In the B lymphocyte lineage, Fas-mediated cell death is important in controlling activated mature cells, but little is known about possible functions at earlier developmental stages. In this study we found that in mice lacking the IgM transmembrane tail exons (μMT mice), in which B cell development is blocked at the pro-B stage, the absence of Fas or Fas ligand allows significant B cell development and maturation, resulting in high serum Ig levels. These B cells demonstrate Ig heavy chain isotype switching and autoimmune reactivity, suggesting that lack of functional Fas allows maturation of defective and/or self-reactive B cells in μMT/lpr mice. Possible mechanisms that may allow maturation of these B cells are discussed.

The development of B lymphocytes is regulated by apoptosis (1). Fas (CD95) is a cell surface protein that transmits apoptotic signals in a wide variety of cell types upon binding of Fas ligand (FasL)3 (2). Activation of the Fas pathway has been implicated in central and peripheral tolerance of T cells and had been shown to be an important mechanism for peripheral tolerance in the B cell compartment (3, 4, 5, 6, 7). Mice lacking functional Fas or FasL expression (lpr/lpr or gld/gld, respectively) develop massive lymphadenopathy and a lupus erythematosus-like autoimmune syndrome (8). In the B lymphocyte lineage, Fas-mediated cell death is important in controlling activated mature cells (7, 9) and elimination of anergic B cells chronically exposed to self Ag (10). In contrast, the contribution of the Fas pathway at earlier stages of B cell development is not known. To test the role of Fas in the elimination of defective B cells, we have introduced the Fas-null, lpr mutation to mice lacking the IgM transmembrane tail exons (μMT mice), in which B cell development is blocked at the pro-B stage (11), to generate μMT/lpr mice. In this study we show that lack of Fas allows significant development and maturation of B cells despite the μMT mutation. Because μMT/lpr mice develop significantly enhanced autoimmunity and autoantibody production, it appears that in the absence of functional Fas, defective B cells can be generated and selected to participate in the autoimmune process.

Normal C57/BL6 (B6), C57/BL6-Faslpr/Faslpr (B6/lpr), C57/BL6-μMT/μMT (μMT), and C57/BL6 gld/gld (gld mice) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice carrying both lpr and μMT homozygous mutations (μMT/lpr) were generated by crossing B6/lpr and μMT mice. μMT/gld mice were the progeny of the F2 generation of a μMT × gld cross. Littermates were typed for μMT, lpr, and gld homozygosity by PCR as previously described (4).

Ab in serum was determined by sandwich ELISA. Sera from 4- to 6-mo-old mice was collected, and total IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM concentrations were measured using specific goat anti-mouse polyclonal reagents (Southern Biotechnology Associates, Birmingham, AL). Purified Ig of each isotype provided the standard curve for calculation of Ab concentration, and results are expressed as micrograms per milliliter. Anti-chromatin IgG in serum was detected by ELISA as previously described (6). Affinity-purified mouse monoclonal IgG anti-dsDNA 3H9 (12) of known concentration served as a positive control and as a reference standard curve. Titers of anti-chromatin Abs for individual mice were calculated using the 3H9 Ab control standard curve and are expressed as micrograms per milliliter of 3H9 equivalent (amount of signal obtained in a tested sample equal to that obtained by a known concentration of the anti-dsDNA 3H9 mAb). In some experiments mice were immunized by OVA emulsified in CFA (100 μg/mouse, administered i.p.) or with dextran (Sigma, St. Louis, MO; 10 μg/mouse, administered i.v.). Mice were bled at various times after immunization, and Ab production was measured by specific ELISA. Dextran immunization and anti-dextran ELISA were performed as previously described (13).

Total RNA was purified from RBC-depleted spleen cells derived from the indicated mice. Samples were reverse transcribed to cDNA and PCR amplified for Vκ-Cκ to detect light chain expression and for Gsα as a control as previously described (14).

Expression of surface molecules was detected using the following Abs: CD43, S7 biotin (PharMingen); κ, goat anti-mouse κ light chain biotin (Southern Biotechnology Associates); TCRβ, H57 biotin (PharMingen, San Diego, CA); CD19, 1D3 FITC (PharMingen), GL-7, GL-7 FITC (PharMingen); and B220, RA3-6B2 PE (Caltag, San Francisco, CA). Biotinylated Abs were visualized with streptavidin-TriColor (Caltag) for three-color analysis. Stained samples were analyzed by FACScan (Becton Dickinson, Mountain View, CA).

In μMT mice, B cell development in the bone marrow is blocked, and cells die at the pro-B stage, resulting in complete deficiency of mature B cells and agammaglobulinemia (Fig. 1,A) (11). This occurs despite the fact that in μMT mice the genes encoding the secreted form of IgM and all other Ig isotypes are intact. In striking contrast to the μMT mice, introduction of the lpr mutation rescues secretion of non-IgM serum Igs in μMT/lpr mice to levels that are similar or even significantly elevated (IgG2a, IgA) compared with those in their IgM-sufficient B6 and B6/lpr counterparts (Fig. 1 A). Careful analysis within the μMT/lpr group revealed a skewed pattern of Ab production in some individual mice. In some mice one Ab isotype was dominantly found, whereas other mice produced high titers of all non-IgM serum isotypes (not shown). Further light chain isotyping revealed that most of the serum Abs were κ, but significant levels (up to 10-fold more than in normal B6 or B6/lpr mice) were λ (data not shown). Mice that showed a severe skewing of IgH isotypes had also a severely skewed λ/κ ratio, which was dominated by either λ or κ light chain (30–90% of the total light chain found in serum). The λ/κ ratio in mice that produced all non-IgM serum Ig was also skewed, but to a lesser extent, ranging from 2 to 25% λ (of the total light chain found in serum).

FIGURE 1.

Production of non-IgM serum Abs in mice lacking functional Fas/FasL. A, Serum samples were collected from 4- to 6-mo-old normal C57/BL6 mice (B6), B6/lpr, μMT, and μMT/lpr mice and were analyzed by ELISA to determine serum concentrations of IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM. Concentrations were determined using the appropriate standard curve, and results are expressed as micrograms per milliliter for individual mice and as group means. Each group contained 5–10 mice. B, μMT/gld mice were the progeny of the F2 generation of a μMT × gld cross. Littermates were typed for μMT and gld homozygosity by PCR. IgG levels in sera from wild-type (carrying at least one functional μH and FasL allele), μMT (μMT/μMT, FasLgld/FasL+ or μMT/μMT, FasL+/FasL+), and μMT/gld (homozygous for both μMT and FasLgld) littermates were analyzed by ELISA at various times after birth. The maternal IgG level and its disappearance are revealed in the μMT group. Each group contained three to five mice.

FIGURE 1.

Production of non-IgM serum Abs in mice lacking functional Fas/FasL. A, Serum samples were collected from 4- to 6-mo-old normal C57/BL6 mice (B6), B6/lpr, μMT, and μMT/lpr mice and were analyzed by ELISA to determine serum concentrations of IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM. Concentrations were determined using the appropriate standard curve, and results are expressed as micrograms per milliliter for individual mice and as group means. Each group contained 5–10 mice. B, μMT/gld mice were the progeny of the F2 generation of a μMT × gld cross. Littermates were typed for μMT and gld homozygosity by PCR. IgG levels in sera from wild-type (carrying at least one functional μH and FasL allele), μMT (μMT/μMT, FasLgld/FasL+ or μMT/μMT, FasL+/FasL+), and μMT/gld (homozygous for both μMT and FasLgld) littermates were analyzed by ELISA at various times after birth. The maternal IgG level and its disappearance are revealed in the μMT group. Each group contained three to five mice.

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To confirm the involvement of the Fas pathway in rescuing Ig secretion, the experiments were repeated using mice deficient of functional FasL (gld). The results in Fig. 1,B indicate that μMT/FasL-deficient (μMT/gld) mice have a phenotype similar to that of μMT/lpr mice. In these experiments serum IgG was measured in (μMT/μMT × gld/gld)F2 mice at various times after birth. As expected, in μMT, FasL-sufficient littermates, IgG levels dropped rapidly after birth due to the loss of maternal Ig, but μMT/gld offspring had high IgG levels that rose to levels similar to those of wild-type littermates by 90 days after birth (Fig. 1 B). These findings suggest that in mice deficient in the Fas/FasL pathway, defective B cells can circumvent developmental check points, such as the block caused by the μMT mutation, mature, and differentiate to Ab-producing cells.

To quantify peripheral B cells in μMT/lpr mice, spleen cells were stained for membrane expression of the pan B cell marker CD19 and the κ light chain and were analyzed by flow cytometry (Fig. 2). As previously shown, CD19+/Ig pro-B cells are found in spleens of μMT mice (Fig. 2,A, right, and Fig. 2,C) (11). In contrast, significant numbers of Ig-bearing B cells, which in several mice reached 10–15% of lymphocytes, were detected in spleens of μMT/lpr mice (Fig. 2,A, middle, and Fig. 2,C). This finding was confirmed independently by the detection of Ig-κ light chain mRNA in μMT/lpr, but not μMT, spleen (Fig. 2,B). In addition, a high frequency of μMT/lpr B cells, which was not different from that found in control B6 and B6/lpr mice, expressed the germinal center marker GL-7 (Fig. 2,C summarizes absolute numbers and frequencies of Ig+, Ig, and GL-7+ B cells in total spleen cells). It is thus suggested that μMT/lpr B cells can undergo cellular activation and are able to participate in an immune response. However, as shown in Fig. 2 C, numbers of Ig+ B cells in the μMT/lpr spleens were still 10- to 20-fold lower than those found in spleens derived from normal B6 or B6/lpr mice.

FIGURE 2.

Detection of Ig+ mature B cells in μMT/lpr mice. The results shown are from individual mice, which are representative of at list five mice from each group. A, Spleen cells from B6, μMT/lpr, and μMT mice were RBC depleted, stained for CD19 and κ-chains, and analyzed by flow cytometry. B, Total RNA was purified from RBC-depleted spleen cells derived from the indicated mice, reverse transcribed, and PCR amplified for Vκ-Cκ to detect light chain expression, or control Gsα expression. C, A table summarizing frequencies and absolute numbers of Ig+, Ig, and GL-7+ B cells in spleens of B6, B6/lpr, mMT, and mMT/lpr mice. Frequencies are shown in parentheses and are of CD19++, CD19+, and B220+/GL7+ cells as analyzed by FACS and gated on all viable cells. Total CD19++, CD19+, and B220+/GL7+ cells were calculated by multiplying each frequency by the total number of viable spleen cells. The results shown are the group mean of five to seven mice from each group ± SEM. D, Spleen cells from μMT and μMT/lpr mice were analyzed by two-color flow cytometry for cell surface expression of the pan B cell marker CD19 and for the expression of Ig-μ, -γ, or -α heavy chain.

FIGURE 2.

Detection of Ig+ mature B cells in μMT/lpr mice. The results shown are from individual mice, which are representative of at list five mice from each group. A, Spleen cells from B6, μMT/lpr, and μMT mice were RBC depleted, stained for CD19 and κ-chains, and analyzed by flow cytometry. B, Total RNA was purified from RBC-depleted spleen cells derived from the indicated mice, reverse transcribed, and PCR amplified for Vκ-Cκ to detect light chain expression, or control Gsα expression. C, A table summarizing frequencies and absolute numbers of Ig+, Ig, and GL-7+ B cells in spleens of B6, B6/lpr, mMT, and mMT/lpr mice. Frequencies are shown in parentheses and are of CD19++, CD19+, and B220+/GL7+ cells as analyzed by FACS and gated on all viable cells. Total CD19++, CD19+, and B220+/GL7+ cells were calculated by multiplying each frequency by the total number of viable spleen cells. The results shown are the group mean of five to seven mice from each group ± SEM. D, Spleen cells from μMT and μMT/lpr mice were analyzed by two-color flow cytometry for cell surface expression of the pan B cell marker CD19 and for the expression of Ig-μ, -γ, or -α heavy chain.

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To further characterize the Ig+ B cells found in spleens of μMT/lpr mice, spleen cells were stained with Abs specific for μ, γ, or α heavy chain constant regions. FACS analysis shown in Fig. 2 D reveal that B cells expressing γ or α heavy chain are found in large numbers in spleens of μMT/lpr mice, but not in Fas-sufficient μMT spleens. As expected, no surface IgM (sIgM) expression was found in μMT or μMT/lpr mice. sIgD expression was not detected in both μMT/lpr and μMT spleens (not shown). The possibility that detection of surface Ig-κ or heavy chain on μMT/lpr spleen B cells reflected coating of the CD19+ cells with serum Ig was excluded by prestaining of μMT spleen cells (containing CD19+/Ig pro B cells) with Ig+ serum collected from μMT/lpr mice (not shown). It therefore appears that in mice deficient of functional Fas or FasL, B cells bearing the μMT mutation can undergo isotype switching, mature, and differentiate to plasma cells. This process, however, may not be efficient as only small number of Ig+ B cells could be found in μMT/lpr spleens relative to control.

To determine whether the previously reported μMT-induced block in the pro-B to pre-B transitional stage of development was breached in μMT mice lacking functional Fas/FasL, bone marrow B cell developmental compartments were analyzed by flow cytometry. In mouse bone marrow, pro-B cells are distinguished from later developmental stages by surface coexpression of CD43 along with low levels of B220 (CD45R; Fig. 3, upper boxes) (15). Pre-B cells lack CD43 and express higher levels of B220 (Fig. 3, lower boxes) (15). As shown in Fig. 3, the transitional block in early B cell development evident in μMT mice is not substantially rescued in μMT/lpr mice. These findings suggest that only few μMT/lpr B cells can circumvent the μMT-induced developmental block by undergoing isotype switching, and that this process does not necessarily occur in the bone marrow.

FIGURE 3.

Analysis of bone marrow B cell compartments. Bone marrow cells from B6, μMT/lpr, and μMT mice were stained for CD43 and B220, then analyzed by flow cytometry as previously described (15 ). Pro-B cells (B220low/CD43+) are distinguished from later stages (B220high/CD43). The results are representative of individual mice from four experiments.

FIGURE 3.

Analysis of bone marrow B cell compartments. Bone marrow cells from B6, μMT/lpr, and μMT mice were stained for CD43 and B220, then analyzed by flow cytometry as previously described (15 ). Pro-B cells (B220low/CD43+) are distinguished from later stages (B220high/CD43). The results are representative of individual mice from four experiments.

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Because B cells have been shown to be critical in the induction and propagation of the lupus-like disease in mice lacking functional Fas or FasL (16, 17, 18), we studied the onset of lupus in μMT/lpr mice. At 8–10 wk of age, μMT/lpr mice developed significant lymphadenopathy (a lupus-like symptom that is a feature of lpr mice) that was more severe than in their IgM-sufficient B6/lpr counterparts, which developed this symptom only at later ages (Fig. 4,A). Also, μMT/lpr mice at this age had significantly exacerbated proteinuria (not shown). Further surface staining for lymphoid markers and FACS analysis revealed that the cell outgrowth in μMT/lpr mice was dominated by TCRβ+/B220+ cells (Fig. 4 B). These cells have been shown previously to accumulate in lymph nodes of old and sick lpr mice causing lymphadenopathy. The results therefore suggest that the partial B cell lymphopenia in μMT/lpr mice exacerbates the lupus-like disease in lpr mice, as has been proposed (6).

FIGURE 4.

Lymphoproliferation in μMT/lpr mice. A, Lymphoproliferation was measured by total leukocyte numbers in one mesenteric and two inguinal lymph nodes from each mouse. Each group contained five to eight mice, and the average cell numbers are shown for each group. B, Lymph node cells from the indicated mice at 8–10 wk of age were tested for B220 and TCRβ expression by two-color flow cytometry.

FIGURE 4.

Lymphoproliferation in μMT/lpr mice. A, Lymphoproliferation was measured by total leukocyte numbers in one mesenteric and two inguinal lymph nodes from each mouse. Each group contained five to eight mice, and the average cell numbers are shown for each group. B, Lymph node cells from the indicated mice at 8–10 wk of age were tested for B220 and TCRβ expression by two-color flow cytometry.

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Nuclear Ags are predominant self-Ags targeted by autoantibodies that develop in lpr mice (6, 8, 17). Anti-chromatin reactivity was measured in serum of young and old μMT/lpr mice and compared with that of age-matched B6/lpr, B6, and μMT mice (Fig. 5,A). μMT/lpr mice developed high titers of chromatin autoantibodies, and these titers increased with age even more rapidly than in B6/lpr mice (Fig. 5,A). No direct correlation between levels of serum Ig and anti-chromatin reactivity was found. Other autoantibodies detected in μMT/lpr sera had anti-cardiolipin specificity (40% of the mice; data not shown). In addition, we tested the ability of μMT/lpr mice to produce Abs in response to exogenous antigenic stimulation. Mice were immunized with OVA (a T cell-dependent Ag) and with dextran (a T-independent Ag), and specific serum Ab responses were measured over time. Results in Fig. 5 B show that μMT/lpr mice failed to respond to each of these Ags and were not different from the Fas-sufficient μMT mice, which completely lack mature B cells and serum Abs. It is thus possible that maturation of B cells in μMT/lpr mice is specifically correlated with reactivity to self-Ags.

FIGURE 5.

Ab response in μMT/lpr mice. A, Serum samples from mice with the indicated genetic background were analyzed by ELISA for IgG anti-chromatin. Titers for individual mice and group means are expressed in equivalent units of anti-dsDNA 3H9 mAb as described in Materials and Methods. B, The indicated mice were immunized by OVA-CFA (100 μg/mouse) administered i.p. or with the T-independent Ag dextran (10 μg/mouse) administered i.v. Mice were bled at various times after immunization, and Ab responses were measured by specific ELISAs. Immunization and ELISA for anti-dextran responses were described in Materials andMethods.

FIGURE 5.

Ab response in μMT/lpr mice. A, Serum samples from mice with the indicated genetic background were analyzed by ELISA for IgG anti-chromatin. Titers for individual mice and group means are expressed in equivalent units of anti-dsDNA 3H9 mAb as described in Materials and Methods. B, The indicated mice were immunized by OVA-CFA (100 μg/mouse) administered i.p. or with the T-independent Ag dextran (10 μg/mouse) administered i.v. Mice were bled at various times after immunization, and Ab responses were measured by specific ELISAs. Immunization and ELISA for anti-dextran responses were described in Materials andMethods.

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These data clearly show that the μMT mutation, which completely blocks B cell developmental in otherwise normal mice, can be circumvented, at least in a proportion of B cells, in mice that lack Fas, rescuing B cell function and promoting autoimmunity. In an interesting contrast to our results, Fas-deficient mice with a targeted deletion of the entire JH locus (JhD/JhD, lpr/lpr mice) remain devoid of B cells and have ameliorated lupus disease (17). The critical difference between JhD/JhD, lpr/lpr and μMT/lpr mice is that in the μMT mice surface Ig expression can be rescued by differential RNA splicing or isotype class switching to heavy chain genes downstream of Cμ, whereas in JhD/JhD, lpr/lpr mice the JH defect is not correctable. Several studies have shown that membrane expression of Ig-δ (19) or transgenic Ig-γ2b (20) chains can support B cell development in the bone marrow. Thus, defective B cells generated as a consequence of the μMT mutation might be rescued by isotype switching, as we have shown in μMT/lpr mice (Fig. 2), which is probably a limiting factor in the survival of these cells. Unlike IgH-γ and IgH-α expression found in μMT/lpr spleen (Fig. 2 D), we were not able to detect significant expression of sIgD in spleen or bone marrow of μMT/lpr mice (not shown). This, however, could be due to the low frequency of IgD-expressing cells and the process of isotype switching in these cells. While we cannot exclude the possibility that isotype switching occurs in μMT B cells, our results clearly show that in a Fas-deficient environment such B cells can mature and differentiate to plasma cells and participate in an autoimmune response.

Breakdown of self-tolerance in mice deficient of functional Fas (MRL-lpr/lpr or lpr/lpr) results in lupus-like disease, which is characterized by production of high titers of autoantibodies such as anti-nuclear Ags (DNA, histone), rheumatoid factors, and cardiolipin (6, 8, 17, 21). Early studies used Ig-Tg/lpr mice to study the role of Fas in central B cell tolerance and showed that Fas-deficient B cells bearing the Tg specificity were efficiently eliminated in the bone marrow or the periphery (6, 21). However, a very small population of variant B cells with different specificities was still generated in these mice, underwent activation and isotype switching, and was able to produce high titers of anti-DNA Abs (6). Similarly, in μMT/lpr (Fig. 2,A) a relatively small subpopulation of class-switched B cells, visible in spleen and lymph nodes, gave rise to a severe autoimmune Ab response (anti-chromatin, Fig. 5,A and anti-cardiolipin; not shown). In both experimental systems the ability of these cells to develop exclusively depends on lack of Fas functionality. It was previously suggested that these autoreactive B cells represent a limited B cell repertoire with anti-nuclear specificities, which in the absence of suppression mediated by nonautoreactive clones exacerbates the disease (6, 22). Supporting this hypothesis are recent findings showing that lack of functional Fas promoted selection of anti-dsDNA B cells into the follicle and production of autoantibodies in anti-dsDNA Ig/Tg MRL/lpr mice (23). In addition, the absence of secreted IgM accelerates IgG autoantibody production and lupus in lpr mice (24). Consistent with all the above is our observation that μMT/lpr mice have high titer of anti-chromatin IgG, which develops more readily than in the B6/lpr control mice (Fig. 5,A), and their failure to respond to external Ags upon immunization (Fig. 5 B). In addition, preliminary experiments of isoelectric focusing separation of μMT/lpr serum IgG suggested the appearance of oligo-monoclonal IgG in these mice (data not shown). Thus, as has been proposed for MRL-lpr/lpr and lpr/lpr mice, a B cell population with a limited repertoire of self-reactivity (anti-histone and rheumatoid factor) may be expanded in the periphery in a T cell-dependent (16, 25) or -independent (6, 26) mechanism, thereby facilitating the development of autoimmunity in μMT/lpr mice. It is also possible that such autoreactive cells are generated in Fas-sufficient mice, but undergo Fas-mediated apoptosis at the outer T cell zone, thus preventing their migration into primary follicles, a paradigm that has previously been suggested by Goodnow and colleagues (10).

An interesting question raised by the present data is how the lack of a functional Fas/FasL pathway facilitates the development of B cells in μMT/lpr mice. A possible explanation is that Fas has a direct role in the elimination of defective and self-reactive B cell precursors. Fas is known to be expressed by developing B cells in the bone marrow (27, 28). The in vivo source of FasL that may regulate the μMT B cells is not clear. Possible sources include circulating T lymphocytes (29) and activated B cells (30). Our bone marrow analysis revealed that the μMT-induced block in B cell development is not substantially circumvented in μMT/lpr mice. Along this line of explanation, our results suggest that in the absence of Fas-FasL signaling the developmental block imposed by the lack of μ-chain, although substantially intact, is rendered somewhat “leaky,” allowing slow accumulation of rare variant B cells in the periphery. Similar accumulation of variant B cells in peripheral organs was described in different B lymphopenic mice (31, 32, 33). However, unlike in μMT mice, the lack of Fas functionality in μMT/lpr mice may allow the specific expansion and survival of autoreactive B cells, perhaps due to the availability of dysregulated, Fas-deficient T cells.

Alternatively, as class switching may rescue μMT B cells, it is also possible that B cell Ag receptor expression and B cell maturation may first occur in the peripheral lymphoid organs of μMT/lpr mice, for example, during the germinal center reaction. It has been suggested that B cells at early stages of development can be recruited to the germinal centers (34, 35) and may undergo Ig gene rearrangements during a germinal center response (reviewed in Ref. 36). Furthermore, ongoing gene rearrangements in germinal center sIg+ cells may generate sIg+ B cells (34, 35), which phenotypically are similar to pre-B cells or μMT B cells found in peripheral organs (Fig. 2 A) that normally fail to participate in an immune response.

Interestingly, a recent study showed that in lpr mice, a sIg B cell generated by a stop mutation in its expressed Ig-κ gene was able to subsequently participate in the germinal center response, acquire self-reactivity through new κ gene rearrangement, and contribute to the autoimmune response (25). The results described here suggest that Fas-deficient mice may be particularly permissive for participation of Ig B cells in Ab responses. Such permissiveness may be accommodated by the lpr T cells, which had been shown to be important in the induction of murine lupus and isotype switching in lpr B cells (37). It is possible, though, that these lpr T cells may be actively involved in the recruitment and activation processes of self-reactive B cells in μMT/lpr mice. Our results showing high frequency of germinal center B cells in μMT/lpr, but not in μMT, mice (GL-7+; Fig. 2 C), are in agreement with this hypothesis. Taken together, our results show that lack of μH expression can be circumvented in mice deficient of functional Fas/FasL by isotype switching, which can take place in the bone marrow or in the periphery, a process that can be regulated by different mechanisms as discussed above. However, the small number of mature cells found in the μMT/lpr mouse suggest that this process is inefficient and may be limited by the availability of T cell activation and the selection process.

We thank Dr. D. Serreze for providing serum samples for analysis and Dr. E. Toubi for screening serum samples for autoantibodies.

1

This work was supported by grants from the Arthritis Foundation (to D.N.) and the National Institutes of Health (RO1GM44809, to D.N.) and by the Loewengart Research Fund (to D.M.). Part of this work was performed at the National Jewish Medical and Research Center (Denver, CO).

3

Abbreviations used in this paper: FasL, Fas ligand; sIgM, surface IgM.

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