig. 3, B and C) and spleen (Fig. 3, D and E) was less in homozygous than in heterozygous Tg lines, indicating that homozygosity of the Tg loci enhances the inhibition of endogenous L chain expression as compared with heterozygosity.

The Size of an Autoreactive B1 Cell Compartment Is Larger in Homozygous Mice than in Heterozygous HϩL Mice.
HϫL mice show almost complete deletion of autoreactive B cells from bone marrow and spleen, whereas a normal level of B-1 cells is found in the peritoneal cavity (18). Bone marrow of both lines of HϩL mice contained some autoreactive B cells (IgM aϩ Id ϩ ), which have B-2 cell phenotypes (CD5 Ϫ B220 high IgM low ), whereas autoreactive B cells in spleen almost completely disappeared in HϩL mice (Fig. 3, B and D). We examined whether homozygosity of the Ig transgene loci influences the size of the autoreactive B-1 cell compartment in the peritoneal cavities of HϩL5 and HϩL6 mice. Peritoneal cells of HϩL mice had a limited number of B220 ϩ Id ϩ cells, almost all of which were IgM aϩ (Fig. 4, A-C and E-G). Almost all Id ϩ cells in the peritoneal cavity were Mac1 ϩ and therefore belong to the B-1 subset (Fig. 4, D and H). Thus, IgM aϩ Id ϩ cells are most likely to be autoreactive B-1 cells (Fig. 4, C and G). These B220 ϩ Id ϩ cells in the peritoneal cavity were much more abundant in homozygous HϩL mice than in heterozygous HϩL mice, indicating that the size of the autoreactive B-1 cell subset is larger in homozygous than in heterozygous HϩL mice. The relative size of the B-1 cell compartment is directly proportional to the B-1 cell number because total cell numbers in the peritoneal cavity did not vary between homozygous and heterozygous Tg mice. These results suggest that a higher expression level of the autoantibody facilitates the increase in autoreactive B-1 cells in the peritoneal cavity.
To test whether autoreactivity of Ig expressed on B cells is crucial to enlargement of the B-1 cell subset, we carried out similar studies on the peritoneal B cells in H3 Tg mice, the vast majority of which are not autoreactive due to the absence of the Tg L chain. The majority of peritoneal B cells expressed the Tg H chain (IgM aϩ ), in agreement with analysis of bone marrow and spleen cells (Fig. 4, I and J). Indeed, B cells expressing endogenous H chains were less in homozygous than in heterozygous H3 mice (Fig. 4 J), probably because of more efficient allelic exclusion due to stronger expression of the transgene product on homozygous B cells (Fig. 1 C). Nonetheless, the percentage of IgM ϩ Mac1 ϩ cells in the peritoneal cavity did not increase in homozygous H3 mice as compared with those in heterozygous H3 mice (Fig. 4 K). These results indicate that increased surface Ig expression of non-auto-reactive B cells did not facilitate enlargement of the B-1 cell subset in the peritoneal cavity. Taken together, enhancement of autoreactive surface Ig expression appears to be crucial to increase the B-1 cell subset in the peritoneal cavity.

Discussion
In this study, we generated new lines of the anti-RBC antibody Tg mice that carry tandem joined H and L chain transgenes, and compared B cell phenotypes between heterozygous and homozygous Tg mice to evaluate quantitatively the effect of surface Ig level on allelic exclusion and Several lines of evidence suggest that surface expression of the chain is critical for H chain allelic exclusion (2,(4)(5)(6). In addition, pre-BCR, which is the H chain paired with the surrogate L chain, is suggested to mediate H chain allelic exclusion through downregulation of recombinationactivating genes (42)(43)(44)(45)(46). Ig␣ and Ig␤, which associate with surface-expressed H chains, have also been shown to trigger the signals inducing allelic exclusion (47)(48)(49). Taken together, expression of the H chain as a pre-BCR on the cell surface may induce signals mediated by Ig␣ and Ig␤, resulting in H chain allelic exclusion. In this study we have demonstrated that homozygosity of the transgene Ig loci increases surface Ig expression on bone marrow B cells and causes stronger allelic exclusion, as compared with heterozygosity. Since allelic exclusion is all or none in each B cell, the present results suggest that there is a threshold of the pre-BCR signal intensity that induces allelic exclusion.
There are several other possibilities to explain our results. First, B cells with allelic inclusion may be negatively selected. However, Sonoda and Rajewsky (50) have shown that B cells with allelic inclusion can normally expand in the periphery using double Ig knock-in mice, indicating the absence of negative selection against allelic inclusion B cells. In addition, we have shown that endogenous only B cells (IgM aϪ IgM bϩ ) are also reduced in homozygotes (Fig.  2). Second, reduction of allelic inclusion B cells could be due to selective expansion of higher surface Ig-expressing cells by a self-antigen. However, the self-antigen (RBC) can kill self-reactive B cells (19). In fact, Id ϩ B cells of HϩL mice decreased in homozygotes as compared with heterozygotes in spleen as well as in bone marrow (Fig. 3,  B and D). In addition, we have shown that non-self-reactive (H chain alone expressing) Tg (H3) B cells also show suppression of endogenous Ig expression (Fig. 2). These results cannot be explained by positive selection of self-reactive Ig expressing B cells by the self-antigen. Third, excess Tg Ig expression on surface may inhibit detection of endogenous Ig expression. It is unlikely that endogenous and Tg Ig molecules compete for surface expression in HϩL5 and HϩL6 spleen cells. This is because the surface Ig levels of homozygous H3, HϩL6, and HϩL5 cells are 909, 251, and 173 (MFI using the anti-IgM a Ab), respectively ( Fig. 1  D), indicating that HϩL5 and HϩL6 B cells have not hit the ceiling of the surface Ig expression level. Sonoda and Rajewsky (50) have shown that there is no inhibitory mechanism for the H chain on the surface as long as it can associate with the L chain. It is inconceivable that the surface expression efficiency differs between H chains derived from the transgene and endogenous gene. The possibility that a lower detection efficiency by anti-IgM b Ab staining in the presence of large amounts of IgM a is also unlikely because the FACS ® profiles of H3 spleen cells expressing large amounts of IgM a clearly show the presence of IgM a IgM b double-positive cells even in homozygotes (Fig. 2 B). Finally, the suppression efficiency of the endogenous locus may be increased in homozygotes simply because the frequency of silencing the transgene locus is reduced by doubling the number of the transgene locus in homozygotes. Assuming that this is the case, the efficiency of silencing of the transgene locus can be calculated from the endogenous H chain only cells shown in Fig. 2 A: H3, 0.06 / (0.06 ϩ 0.11 ϩ 6.50) ϭ 0.009 in heterozygotes and 0.00144 ϭ 0.01 / (0.01 ϩ 0.03 ϩ 6.9) in homozygotes. The value in homozygotes is ‫81ف‬ times of the expected value (0.009 ϫ 0.009 ϭ 0.000081) based on the simple statistics. Similar discrepancy was seen in the HϩL5, HϩL6, and H3 spleen cells (Fig. 2 B). In addition, homozygosity alters the number of Tg Id ϩ B cells in spleen and peritoneal cavity to the opposite direction. These considerations make the final possibility unlikely.
We have shown that HϩL Tg mice have B cells expressing endogenous L chains together with the Tg H chain. These cells, expressing endogenous L chains in HϩL mice, may be generated either by incomplete allelic exclusion or by receptor editing because the B cells which express the transgenes are self-reactive (51,52). Nemazee and colleagues (53)(54)(55) have suggested that interaction with autoantigens leads IgM low IgD Ϫ bone marrow cells to undergo receptor editing but IgM high IgD ϩ cells to undergo rapid apoptosis. On the other hand, Rusconi et al. (16) generated B cell hybridomas from anti-trinitrophenol antibody (antinon-self) Tg mice that carried tandem joined H and L chain transgenes and demonstrated that the B cell hybridomas secreting the Tg antibody expressed the Tg L chain at about one-tenth of the level of coexpressed endogenous L chains. Endogenous L chain expression in their Tg mice is probably due to incomplete allelic exclusion because these B cells are unlikely to receive BCR stimulation by self-antigens to trigger receptor editing. Although we cannot exclude the possibility that receptor editing is involved in the appearance of B cells with endogenous L chains in our Tg mice, we think it less likely because of the following reason. If the expression of endogenous L chain is due to receptor editing induced by stimulation with the self-antigen, the mechanism to reduce the number of B cells with endogenous L chains by enhanced expression of self-reactive Ig should be clonal deletion. However, when B cells expressed more self-reactive Ig on surface, B cells with both Tg and endogenous L chains (IgM aϩ Id low ) are more efficiently reduced than Tg only B cells (IgM a Id ϩ ), which are most likely eliminated by clonal deletion (Fig. 3). This observation is somewhat opposed to the expected efficiency of clonal deletion by the self-antigen because stronger BCR signaling will be induced in IgM a Id ϩ cells than Ig-M a Id low cells.
Although it is still controversial whether B-1 cells belong to an ontogenetically different B cell lineage from conventional B cells (34)(35)(36)(37)(38)(39)(40), B-1 and B-2 cells clearly constitute different subsets of B cells. In this study we have shown that the size of the Tg B-1 cell compartment is larger in homozygous than in heterozygous HϩL mice (Fig. 4).
Since Tg B-1 cells in heterozygous and homozygous mice show the same antigen specificity, our results suggest that the level of surface Ig expression directly influences the size of the Tg B-1 cell compartment. Our findings are consistent with the previous observations that defects of BCR signaling cause reduction of the B1 cell (56,57), and loss of a BCR inhibitory molecule, SHP-1, increases the B1 cell number (58)(59)(60)(61)(62). It is important to note that increased levels of autoreactive BCR induced augmented clonal deletion of B-2 cells in bone marrow and spleen but expansion of B-1 cells in the peritoneal cavity (Fig. 4). Increased expression of nonautoreactive Ig (H3) enhanced neither clonal deletion of B-2 cells nor expansion of B-1 cells. These results suggest that there are at least three levels of BCR signaling that regulate self-reactive B1 and B2 cell differentiation. At a lower level, self-reactive B-2 cells can be stimulated to induce receptor editing or to become anergic (53)(54)(55)63). At an intermediate signaling level, B-2 cells are clonally deleted and B-0 (40, 41) and/or B-2 cells are induced to differentiate into B-1 cells, which migrate into the peritoneal cavity. At a strong signaling level, B-1 cells are also clonally deleted (19). It is tempting to speculate that at least a sizable fraction of peritoneal B-1 cells originate and expand from autoreactive B cells that are stimulated by self-antigens to a level strong enough to be activated but weak enough to avoid apoptosis.
We thank Ms. Y. Kobayashi and Ms. T. Taniuchi for their technical assistance, and Ms. Y. Takahashi for manuscript preparation.
This work was supported by grants from the COE program from the Ministry of Education, Science, Sports and Culture of Japan.