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Genetic Dissection of Systemic Lupus Erythematosus Pathogenesis: Evidence for Functional Expression of Sle3/5 by Non-T Cells¹

Eric S. Sobel^2,*,, Laurence Morel^*,, Raquel Baert^*, Chandra Mohan, Joel Schiffenbauer^3,*, and Edward K. Wakeland

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

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
On the non-autoimmune C57BL/6 (B6) background, the chromosome 7-derived lupus susceptibility loci Sle3 and Sle5 have been shown to mediate an elevated CD4:CD8 ratio with an increase in activated CD4⁺ T cells, decreased susceptibility to apoptosis, and a break in humoral tolerance. Development of subcongenic strains has subsequently shown that the elevated CD4:CD8 ratio is due to Sle3 but that both loci contribute to the development of autoantibodies. To elucidate the functional expression patterns of these loci, adoptive transfer experiments were conducted. All possible combinations of bone marrow reconstitution, including syngenic, were conducted between the congenic B6 and B6.Sle3/5 strains. It was found that the Sle3/5 locus was functionally expressed by bone marrow-derived cells, but not by host cells, and that the elevated CD4:CD8 phenotype could be reconstituted in radiation chimeras. Using Ly5-marked congenic strains and B6 host mice, additional experiments surprisingly demonstrated that the elevated CD4:CD8 ratio was neither an intrinsic property of the T cells nor of single positive thymocytes. Allotype-marked chimeras indicated that autoantibody production by B cells was also an extrinsic property, as shown by the fact that B cells without the Sle3/5 interval contributed to autoantibody production. These experiments strongly suggest that a gene within the B6.Sle3/5 interval was expressed by a bone marrow-derived, nonlymphocyte population in the thymus and periphery and was affecting T cell selection and/or survival.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The development of genomewide scans has provided new tools for dissecting the pathogenesis of multigenic diseases (1, 2, 3, 4). Several groups have used this approach to identify the positions of loci contributing to lupus susceptibility. Thus far, 22 genomic intervals associated with susceptibility to systemic lupus erythematosus (SLE)⁴ or to SLE-related phenotypes have been identified by linkage analyses in the NZB/W F₁ model and its derivative NZM2410 (5, 6). Our group has concentrated on the NZB/NZW-derived NZM2410 strain (7), in which we have identified three major genomic intervals linked to early onset glomerulonephritis (4). Each of these SLE susceptibility intervals was subsequently transferred individually onto the non-autoimmune C57BL/6 genome using marker-assisted selection to produce "speed congenics" (8, 9). Two of the most critical regions are on chromosomes 1 and 7. B6.Sle1 (bearing Sle1 on chromosome 1 and previously called B6.NZMc1) mice spontaneously produced IgG autoantibodies specific for subnucleosome components of chromatin. They also developed spontaneous autoreactive T cells responding to histone epitopes (10, 11) and an increased expression of the early activation marker CD69 (11). The B6.Sle3/5 strain (bearing Sle3 and Sle5 on chromosome 7 and previously called B6.NZMc7) had an elevation of the CD4:CD8 ratio, particularly in the spleen (12). In vitro, CD4⁺ T cells showed increased resistance to apoptosis (12). In addition, loss of tolerance to chromatin and dsDNA has been a feature (12). Compared with Sle1, the autoantibody titers to subnucleosome structures were of lower titer and penetrance but with more evidence of epitope spreading, and a small percentage of these mice developed clinical glomerulonephritis (12). Two subinterval congenics were subsequently derived. It was found that Sle3 was responsible for the elevated CD4:CD8 ratio and the relative resistance to apoptosis but that both intervals contributed to the development of humoral autoimmunity (13). The critical nature of both loci was exemplified by the bicongenic strain B6.Sle1.Sle3/5, which showed a much more robust phenotype, including the development of glomerulonephritis (14, 15).

In a previous report, we demonstrated by adoptive transfer of bone marrow that Sle1 was functionally expressed in bone marrow-derived cells and that expression by B cells was essential to break tolerance to nuclear autoantigens and to develop humoral autoimmunity (16). Subsequent experiments indicated that the increased percentage of CD4⁺CD69⁺ T cells was also an intrinsic property of Sle1-expressing T cells (36). In the present report, analogous experiments have been performed with B6.Sle3/5 mice. Similar to Sle1, Sle3/5 was found to be functionally expressed in cells of hemopoietic but not host origin. However, in striking contrast, the elevated CD4:CD8 ratio was not found to be an intrinsic property of Sle3-bearing T cells. When mice were reconstituted with a combination of B6.Sle3 and the congenic B6.Ly5^a strain, both donor populations contributed equally to the elevation. In separate experiments using allotype-marked B6 congenic strains of

mice, informative mixed chimeras that showed an elevated IgG2a antichromatin titer also had significant contributions by both donors. Taken together, these data suggest that Sle3/5 was functionally expressed by bone marrow-derived, non-B cell APC and that these cells were responsible for a break in tolerance.

	Materials and Methods

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Mice

C57BL/6 (B6), B6.SJL-Ptprc^{a Pep3b}/BoyJ (B6.Ly5^a), and B6.PL-Thy1^a/Cy (B6.Thy1^a) mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and bred in our animal colony. The B6.C20 strain (B6.Igh^a) was originally obtained from G. Bosma (Institute for Cancer Research, Philadelphia, PA). The B6.Ly5^a strain is allelic for an isoform of CD45 expressed by all leukocytes. The development of the B6.Sle3/5 congenic strain has been previously described (8, 12). This interval, defined by the markers D7 MIT31 and D7 MIT178, contains the 95% confidence limits for inclusion of Sle3 and is of NZW origin (4, 8). The subcongenic strain B6.Sle3 (B6.Sle3) contains the telomeric 12.5 cM interval of B6.Sle3/5 as defined by the markers D7 MIT69 and D7 MIT147 (13, 17).

Preparation of chimeras

Production of mixed bone marrow chimeras was performed at a 1:1 ratio for the two donors. In all other respects, the chimeras were prepared as previously described (16). In brief, mice were lethally irradiated with two doses of 525 rad gamma irradiation (3 h apart) in a Gammacell 40 ¹³⁷Cs apparatus (Atomic Energy of Canada, Ottawa, Canada). Donor bone marrow cells were depleted of mature T and B cells by a mixture of anti-CD4, anti-CD8, anti-Thy-1.1, anti-Thy-1.2, and anti-I-A^b (clone D3-137.5), followed by complement. Ten million cells were given to each mouse by tail vein injection.

Flow cytometry

Single-cell suspensions of spleen cells, lymph node cells, and thymocytes were prepared, followed by lysis of RBC in 0.83% NH₄Cl. For lymph node cells, only inguinal, axillary, and periaortic lymph nodes were used. Cells were first blocked with staining medium (PBS with 5% FCS and 0.05% NaN₃) supplemented with anti-CD16/32 (clone 2.4G2; American Type Culture Collection, Manassas, VA). All reagents were purchased from BD PharMingen (San Diego, CA), unless otherwise stated. For mice receiving a combination of B6.Igh^a and either B6 or B6.Sle3/5 bone marrow, three-color flow cytometry to characterize T and B cells was performed. For T cell phenotypic determination, directly fluoresceinated anti-CD90.2 (also called anti-Thy 1^b) or anti-CD90.1 (also called anti-Thy 1^a) was used along with CyChrome C-conjugated anti-CD4 (clone H129.19) or anti-CD8 (clone 53-6.7). For the third color, biotinylated anti-CD69 (clone H1.2F3) or anti-CD25 (7D4) was used, followed by avidin-PE. For phenotypic analysis of B cells, the combination of either directly fluoresceinated anti-IgM^a (clone DS1) or anti-IgM^b (clone AF6.78) and CyChrome C-conjugated anti-B220 was used. As the third color, either biotinylated anti-CD69, anti-CD25, anti-CD80 (clone 16-10A1), or anti-CD86 (GL1) was added, again followed by avidin-PE. Cells were fixed in 1% paraformaldehyde.

For mice receiving a combination of B6.Ly5^a and either B6 or B6.Sle3, there was no allelic marker for CD90 or IgM. Instead, the donor origin of T or B cells was identified by expression of CD45. For the a allele of CD45 (also called CD45.1), clone A20 was used. For the b allele (CD45.2), clone 104 was used. Cells were analyzed using a FACScan (BD Biosciences, San Jose, CA). Dead cells were excluded by forward angle and side scatter profiles. At least 20,000 cells were collected per sample. Data were processed by FCS Express version 1.0 (De Novo Software; Thornhill, Ontario, Canada) and figures were created with WinMDI version 2.8 (http://facs.scripps.edu/software.html).

Ig assay by ELISA

Total IgM and IgG2a determinations were performed as previously described (16). The allotype nonspecific IgG2a antichromatin ELISA was performed as described previously for the subnucleosome assay (16), substituting chicken chromatin (18) for the dsDNA-H2A-H2B complex. When necessary, allotype-specific IgM and IgG2a determinations were conducted for the above specificities, also as previously described (16). The a and b allotype titers were compared with each other by using serial dilutions of standard sera from a B6/lpr-Igh^a (allotype a) and a B6/lpr mouse adjusted in dilution to give equivalent OD readings when developed by the allotype-nonspecific reagent.

Statistics

Comparisons between two groups were performed using Student’s t test. Where indicated, a paired t test was used. Comparisons among groups were by one-way ANOVA with pairwise testing of all combinations performed by the Student-Newman-Keuls test. Correlations were determined by the Pearson product correlation technique. Results were calculated with SigmaStat for Windows version 1.02 (Jandel Scientific, San Rafael, CA). All p < 0.05 were considered to be statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
B6.Sle3/5 had elevated CD4:CD8 ratios compared with other congenic B6 strains

One of the main phenotypes of B6.Sle3/5 is an alteration in the CD4:CD8 ratio. Although previous chimera studies have used B6.Thy1^aIgh^aGpi1^a as the normal donor and this strain provides the advantage of simultaneously tracking T and B cells, the Gpi1 gene maps to chromosome 7 at 11 cM within the introgressed NZM2410 interval. Therefore, the single congenic B6.Thy1, B6.Igh^a, and B6.Ly5^a strains were used. The B6.Ly5^a strain has the a allele for the pan-leukocyte marker CD45 and maps to chromosome 1 and does not overlap with Sle1. B6.Thy1^a has a different allele for CD90, which is expressed on T cells and maps to chromosome 9, while B6.Igh^a differs at the Ig H chain locus and maps to chromosome 12. It was still important to verify that the elevated CD4:CD8 ratio phenotype was specific for the chromosome 7 interval derived from NZM2410. To test this, the three congenic strains of B6 mice used as normal donors were compared with B6.Sle3/5 and B6. As shown in Fig. 1, the three congenic strains did not differ statistically from B6 (p > 0.05 by ANOVA) and all were clearly different from B6.Sle3/5 (p = 0.004). Previous studies had also shown that the Ig H chain a allele was not intrinsically predisposed to producing autoantibodies on the B6 background (16, 18).

View larger version (16K): [in this window] [in a new window]   FIGURE 1. B6.Sle3/5 mice showed an elevated splenic CD4:CD8 ratio compared with other B6 congenic strains. Unmanipulated congenic mice of the indicated strains were grown for 6 mo, and the phenotype of the spleen cells was analyzed by FACS analysis.

The first set of adoptive transfer experiments was designed to determine whether Sle3/5 was functionally expressed by cells of bone marrow origin or on a radioresistant host population (or both). To accomplish this, B6.Sle3/5 and B6 host mice were lethally irradiated and given either B6.Sle3/5 or B6 bone marrow. Syngenic reconstitutions of B6 and B6.Sle3/5 were performed to provide negative and positive controls, respectively, for the possible modulating effects of radiation. Results from the analysis of mice at 12 mo of age are shown in Fig. 2. As expected, syngenic reconstitution of normal B6 mice resulted in a low CD4:CD8 ratio in the spleen compared with syngenic reconstitution of B6.Sle3/5 (p < 0.005). As shown by the comparison of the remaining two groups, bone marrow derived from B6.Sle3/5 mice was both necessary and sufficient to mediate the elevated CD4:CD8 ratios in B6 host mice. In contrast, B6 bone marrow did not develop elevated CD4:CD8 ratios in B6.Sle3/5 hosts. Comparison of the two groups receiving B6.Sle3/5 bone marrow showed no statistically significant host effect.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. The elevated CD4:CD8 ratio in the spleen was an intrinsic property of cells of bone marrow origin. B6 or B6.Sle3/5 host mice were lethally irradiated and reconstituted with T cell-depleted bone marrow as indicated. Mice were aged for 12 mo and spleen cells were tested for CD4 and CD8 expression by flow cytometry. Mean ± SD are shown with error bars. Both groups receiving bone marrow from B6.Sle3/5 donors showed statistically significant differences from the two groups receiving bone marrow from B6 donors (p < 0.05).

To examine whether the elevated CD4:CD8 ratio was an intrinsic property of B6.Sle3/5 T cells, mixed chimeras were produced in which T cell-depleted B6.Thy1^a bone marrow was mixed with either B6 or B6.Sle3/5 bone marrow and injected into lethally irradiated B6 hosts. Again, mice were sacrificed 1 year after reconstitution and the CD4:CD8 ratio was evaluated by flow cytometry in the spleen (Fig. 3A). As seen in the first chimera experiment, mice receiving only normal (B6.Thy1^a and B6) bone marrow had a relatively low splenic CD4:CD8 ratio, whereas mice receiving a combination of B6.Thy1^a and B6.Sle3/5 bone marrow had an elevated ratio (p < 0.005). By three-color flow cytometry and CD90 Abs specific for either the a or b allele, it was possible to determine the CD4:CD8 ratio by donor origin. The results showed that T cells derived from both donors were contributing to the elevated CD4:CD8 ratio. Overall, these data were consistent with a secondary phenomenon in which a bone marrow-derived cell population mediated an increased CD4:CD8 ratio.

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Mixed chimeras prepared with donors differing by expression of the CD90 allele revealed an increased CD4:CD8 ratio in the spleens of mice given bone marrow expressing Sle3/5 but also had a significant percentage of CD4+ T cells negative for CD90 expression. A, Mice were prepared as in Fig. 2, and a combination of anti-CD4, anti-CD8, and anti-CD90.1 (a allele) or anti-CD90.2 (b allele) was used for FACS analysis. The overall CD4:CD8 ratio gated only by size is shown under the None column. T cells of B6.Thy1a origin were identified by gating on CD90.1+ events, while cells of either B6 or B6.Sle3/5 origin are identified by gating on cells expressing CD90.2. Means ± SD are shown with error bars. B, Cells were stained for the combination of anti-CD90.2, anti-CD90.1, and anti-CD4. After gating on CD4+ events, the CD90.2 vs CD90.1 profile was displayed. The lower left quadrant shows the percentage of CD90- T cells.

One potential confounder in interpretation of the first mixed chimera experiment was the identification of a subset of CD4⁺ (Fig. 3B) and CD8⁺ cells (data not shown) negative for CD90 expression. These cells accounted for as much as 20% of the total CD4⁺ and 10% of the CD8⁺ population. By three-color flow cytometry, these cells were conventional T cells, as shown by TCR expression (data not shown). Therefore, the true CD4:CD8 ratio contributed by each donor was not fully accounted for using CD90 allelic expression.

Use of B6.Ly5^a bone marrow confirmed that the elevated CD4:CD8 ratio in the spleen was an extrinsic property of T cells

Because of the inability of CD90 to identify the donor origin of all T cells, an experiment was performed using B6.Ly5^a mice as the "normal" codonor. This strain has the a allele of the pan-leukocyte isoform of CD45 (also called CD45.1). This experiment also differed from earlier ones in that the subinterval-congenic B6.Sle3 strain was used, as it had previously been shown that the elevated CD4:CD8 ratio was a property of this locus (13). Mice were prepared as before and allowed to grow for 1 year. In contrast to the CD90 marker, which could miss 15% or more of the CD4⁺ T cells, all lymphocytes expressed this isoform of CD45. This is shown most clearly in the top right panel of Fig. 4, where there were essentially no CD45.2^- cells in mice receiving only B6.Sle3 (CD45.2⁺) bone marrow. Using a combination of CD45.1- and CD45.2-specific mAbs confirmed that donor origin of all lymphocytes in the other groups was also being identified (data not shown). Representative contour plots are shown for spleen cells (Fig. 4), and the results for the spleen, lymph nodes, and thymus are compiled in Fig. 5. Similar to the B6.Thy1^a donor, B6.Ly5^a-derived cells did not have an elevated CD4:CD8 ratio when paired with normal B6 codonor marrow. When paired with B6.Sle3 donor marrow, the B6.Ly5^a-derived T cells showed a comparable elevation. Similar results were seen in the lymph node and thymus. Overall, these data confirm the secondary nature of the elevated CD4:CD8 ratio and also indicated that at least some of the elevation occurred during thymic selection. The use of CD45 also permitted the relative contribution of the CD90^- T cell subset to be assessed. Although the percentage of CD4⁺CD90^- T cells was decreased from the first experiment, the pattern was similar. As shown in Table I, each donor contributed proportionally to this population, again suggesting the NZMc7-derived susceptibility locus need not be expressed by the cells demonstrating the phenotype.

View larger version (58K): [in this window] [in a new window]   FIGURE 4. Use of B6.Ly5a bone marrow eliminated T cells of indeterminate donor origin and confirmed that the elevated CD4:CD8 ratio in the spleen was an extrinsic property of T cells. Spleen cells from chimeric mice of the indicated donor composition were analyzed for CD45.2 expression (top row). Mice given only B6.Sle3 bone marrow had very few CD45.2- cells within the lymphocyte gate (top right histogram). Representative CD4 vs CD8 profiles are shown for size-gated only (second row), CD45.2+-gated (third row), and CD45--gated cells (fourth row) samples. The calculated CD4:CD8 ratio is displayed in the top right quadrant of each contour plot.

View larger version (27K): [in this window] [in a new window]   FIGURE 5. Mixed chimeras revealed an increased CD4:CD8 ratio in the spleen, lymph nodes, and thymi of mice given bone marrow expressing Sle3. Mixed chimeras revealed an increased CD4:CD8 ratio in the spleens (A), lymph nodes (B), and thymi (C) of mice given bone marrow expressing Sle3. Mice were prepared as in Fig. 2, and a combination of anti-CD4, anti-CD8, and anti-CD45.1 (a allele) or anti-CD45.2 (b allele) was used for FACS analysis. The overall CD4:CD8 ratio gated only by size is shown under the None column. T cells of B6.Ly5a origin are identified by gating on CD45.1+ events, while cells of either B6 or B6.Sle3 origin are identified by gating on cells expressing CD45.2.

The allele-specific expression of a number of T and B cell markers was systematically examined in the mixed chimeras. With size-gating alone, CD4⁺ spleen cells from B6 mice receiving B6.Sle3/5 bone marrow, either alone or in combination with B6.Ly5^a, showed an increased percentage of CD69 expression (Table I). This was statistically significant for comparison between combinations of B6.Ly5^a and B6 vs B6.Ly5^a and B6.Sle3 (p < 0.03) and B6.Ly5^a and B6 vs B6.Sle3 (p = 0.05). Compared with the B6.Ly5^a and B6 groups, the increased expression of CD69 seen in the B6.Ly5^a and B6.Sle3 mixed chimeras was present in both donor populations and demonstrated that increased expression of CD69 was also a secondary phenomenon. In contrast to CD69 expression on CD4⁺ T cells, CD25 expression showed no consistent differences among the different groups (Table I).

T cell subset analysis showed a relative decrease of CD8⁺ T cells as the cause for the elevated CD4:CD8 ratio in the spleen

Lymph node cells from mice receiving B6.Sle3 bone marrow tended to show both a modest increase in CD4⁺ and a decrease in CD8⁺ T cells (Table II). In contrast, the spleen cells tended to show mostly a relative decrease in CD8 T cells in the those mice receiving B6.Sle3 bone marrow. Interestingly, the increased ratio in the thymus was mostly due to an increase in CD4⁺ cells. Overall, it was the altered CD4:CD8 ratio that was the most consistent finding in the mixed chimera experiments.

Neither the B6.Thy1^a nor the B6.Ly5^a mixed chimera experiments were capable of revealing the donor source of autoantibody production. For this, the B6.Igh^a strain was used. Because of the relatively low penetrance of antichromatin in unmanipulated B6.Sle3/5 mice and the additive nature of Sle3 and Sle5 in this phenotype, mixed chimeras with B6.Sle3/5 were used. Adequate reconstitution of the B cell compartments was necessary to interpret results. The B6.Igh^a-derived B cells comprised 47 ± 12% of the total B220⁺ B cell population when paired with either B6 or B6.Sle3/5 bone marrow at a 1:1 infusion ratio. This correlated well with the serum total IgM and serum total IgG2a (data not shown), both determined by allotype-specific ELISA. There was little evidence of allotype suppression, which has been seen in other adoptive transfer experiments (16, 19).

Examination of B cell activation and costimulatory markers failed to show any consistent differences in mixed chimeras

It was possible that the elevated CD4:CD8 ratio seen in mixed chimeras was the result of selection by abnormal Sle3/5-expressing B cells. If so, it might be expected that this would be reflected by differential expression of either activation or costimulatory markers. In contrast to CD69 expression on T cells, there was no statistically significant difference between CD69 expression in B cells (Table I). In addition, expression of the costimulatory molecules CD80 and CD86 showed no statistically significant elevation.

Mixed chimeras from antichromatin-positive mice had autoantibodies of both allotypes

B6.Sle3/5 mice produce autoantibodies with a penetrance of 20–30%. In contrast to B6.Sle1, which is limited to the H2A/H2B/dsDNA subnucleosome particle, B6.Sle3/5 mice showed epitope spreading to include dsDNA and intact chromatin. Sera at 9 mo after reconstitution were tested by IgG2a allotype-specific antichromatin ELISA. As shown in Fig. 6, mice given a combination of normal B6.Igh^a and B6 bone marrow produced little antichromatin of either allotype. In contrast, 6 of 14 of the host mice receiving B6.Sle3/5 in combination with B6.Igh^a produced total antichromatin >3 SD above the normal mixed chimera control group. This compares with 6 of 19 mice receiving B6.Sle3/5 alone that produced elevated titers. More importantly, of the six mixed chimeras with elevated titers of antichromatin, five had a significant proportion produced by the normal B6.Igh^a-derived B cells. There was no consistent difference in the degree of mixed chimerism between autoantibody-positive and -negative mice (data not shown). These data strongly indicate that the susceptibility locus (loci) in the Sle3/5 interval need not be expressed by the autoantibody-producing B cells.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. Mixed chimeras from antichromatin-positive mice had autoantibodies of both allotypes. Sera from mixed chimeras containing donor bone marrow, as indicated, were analyzed 1 year after reconstitution by allotype-specific ELISA. For these experiments, the B6.Igha strain was used as the normal a allotype donor. Antichromatin response between groups receiving B6.Sle3/5 and groups receiving congenic B6 bone marrow differed significantly (p < 0.01 by ANOVA). By paired t test, no differences were seen between donors within individual groups. The dotted line represents the mean plus 3 SD of the total antichromatin for the B6.Igha plus B6 group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
As part of efforts to understand the genetics murine lupus, we have created a panel of congenic strains of mice on the B6 background, each harboring at least one susceptibility locus, and have identified phenotypes indicating an immune perturbation potentially relevant to SLE. An important part of the characterization process has been the systematic determination of the functional expression profile of each locus using the techniques of adoptive bone marrow transfer. In a previous publication, it was shown that Sle1 was functionally expressed by B cells (16). More recent experiments have indicated that Sle1 is also functionally expressed by T cells.⁵ In the present work, we have extended these observations to the Sle3 locus. Similar to Sle1, our initial reciprocal transfer experiments between B6.Sle3/5 and B6 showed that Sle3/5 was expressed by cells of hemopoietic origin, at least for the most robust phenotype of an elevated splenic CD4:CD8 ratio (Fig. 2). Attempts to determine whether glomerulonephritis, a feature seen in B6.Sle3/5 with a penetrance of 20% (12), were thwarted by radiation artifacts seen in all chimeras (data not shown). Autoantibodies to chromatin, also a feature of unmanipulated B6.Sle3/5 mice, were also preferentially seen in mice receiving B6.Sle3/5 bone marrow (data not shown).

Although an important first step, these initial studies could not delineate which hemopoietically derived cells were responsible for the phenotype. To assess this, additional experiments were conducted in which congenic normal and B6.Sle3/5 bone marrow were coinfused into lethally irradiated young B6 mice. Over time, the T and B cells derived from each donor would all be positively and negatively selected under identical conditions, reconstituting an intact immune system. Any differences in phenotype between the donor T and B cells should be attributable as an intrinsic property of that cell population conferred by expression of the Sle3/5 interval. In marked contrast to Sle1, Sle3 appeared to be functionally expressed by a nonlymphocytic population of cells of hemopoietic origin. For T cells, this was shown most clearly by the fact that splenic T cells of B6.Thy1^a origin had a more elevated CD4:CD8 ratio when these cells differentiated in the presence of bone marrow cells of B6.Sle3/5 origin (Fig. 3). Likewise for B cells, this was shown by the tendency of B cells B6.Igh^a origin to also break tolerance when they differentiated in the presence of bone marrow cells of B6.Sle3/5 origin (Fig. 6).

Our initial experiments also suggested that the Thy-1 (or CD90) allelic marker did not identify all T cells (Fig. 3B). To address this issue, we turned to the B6.Ly5^a congenic strain. This strain, with an allelic form of the CD45 isoform expressed by virtually all cells of hemopoietic origin, has been very useful in a number of adoptive transfer experiments. An analogous set of adoptive transfer experiments was performed, substituting B6.Ly5^a for B6.Thy1^a. In addition, since the elevated CD4:CD8 ratio was a property only of the telomeric region of chromosome 7 (13), the subcongenic B6.Sle3 strain was used instead of the larger interval contained in B6.Sle3/5. Comparable to our earlier results, an elevated CD4:CD8 ratio was again seen in T cells derived from the "normal" congenic strain when bone marrow cells of B6.Sle3 were coinfused. In contrast to our results with B6.Thy1^a, all T cells could be phenotyped by allelic CD45 expression, leaving an undetectable number of T cells of indeterminate donor origin. Together, these data strongly indicate that control of the peripheral CD4:CD8 ratio was mediated by extrinsic expression of Sle3.

It had originally been reported that the CD4:CD8 ratio was unaltered in the thymus of B6.Sle3/5 mice (12). Additional studies have indicated that the ratio is increased (our unpublished data), a finding also seen in the mixed chimeras (Fig. 5). Moreover, coinfusion of bone marrow cells containing the Sle3 interval resulted in an increased CD4:CD8 ratio in the single positive populations. Consistent with the results in the spleen and lymph nodes, the increased ratio was seen in both donor populations. It is concluded that the CD4:CD8 ratio in the thymus is also a secondary effect relative to the thymocytes. In addition, the correlation between thymus and spleen and thymus and lymph node (Table III) indicate that the initial ratio as determined within the thymus persists and is perhaps even accentuated in the periphery.

It has been known for quite some time that the CD4:CD8 ratio in the periphery is under complex genetic control in mice and humans (20, 21). Two strains with widely different ratios are B6 and DBA/2 (22). Similar to our results, it was shown by MacDonald and colleagues (22) that the difference in ratios originated in the thymus and appeared to be accentuated in the periphery. They also demonstrated that the CD4:CD8 ratio was relatively independent of the host (22). However, in sharp contrast to the present results, mixed chimera experiments between MHC-matched B10.D2 and DBA/2 indicated that control of lineage commitment was due to a thymocyte-intrinsic factor (22). That is, regardless of host, the thymocytes of B10.D2 origin maintained a low ratio and thymocytes of DBA/2 origin maintained a high ratio. Additional experiments suggested that the mature CD8⁺ thymocyte but not the CD4⁺ population was under homeostatic control (23), perhaps by competing for a limited number of selectable niches (24). This was also reflected in steady-state analysis, where the difference in thymic CD4:CD8 ratio was largely due to a decrease in the percentage of CD8 positive cells. These niches are thought to be provided by thymic stromal cells, which mediate positive selection and are radioresistant (25, 26). This would suggest that in the mixed chimera experiments performed by MacDonald’s group (22), thymocyte-intrinsic factors may have allowed for better competition for the limiting thymic stromal cells. In contrast, our data indicate that the CD4:CD8 ratio as determined by Sle3 was mediated by a thymocyte-extrinsic radiosensitive population. Further evidence for a different mechanism is seen in the composition of the single positive population, where either increased CD4⁺, decreased CD8⁺ cells, or a combination of the two could be seen (Table I).

Our studies and those of MacDonald (22) also contrast sharply with recently published findings in the rat, where adoptive bone transfers between MHC-disparate strains revealed a controlling role for radioresistant thymic stromal cells (27). In this study, the CD4:CD8 ratio was found to be critically dependent on the host MHC, with little effect by source of thymocytes. A recent study in mice used genomewide scanning techniques in an F₂ cross between B6 and BALB/c. (28). They mapped a locus close to the MHC class II Ea gene that accounted for 60% of the effect. In B6 mice, this gene is defective and results in no I-E expression. It was hypothesized that the decreased CD4 single positive compartment found in B6 mice was due to reduced positive selection on class II. In our studies in the mouse, all mice were of the H-2^b haplotype, and it may be that in the absence of MHC differences, more subtle effects could be detected.

Although it is unclear whether the gene(s) within the Sle3 interval causing an elevated CD4:CD8 ratio is contributing directly to autoimmunity, the control of lineage commitment to CD4 or CD8 T cells is a complicated issue that has received considerable attention recently. Control at both the level of thymic development (22, 29, 30) and in the periphery (31, 32, 33) has been demonstrated. A number of different molecules have been shown to affect the CD4:CD8 ratio, including Notch1 (30, 34) and one of its ligands, Jagged1 (29, 33). IL-7 has also been shown to alter the CD4:CD8 ratio in vivo, in part by causing increased proliferation of peripheral CD8⁺ T cells (32). None of these molecules maps to chromosome 7, making them poor candidates for Sle3. Moreover, the pattern of expression of the Notch/Jagged system is inconsistent with our results in as much as Notch1 is expressed by thymocytes and Jagged1 by thymic stromal cells (29). The role of Notch1 signaling is still unclear. Although gain-of-function experiments demonstrated a potential role, inducible inactivation of Notch1 had no effect on the CD4:CD8 ratio (35). It may be that as yet unidentified related members of this family exert effects at physiologic levels of expression.

Based on these comparisons, we conclude that the control of the CD4:CD8 ratio can involve all three cellular compartments (i.e., thymocytes, bone marrow-derived APC, and radioresistant host-derived APC). Although we cannot formally exclude the possibility that a Sle3-encoded factor expressed by T cells was exerting both an autocrine and a paracrine effect, the most straightforward explanation is that Sle3 is mediating its effect through the second compartment, the non-B cell professional APC. If so, we would predict that an elevated CD4:CD8 ratio would still be seen in mixed chimeras when bone marrow from B6.Sle3/5 mice with a targeted deletion of T and B cells is used. We would also predict that CD69 expression would also be elevated. Efforts are underway to test these hypotheses.

	Acknowledgments

 
We thank Justin Kearse and Aimee Young for technical assistance.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants P01 AI39824, R01 AR44894, and R01 AI043454.

² Address correspondence and reprint requests to Dr. Eric S. Sobel, Department of Medicine, University of Florida, Box 100221, J. Hillis Miller Health Center, Gainesville, FL 32610-0221. E-mail: sobeles{at}medicine.ufl.edu

³ Current address: Food and Drug Administration, Rockville, MD 20850.

⁴ Abbreviation used in this paper: SLE, systemic lupus erythematosus.

Received for publication September 6, 2001. Accepted for publication July 22, 2002.

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