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The Major Murine Systemic Lupus Erythematosus Susceptibility Locus Sle1 Results in Abnormal Functions of Both B and T Cells¹

Eric S. Sobel^*, Minoru Satoh^*, Yifang Chen, Edward K. Wakeland and Laurence Morel^2,*,

^* Division of Rheumatology and Clinical Immunology, Department of Medicine, and Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, FL 32610; and Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75235

	Abstract

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Sle1 is a major susceptibility locus in the NZM2410 murine model of systemic lupus erythematosus. When isolated on a C57BL/6 background in the B6.Sle1 congenic strain, Sle1 results in the production of high levels of anti-chromatin IgG Abs, histone-specific T cells, and increased B and T cell activation. We have shown by mixed bone marrow chimeras with allotypic markers that Sle1 is expressed in B cells. Using the same technique, we now show that it is also expressed in T cells. To assess whether Sle1 results in intrinsic defects in B or T cells, we have bred the µMT and Tcr^-/- mutations onto B6.Sle1 resulting in the absence of circulating B cells and T cells in B6.Sle1.µMT and B6.Sle1.Tcr^-/-, respectively. The immune phenotypes in these two strains were compared with that of B6.Sle1 and B6.µMT or B6.Tcr^-/-. Sle1-expressing B cells broke tolerance to chromatin in the absence of T cells, as shown by high levels of anti-ssDNA IgM Abs in B6.Sle1.Tcr^-/- mice, and had an increased expression of activation markers. Conversely, increased expression of activation markers and increased cytokine production were observed in Sle1-expressing T cells in the absence of B cells in B6.Sle1.µMT mice. However, the production of IgG antinuclear Abs required the presence of both T and B cells. These experiments showed that Sle1 expression results in both B and T cells intrinsic defects and demonstrate that the documented involvement of each cell compartment in the production of anti-chromatin Abs corresponds to genetic defects rather than bystander effects.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
One of the hallmarks of systemic lupus erythematosus (SLE),³ both in humans and mice, is the loss of tolerance to nuclear Ags, resulting in the production of antinuclear autoantibodies (ANA), including anti-chromatin and anti-DNA (1). It has been clearly demonstrated that functional defects in both T cells and B cells were necessary for ANA production (2, 3, 4). We have used the NZM2410 mouse (5) to identify the genetic loci responsible for SLE susceptibility and to characterize their contribution to the systemic autoimmune pathology (6). Among the four loci that we have identified (7), the strongest one was Sle1 on telomeric chromosome 1, mapping to a region that has been implicated in all human linkage studies of SLE and in multiple murine studies (8, 9, 10, 11, 12).

The characterization of the phenotypes of B6.Sle1 mice, a congenic strain that carries the Sle1 NZM susceptibility interval on a C57BL/6 (B6) background (13), has shown that this locus is associated with a loss of tolerance to chromatin. B6.Sle1 mice produce large quantities of autoantibodies directed against chromatin, specifically against H2A/H2B/DNA subnucleosomes, and generate histone-specific T cells (14, 15). The H2A/H2B/DNA particle constitutes the most exposed chromatin epitope. Autoantibodies with that specificity are probably the first to be generated as a result of a loss of tolerance to chromatin (16). Sle1-expressing lymphocytes have a spontaneously activated phenotype, as indicated by an increased expression of B7-2 and CD69 on B and T cells, respectively (15, 17). Despite a large amount of autoantibodies and abnormal lymphocyte activation, B6.Sle1 mice do not develop clinical nephritis. In many respects, this strain reflects what is seen in drug-induced lupus, which is also characterized by H2A/H2B/DNA autoantibodies in the absence of renal disease (18).

By combining the Sle1, Sle2, and Sle3 loci into a triple congenic strain, we have shown that Sle1 is a necessary step at the root of the SLE pathogenic cascade (19). In addition, we have identified a series of NZW-derived negative epistatic modifiers of Sle1. The most potent one, Sles1, specifically turns off all the Sle1 immune phenotypes, leading to the suppression of the entire autoimmune pathological process triggered by Sle1 interactions with other Sle loci (20). In summary, Sle1 is a potent SLE susceptibility locus whose primary defect is a break of tolerance to nuclear Ags. Because of its place at the initiation of the autoimmune pathogenic process, and the existence of Sle1 suppressor loci, the identification of the Sle1 gene(s) and their functional characterization constitute an important piece in solving the puzzle of SLE pathogenesis.

This study addresses which cellular compartment is affected by Sle1 expression. This is an important question to understand how this locus contributes to SLE pathogenesis, and to narrow down the field of candidate genes in our ongoing effort toward Sle1 gene identification. By using mixed bone marrow-mixed chimeras, we have demonstrated that Sle1 is functionally expressed in B cells (17). In this study, using mixed chimeras again, we show that Sle1 is also functionally expressed in T cells. However, these functional defects could result from Sle1-B cells effects on T cells or conversely from Sle1-T cell effects on B cells. To evaluate Sle1 contribution to intrinsic B cell defects, we combined Sle1 with the Tcr^-/- targeted mutation that eliminate peripheral T cells (21, 22). Analysis of B6.Sle1.Tcr^-/- mice showed that Sle1-expressing B cells are abnormal independently of T cell help, producing anti-ssDNA Ab and increased levels of activation markers comparable to that of intact B6.Sle1 mice. Conversely, to evaluate Sle1 contribution to intrinsic T cell defects, we bred Sle1 to the Igh-6 or µMT mutation that eliminates peripheral B cells (23), and showed that increased activation is an intrinsic defect of Sle1-expressing T cells, independently of the presence of B cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

C57BL/6J (B6), C57BL/6.Thy1^aIgh^aGpi^a (B6.TC), B6.129S2-Igh-6^tm1Cgn (B6.µMT), and C57BL/6J-Tcra^{tm1 Mom} (B6.Tcr^-/-) mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and subsequently bred at the University of Florida (Gainesville, FL). The production of the B6.Sle1 strain has been previously described (13). Mice used in the bone marrow chimera experiments were maintained in conventional housing. B6.Sle1.µMT, B6.Sle1.Tcr^-/-, B6.µMT, B6.T^-/-, and B6, B6.Sle1 control mice were produced and maintained in specific pathogen-free conditions. B6.Sle1.µMT and B6.Sle1.Tcr^-/- were produced by intercrossing B6.Sle1 and B6.µMT or B6.Tcr^-/-. Homozygozity at the Sle1 interval was selected by PCR genotyping on tail biopsies with the D1Mit47, D1Mit15, and D1Mit17 markers, as previously described (19). The absence of B cells or Tcr⁺ cells was first screened by PCR for the presence of the neomycin resistance marker (forward primer: CTT GGG TGG AGA GGC TAT TC, reverse primer: AGG TGA GAT GAC AGG AGA TC), then by flow cytometry on PBLs stained with anti-CD19 or anti-CD3, respectively. Positive controls B6.Sle1.µMT and B6.Sle1.Tcr^-/- were included for each typing session. The level of B220⁺ PBL was <1% in B6.Sle1.µMT mice, while B6.Sle1.Tcr^-/- had 5–7% CD3⁺ PBL. We have verified, as it has been shown previously (21), that these CD3⁺ cells were mostly to or TCR T cells (see Results). All mice were aged up to 12 mo and an approximately equal number of males and females were used. Mice carrying the Tcr^-/- have a reduced life span, mostly due to inflammatory bowel disease (24). Accordingly, these mice and their controls were used at a younger age (7–10 mo) than the µMT mutants. Mutant and control mice used in a same experiment were aged concurrently.

Production of chimeras

Chimeric mice were prepared as previously described (17). Briefly, young female mice were placed in sterilized tap water and given lethal doses of gamma radiation (two doses of 525 rad separated by 3 h) a day before cell transfer. Each mouse received a total of 10⁷ T and B cell-depleted bone marrow by tail vein injection from young sex-matched donors. For mixed chimeras, the mice received a mixture of donor marrow at a ratio of 3:1 in favor of the B6.TC donor. This has been found necessary to achieve adequate peripheral reconstitution. The mice were aged for 1 year with monthly bleeds starting at 5 mo of age.

Serology

Sera from B6.Sle1, B6.Sle1.Tcr^-/-, B6.T^-/-, and B6 mice were screened for anti-dsDNA IgG, and anti-ssDNA IgM Ab between 7 and 10 mo of age by ELISA at 1/100 serum dilution as previously described (14). For interplate comparisons, a serial dilution of a B6.Sle1 serum was included on each plate to construct a standard curve. A value of 100 U was assigned to the OD reading corresponding to the 1/100 dilution of this standard sample. Anti-chromatin Ab was screened similarly as for anti-DNA, but in addition, the isotypic profile was evaluated with secondary Abs from Southern Biotechnology Associates (Birmingham, AL). A serial dilution of an MRL/lpr serum was used as standard, with a dilution of 1/250 corresponding to 1000 U. The limit of detection of the assay was 1/250,000 or 1 U. Serum samples corresponding to <1 U were deemed negative. Total serum IgM and IgG were also assayed on 9-mo-old sera by capture ELISA as previously described, at a 1/200,000 serum dilution (14, 25).

ANA immunofluorescent assay

Immunofluorescence assays for ANA detection were performed as described previously (26). Briefly, Hep-2 cell-coated slides were incubated with sera at a 1/40 dilution for 30 min at room temperature, washed with 1% BCS in PBS, and developed with a 1/40 solution of FITC-conjugated anti-mouse IgG (-chain specific) or anti-mouse IgM (µ-chain specific; Southern Biotechnology Associates), and then viewed with a fluorescent microscope. Sera used in this assay were collected from 8- to 10-mo-old mice.

Flow cytometry

Splenocytes were depleted of RBCs with 0.83% NH₄Cl, and single-cell suspensions were prepared. FACS analysis was performed as previously described (27). All primary Abs (CD90.1 (OX-7), CD90.2 (30-H12), CD45R/B220 (RA3-6B2), CD3 (145-2C11), TCR (GL3), TCR (H57-597), CD22 (Cy34.1), CD23 (B3B4), CD24 (M1/69), CD25 (7D4), CD4 (RM4-5), CD69 (H1.2F3), and CD86/B7.2 (GL1), CD44 (IM7), CD62L (MEL-14), IgM^b (Igh6), and IgD^b (Igh5)) were purchased from BD PharMingen (San Diego, CA) and used at pretitrated dilutions. Cells were first blocked on ice with staining medium (PBS, 5% horse serum, 0.05% sodium azide) containing 10% rabbit serum. Cells were then stained with optimal amounts of conjugated primary Abs diluted in staining medium for 30 min. After two washes, biotin-conjugated Abs were revealed using streptavidin-Quantum red (Sigma-Aldrich, St. Louis, MO). Cell staining was analyzed using a FACScan (BD Immunocytometry Systems, Mountain View, CA). Dead cells were excluded based on scatter characteristics, and at least 10,000 events were acquired per sample. Positive staining for each given primary Ab was determined relative to the isotype controls, all purchased from BD PharMingen. Analyses were conducted on 7- to 12-mo-old mice.

Intracellular cytokine analysis

Anti-IL-2, anti-IFN-, and anti-IL-4 were purchased from BD PharMingen and used for intracellular staining according to the recommended protocols with the Golgi Plug kit. For the IL-2 and IFN- staining, RBC-depleted splenocytes were activated with PMA and ionomycin for 4 h at 37°C in RPMI at a density of 10⁶ cells/ml. After fixation and permeabilization, cells were then stained with FITC-conjugated anti-CD4 Ab and PE-conjugated anti-IL-2 or anti-IFN- in the presence of FcR block (2.4G2). Positive staining was determined relative to the anticytokine isotype control. For Il-4, 10⁵ splenocytes were first activated on plate-bound anti-CD3 with anti-CD28 (1 µg/ml) and murine IL-2 (10 ng/ml) and murine IL-4 (50 ng/ml) for 2 days at 37°C in RPMI, followed by an activation with IL-2 and IL-4 only for 3 days. The cells were then activated with PMA and ionomycin and stained as described for the two other cytokines.

Histone-specific T cell assay

T cell proliferation assays in response to histone were adapted from previous work (15). RBC-depleted splenocytes from 9- and 12-mo-old B6.Sle1.µMT, B6.Sle1, B6.µMT, and B6 were treated with anti-B220-coated Dynabead magnetic beads (Dynal Biotech, Lake Success, NY) according to the manufacturer’s instructions, leading T cells with >90% purity. Irradiated APC (2000 rad, 5 x 10⁵/well) provided by RBC-depleted B6.T^-/- splenocytes (from 2- to 3-mo-old mice) were incubated with total histone (Boehringer Mannheim, Indianapolis, IN) at 1 µg/ml in PBS for 1 h at 37°C, washed, then cocultured with T cells (5 x 10⁵/well) in 200 µl cultures in serum-free HL-1 medium (Biowhittaker, Walkersville, MD). Negative and positive controls were provided with APC/T cell cocultures with no Ag added, and with APC/T cells cultured with Con A (Sigma-Aldrich) or anti-CD3 mAb (17A2) (BD PharMingen) at 1 µg/ml, respectively. Supernatants were collected after a 48-h culture and assayed for IFN- with an OptiEIA kit (BD PharMingen) according to the manufacturer’s instructions. Mann-Whitney U tests were performed between groups after subtracting for each mouse the amount of IFN- produced in absence of stimulation.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sle1 is functionally expressed by T cells

It has previously been reported that Sle1 is functionally expressed by B cells (17). These experiments also suggested that CD69 expression was preferentially up-regulated in Sle1-bearing CD4⁺ T cells, but the number of informative mice was too small to achieve statistical significance. The experiment has subsequently been repeated, and the results displayed in Fig. 1. Consistent with the earlier results, an increased percentage of CD4⁺ T cells expressing CD69 was seen in the spleen of mice receiving B6.Sle1 bone marrow. Mice receiving bone marrow from a combination of B6.TC and B6 bone marrow had consistently lower levels of expression, and there was no difference based on the origin of the donor marrow (p = 0.45 by paired t test). This contrasted sharply with the results for mice receiving a combination of B6.TC + B6.Sle1 bone marrow. Cells of normal B6.TC origin expressed levels of CD69 comparable to the negative control group, while CD4⁺ T cells of B6.Sle1 origin had levels comparable to the positive control group. The differences in expression were statistically significant by the paired t test (p = 0.02). Overall, it is concluded that Sle1 is functionally expressed on CD4⁺ T cells, leading to an increased percentage of activated cells in the spleen.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Sle1 is functionally expressed by CD4+ T cells. B6 mice were lethally irradiated and reconstituted with the combination of T cell-depleted bone marrow as indicated. Twelve months later, the mice were killed, and spleen cells examined by flow cytometry. After gating on the lymphocyte population and either CD90.1+ (B6. TC origin) or CD90.2+ (B6 or B6.Sle1 origin) events, the percentage of CD4+ T cells positive for CD69 was determined. Individual values and mean ± SD are shown for each group.

To assess the impact of Sle1 on B cell functions independently from T cell help, we compared B cell phenotypes between B6.Sle1.Tcr^-/- and B6.Sle1 mice. As a control for the effect of the Tcr^-/- mutation itself, we also compared B6.Tcr^-/- to B6. As was previously reported on a BALB/c background (21), the absence of T cell does not result in a drastic reduction in spleen weight, mostly due to B2 cell expansion (Table I). Interestingly, the percentage of T cells in the spleen was significantly higher in B6.Sle1.TCR^-/- than B6.TCR^-/- (p = 0.004). This increase involved all CD3⁺ subsets (NK1.1⁺, ⁺, TCR) which did not differ in proportion from B6.TCR^-/- (data not shown).

Sle1.Tcr

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View larger version (19K): [in this window] [in a new window]   FIGURE 2. Sle1 affects B cells phenotypes independently of the presence of T cells. Each data point represents a single 7- to 10-mo-old mouse and horizontal bars show the average value in each group. A, Anti-ssDNA IgM production is significantly higher (t tests) in Sle1-expressing mice than in B6 controls, in both intact mice (p = 10-5) and in the absence of T cells (p = 0.001). The absence of T cells also contributes to anti-ssDNA Ab since B6.Sle1. Tcr-/- produce significantly more autoantibodies than B6.Sle1 mice (p < 0.01). B, Anti-chromatin Ab production in B6.Sle1. Tcr-/- and B6.Tcr-/- median. Median unit values, 30 mice per strain tested at 7 mo of age.

View larger version (21K): [in this window] [in a new window]   FIGURE 3. Immunofluorescence pattern of sera collected from B6.Sle1 (top panels) or B6.Sle1.Tcr-/- mice (bottom panels) detected with an anti-IgG (left panels) or anti-IgM (right panels) FITC-conjugated Ab. Photographs of representative samples from >10 in each group are shown.

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View larger version (23K): [in this window] [in a new window]   FIGURE 4. Surface expression of B cell activation markers is significantly different in Sle1-expressing B cells in the presence or absence of T cells. Representative histograms for B6.Sle1 in filled gray and B6 in open histograms, wild type in the left column, and Tcr-/- in the right column. All histograms were gated on B220+ splenocytes. Ten 6- to 7-mo-old mice were tested per strain, and differences between B6.Sle1 and B6 or B6.Sle1. Tcr-/- and B6.Tcr-/- reached a significance level of at least p < 0.05 by t test.

The µMT mutation resulted in a dramatic reduction in spleen size in both B6.Sle1.µMT and B6.µMT mice (Table I). We did not observe any difference in survival, spleen weight, or lymphocyte populations between these two strains (Table I). Pathogen serological screen and oropharyngeal and fecal bacterial cultures did not show any difference between the µMT and normal mice. Moreover, survival was comparable among all four strains, indicating the absence of a major interaction between the Sle1 locus and the µMT mutation.

We have shown that Sle1 is associated with a significant increase of T cell activation markers such as CD69 and CD44 (15, 27). In this study, we showed that this increased activation is a result of an intrinsic defect of Sle1-expressing T cells. The percentage of CD4⁺CD62L^lowCD44^high splenocytes was similar between B6.Sle1.µMT and B6.Sle1 mice, and the difference with either B6.µMT or B6 was significant (Fig. 5). Similar results were obtained for CD69 expression (data not shown). No significant increase of either CD44 or CD69 was observed in B6.µMT CD4⁺ T cells, indicating that the µMT mutation by itself does not induce significant changes in T cell activation.

View larger version (12K): [in this window] [in a new window]   FIGURE 5. Sle1 induces a greater accumulation of CD4+CD62LlowCD44high T cells in the spleen independently of the presence of B cells. t tests showed a significant difference both between B6.Sle1 and B6 (p = 0.03) and between B6.Sle1.µ MT and B6.µMT (p < 0.01).

View larger version (11K): [in this window] [in a new window]   FIGURE 6. Histone-specific T cells are induced both by Sle1 expression and the absence of B cells. In response to histone stimulation, T cells from B6.Sle1 mice produced significantly more IFN- than B6 mice (p = 0.03), and there is no significant difference between B6.Sle1 and B6.Sle1.µMT T cells. However, T cells from B6.µMT mice produced significantly more IFN- than B6 mice (p = 0.001) in response to histone.

View larger version (34K): [in this window] [in a new window]   FIGURE 7. IL-2, IFN-, and IL-4 production in stimulated CD4+ T cells, showing B6.Sle1 (gray histograms) compared with B6 (open histograms) on the left column, and B6.Sle1. µMT (gray histograms) compared with B6.µMT (open histograms) on the right column. Values on the top of each graph indicate the percentage of cytokine positive cells relative to the isotype control. Representative histograms of three experiments conducted with two 6-mo-old mice for each strain.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Although our bone marrow chimera experiments have shown that Sle1 is functionally expressed in both B and T cells, they did not allow to address whether expression in both cellular compartments was necessary to produce Sle1 phenotypes. Alternatively, Sle1 could confer independent intrinsic defects in either B or T cells, or both. To address this question, we combined Sle1 with mutations engineered to eliminate peripheral B cells (µMT) or T cells (Tcr^-/-) to produce B6.Sle1.µMT and B6.Sle1.Tcr^-/- mice, respectively. The µMT and Tcr^-/- mutations have been used extensively to characterize the role of the corresponding cellular compartments in the immune response in general, and in autoimmune diseases in particular. µMT is characterized by the complete absence of mature B cells and serum Abs, but has not been associated with specific pathology in specific pathogen-free conditions. Tcr^-/- mice carry nearly normal levels of T cells (21), produce class-switched Abs (31), and develop germinal centers containing CD4⁺TCRT cells (32). The Tcr^-/- mutation is associated with inflammatory autoimmune manifestations, such as inflammatory bowel disease (24), most likely due to the absence of regulatory T cells. Our experiments included both mutants and intact mice, with and without Sle1, allowing detection of phenotypes associated with either Sle1, or the µMT and Tcr^-/- mutations.

Analysis of B6.Sle1.Tcr^-/- mice demonstrated that Sle1 induced autoimmune phenotypes in B cells in the absence of conventional T cell help. B cells in B6.Sle1.Tcr^-/- mice lost tolerance to ssDNA and chromatin, resulting in IgM anti-ssDNA and anti-chromatin Abs, and elevated total serum IgM. Anti-chromatin IgG Abs were found at very low levels in both B6.Sle1.Tcr^-/- and B6.Tcr^-/-, but with a different isotype distribution pattern. B6.Sle1.Tcr^-/- was associated with more IgG1 anti-chromatin Abs while B6.Tcr^-/- was more skewed toward T-independent IgG3. This pattern did not correspond to the anti-chromatin Abs found in T cell intact mice, which are overwhelmingly IgG2a and IgG2b. Increased IgG1 autoantibodies in B6.Sle1.Tcr^-/- may result from Sle1 expression in non- T cells present in these mice. This population was increased 2-fold in B6.Sle1.Tcr^-/- as compared with B6.Tcr^-/-. Phenotypes of these Sle1-expressing T cells are currently under investigation. Expression of surface markers also showed that the activated phenotype associated with Sle1 in B cells was independent from the presence of conventional T cells. An intrinsic loss of tolerance to nuclear Ags has been shown in NZW-derived B cells by transferring bone marrow-derived pre-B cells in immunodeficient mice (33). We have shown that Sle1 is NZW-derived (13), and we propose that Sle1 plays a major role in this B cell defect.

These autoimmune phenotypes of the B6.Sle1.Tcr^-/- mice could not be attributed to the Tcr^-/- mutation by itself, because there was no significant difference between B6.Tcr^-/- and B6 mice. In fact, B6.Tcr^-/- mice did not present the generalized autoimmunity that was described in BALB/c.Tcr^-/-, although they showed a similar expansion of the B cell compartment (30). A similar absence of autoimmune manifestations directly associated with the Tcr^-/- mutation was also recently observed on a BXSB background (34). This discrepancy is likely due to the different genetic backgrounds, which are known to significantly affect the phenotypic expression of targeted mutations in general (35), and also in SLE susceptibility loci (36).

Although class-switching occurs in Tcr^-/- mice (31), B6.Sle1.Tcr^-/- mice failed to produce IgG Ab levels comparable to that of B6.Sle1. More importantly, IgG antinuclear Abs were detected either at very low levels by ELISA, or not at all by immunofluorescence. Therefore, conventional T cell help is necessary for this Sle1 phenotype. It is not clear at this point whether this help requires Sle1 to be expressed in the T cells. Indeed, it has been shown recently that although lupus nephritis requires CD4⁺ T cells in the BXSB.Yaa model, Yaa expression in these T cells is not required (34). Bone chimera experiments have been initiated to answer this question for the Sle1 locus.

T cells play a key role in the loss of tolerance to nuclear Ags (2, 4), and we have shown that B6.Sle1 mice accumulate histone-specific T cells (15). The chimera experiments presented in this study showed that Sle1 is functionally expressed in T cells. However, the increased activation levels of Sle1-expressing T cells could result, at least in part, from cotransferred Sle1-expressing hemopoietic cells. B cells are unfortunately the only cell types to which effects on T cells can be directly addressed, thanks to the availability of genetically engineered mice lacking B cell expression. Indeed, B cells play an essential role beyond their production of autoantibodies in autoimmune diseases such as diabetes (37) and SLE (38), most likely through their Ag presentation function (39, 40, 41, 42). More specifically, B cells are essential in promoting T cell activation and expansion in the MRL/lpr model (43). Nevertheless, we found that Sle1-expressing T cells express a higher level of activation markers, and produce more cytokine in response to stimulation, even in the absence of B cells. These results demonstrate a direct effect of Sle1 on T cell functions. It should be noted that both IFN- and IL-4 production are increased by Sle1, which indicates that Sle1 does not participate in a polarization toward Th1 or Th2. A similar conclusion was reached from the absence of isotypic skewing in Sle1 IgG repertoire (our unpublished observations).

Another Sle1-associated T cell phenotype, proliferation in response to histone Ags, was not informative in the context of the µMT mutation, because the mutation itself was associated with T cells proliferating to histone. Interestingly, cytokine production was markedly increased in T cells in the absence of B cells, even without stimulation. B cells are not required for self tolerance of T cells in the hen egg lysosome transgenic system (44). However, it has been shown that B cells regulate dendritic cells capacity to promote IL-4 production, leading to alterations of Th2 responses in B6.µMT mice (45). To our knowledge, T cell loss of tolerance to nuclear Ags has not been associated with the absence of B cells. However, it has been shown recently that CD4⁺CD25⁺ regulatory T cells are markedly reduced in the absence of B cells (46). Absence of regulatory T cells could explain the presence of histone-specific T cells and increased effector functions in µMT mice. This possibility is currently under investigation.

In parallel with the experiments presented in this study, we have shown that Sle1 corresponds to at least three loci, Sle1a, Sle1b, and Sle1c (27). Each of these loci independently result in the production of anti-chromatin Abs, with Sle1b being associated with an earlier onset, higher penetrance, and higher levels of Abs. Comparisons of lymphocyte surface markers showed Sle1a mostly affected T cell phenotypes while Sle1b mostly affected B cell phenotypes. In addition, we have proposed that mutations in the complement receptor 2 gene, Cr2, are responsible for the Sle1c phenotypes (47). Taken together, these results suggest that Sle1 effects on B and T cells may be mediated by different loci. It is tempting to speculate that Sle1b and Sle1c will be expressed in B cells, while Sle1a will be expressed in T cells. Additional experiments such as bone marrow chimeras or breeding of the µMT and Tcr^-/- mutations to the individual loci will be required to provide a definitive answer.

	Acknowledgments

 
We thank Dr. Byron Croker for stimulating discussions, and Elisabeth Basco, Kim Blenman, Guangling Huang, Raquel Baert, and Krista Behney for excellent technical help.

	Footnotes

 
¹ This work was supported by grants by the National Institutes of Health (R01 AI043454 and R01 AI45050).

² Address correspondence and reprint requests to Dr. Laurence Morel, Department of Medicine, University of Florida, Box 100275, Gainesville, FL 32610-0275. E-mail address: morel{at}ufl.edu

³ Abbreviations used in this paper: SLE, systemic lupus erythematosus; ANA, antinuclear autoantibodies.

Received for publication September 14, 2001. Accepted for publication June 26, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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