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Autoreactive T Cells Revealed in the Normal Repertoire: Escape from Negative Selection and Peripheral Tolerance¹

Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520

	Abstract

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Self-reactive T cells are known to be eliminated by negative selection in the thymus or by the induction of tolerance in the periphery. However, developmental pathways that allow self-reactive T cells to inhabit the normal repertoire are not well-characterized. In this investigation, we made use of anti-small nuclear ribonucleoprotein particle (snRNP) Ig transgenic (Tg) mice (2-12 Tg) to demonstrate that autoreactive T cells can be detected and activated in both normal naive mice and autoimmune-prone MRL lpr/lpr mice. In contrast, autoreactive T cells of nonautoimmune Tg mice are tolerized by Tg B cells in the periphery. In adoptive transfer studies, autoreactive T cells from MRL lpr/lpr mice can stimulate autoantibody synthesis in nonautoimmune anti-snRNP Tg mice. Transferred CD4 T cells migrate to regions of the spleen proximal to the B cell follicles, suggesting that cognate B cell-T cell interactions are critical to the autoimmune response. Taken together, our studies suggest that anti-snRNP B cells are important APCs for T cell activation in autoimmune-prone mice. Additionally, we have demonstrated that anti-snRNP B cell anergy in nonautoimmune mice may be reversed by appropriate T cell help.

	Introduction

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T cell development in the thymus undergoes both positive and negative selective processes, in which they are programmed for self-MHC restriction and elimination of potentially self-reactive cells (1, 2, 3, 4). However, studies in animal models of T cell-mediated autoimmunity show that these processes in the thymus may not be sufficient for controlling self-reactive T cells. For example, analysis of self-reactive TCR transgenic (Tg)³ mouse strains have established that as many as 25–40% of autoreactive T cells escape clonal deletion even in the presence of the deleting ligand (5). Studies of organ-specific autoimmune diseases such as multiple sclerosis and type-1 diabetes mellitus reveal that autoimmune and potentially pathogenic self-reactive T cells are present in the normal peripheral T cell repertoire (6, 7). Therefore, these autoreactive T cells are not efficiently deleted during normal development in the thymus and/or periphery. However, one important implication of this concept is in whether the remaining autoreactive T cells may be activated and functionally competent to create autoimmunity. In addressing this hypothesis, we investigated the physiological status of autoreactive T cells in the normal repertoire of naive mice. In essence, what is the clinical significance of self-reactive T cells in the normal repertoire, and to what extent can these cells be activated and cause autoimmune disease?

The importance of B cells as autoantigen presenting cells has been extensively studied in our laboratory and by other investigators (8, 9, 10, 11, 12, 13, 14, 15, 16, 17). In B cell-deficient autoimmune-prone MRL lpr/lpr mice, populations of activated T cells are virtually eliminated as compared with wild-type (wt) MRL lpr/lpr mice (12), suggesting that B cells play a central role in the activation of autoreactive T cells. In the present studies, we have used Ig Tg mice, in which B cells have specificity for a target of lupus autoimmunity, the small nuclear ribonucleoprotein particle (snRNP; Ref. 18). We find that autoreactive snRNP-specific T cells can be activated from normal naive mice and MRL lpr/lpr mice. Anti-snRNP B cells tolerize T cells in Tg B10.A mice, but activate T cells in MRL lpr/lpr mice. Most importantly, we find populations of autoreactive T cells in the thymus of Tg B10.A mice while peripheral T cells are anergized, suggesting that tolerance induction is mediated by B cells in the periphery. Finally, autoreactive T cells from MRL lpr/lpr mice could provide a source of in vivo help for anti-snRNP B cells to make autoantibodies in Tg B10.A strains of mice. Thus, this system provides us the unique opportunity to examine how B cells can either activate or tolerize autoreactive T cells in the normal and autoimmune-prone repertoire.

	Materials and Methods

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Mice

Anti-snRNP Ig Tg mice were derived using the rearranged VDJ segment of the 2-12 anti-snRNP hybridoma cloned upstream in a vector containing the Cµ region gene segment (18). Tg mice have been backcrossed for >10 generations to C57BL/6, B10.A, or MRL lpr/lpr mice. The presence of the transgene was identified by PCR analysis of tail DNA using 2-12-specific primers 5'-GAGGTCCAGCTGCAGTCTGGA-3' in the first coding region of the V region, and 5'-CGCTCCACCAGACCTCTCTAGA-3' complementary to the XbaI site of JH4. Prior studies demonstrated that 92% of B cells possessed the transgene H chain (Ref. 18 and our unpublished observations). Animals are age- and sex-matched in all experiments, and are housed in a conventional facility at Yale University (New Haven, CT).

Purification of recombinant snRNP D protein (rSm-D) fusion protein

rSm-D expressed in Escherichia coli was used as stimulating Ag in T cell proliferation assays (19). rSm-D was purified by anion and cation exchange chromatography as previously described (20), and was absorbed for potential mitogens by anti-LPS column chromatography using agarose beads coated with polymyxin B (Pierce, Rockford, IL). In some experiments, rSm-D was labeled with biotin for FACS staining or tissues-section staining.

T cell proliferation assays

Splenic CD4 T cells were purified by negative selection, using anti-mouse B220, anti-mouse MHC class II, anti-mouse CD11b, and anti-mouse CD8 microbeads (Miltenyi Biotec, Auburn, CA), or positive selection using anti-mouse CD4 microbeads. Thymic CD4 T cells were purified by two-step selection. In brief, thymocytes were incubated with anti-CD8 microbeads and ran through the LS column. The depleted fraction was incubated with anti-CD4 microbeads. The thymic single-positive CD4 cells were positively selected by magnetic separation. B cells were purified by negative selection, using anti-mouse CD43 and anti-CD90 microbeads. The purity of CD4⁺ T cells or B cells was >94% as assessed by flow cytometry.

Conventional T cell proliferation assays were performed with CD4⁺ T cells (2 x 10⁵/well) and irradiated purified B cells (500 rad) as APCs at 5 x 10⁵/well. All assays were performed with triplicate samples and incubated with or without Ag for 3 days. Lymphocyte proliferation was assessed by [³H]thymidine incorporation (1.0 µCi/well; ICN Chemicals, Irvine, CA) during the last 18 h of culture. Sample wells were harvested onto filters, and incorporated radioactivity was counted in a Betaplate liquid scintillation counter (Wallac, Gaithersburg, MD).

In vitro studies or in vivo administration of CFSE-labeled CD4 T cells

CD4 T cells purified from B10.A or MRL lpr/lpr (>16-wk-old) Tg or non-Tg mice were labeled with the fluorescent dye CFSE (Molecular Probes, Eugene, OR; Ref. 21). Briefly, the cells were suspended at 5 x 10⁷/ml in prewarmed PBS containing a final concentration of 10 µM CFSE and incubated for 30 min at 37°C. The cells were washed once in FCS and twice in PBS before transfer. CD4 T cells (20 x 10⁶) were transferred i.v. via tail-vein injection. At 24 h or 5 days after transfer, spleens and lymph nodes were harvested from the recipients for tissue immunofluorescence and flow cytometry. For in vitro studies, CFSE-labeled CD4 T cells were cocultured with 2-12 Tg B cells at 5% CO₂ 37°C incubator. After 3 days incubation, cells were harvested for FACS analysis.

Tissue immunofluorescence

Spleens were suspended in OCT, frozen in 2-methyl-butane cooled with dry ice, sectioned, and fixed with acetone. The sections were blocked using PBS/3% BSA, and then stained with anti-B220-PE overnight at 4°C to detect follicular B cells. Following three washes with PBS/1% BSA, sections were mounted with Fluoromount-G and then visualized by laser scanning confocal fluorescence microscope (Zeiss Axiovert 100 M; Zeiss, Oberkochen, Germany).

Flow cytometry

Cells (1 x 10⁶) were surface-stained with various mAbs by conventional methodology. The following Abs were used: anti-B220-cy5, anti-heat-stable Ag-FITC, anti-CD4-PE, anti-B7-1-FITC, anti-B7-2-PE, and anti-IgM-FITC (BD PharMingen, San Diego, CA). All samples were analyzed on a FACSCalibur flow cytometry (BD Biosciences, Mountain View, CA) using CellQuest software. Between 10,000 and 20,000 events were collected within a live lymphocyte gate set based on forward and side scatter.

	Results

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Anti-snRNP 2-12 B cells are not deleted in either normal or autoimmune-prone mice

The 2-12H transgene construct used to generate the Tg mice consists of the 2-12 VDJ cloned upstream of the Igh^a allotype Cµ exon. An anti-snRNP B cell hybridoma, designated 2-12, was originally cloned from an MRL lpr/lpr mouse. Abs from the 2-12 hybridoma are specific for the D protein of murine snRNPs and to denatured DNA (22). To determine whether anti-snRNP autoreactive B cells develop and reach the periphery, three-color immunofluorescence of spleen cells with anti-B220, anti-IgM, and biotinylated rSm-D was performed. The percentage of IgM⁺ cells that stain with rSm-D ranges from 15–30% in B10.A, C57BL/6, and MRL lpr/lpr 2-12 Tg mice (Fig. 1), consistent with our previous studies performed in C57BL/6 mice (18). The specificity of the Tg B cells binding to snRNP D protein was confirmed with cold Ag competition (data not shown). In such assays, unlabeled snRNP reduces the binding of biotin-rSm-D by >80%. Control recombinant fusion protein failed to block snRNP D-specific binding to B cells. Thus, anti-snRNP Tg mice have significant numbers of snRNP D-specific B cells in the periphery, demonstrating that these cells are not actively deleted in either normal mice or in autoimmune-prone MRL mice.

View larger version (40K): [in this window] [in a new window]   FIGURE 1. Anti-snRNP Tg mice do not delete self-reactive B cells. Spleen cells were prepared from anti-snRNP Tg or wt mice and costained with anti-mouse IgM-FITC, anti-B220-Cy5, and biotinylated D protein of the snRNP particle. Dot plots are gated on B220+ cells.

It has been suggested that B cells could promote T cell activation and infiltrative disease by several mechanisms. Because cognate T-B interactions are crucial for the development of lupus autoimmunity (23, 24, 25, 26, 27), we hypothesized that B cells may exert their effects on CD4⁺ T cells through autoantigen presentation. Toward this end, we used anti-snRNP B cells as APCs to examine the status of autoreactive T cells inhabiting the repertoire of normal and autoimmune-prone mice. T cell proliferation was examined with B cell APCs from anti-snRNP Ig Tg animals.

As illustrated in Fig. 2A, CD4 T cells from B10.A wt mice did not respond to rSm-D protein presented by autologous non-Tg APCs, but had a significant response to 2-12 Tg B cells either with or without rSm-D protein. This observation indicated that self-reactive T cells inhabit the normal repertoire, and can be activated by appropriate snRNP-presenting APCs. The ability of wt B10.A T cells to respond to Tg B cells is likely due to specific Ag-processing functions of these cells. We hypothesize that Tg B cells, by virtue of their surface receptor specificities, present a unique group of snRNP D peptides which T cells in the non-Tg mice have never contacted and which have never been deleted. CD4 T cells originating from B10.A Tg mice failed to respond to peptide presented by either non-Tg or Tg B10.A B cells, suggesting that these autoreactive T cell subsets have been anergized in Tg mice, presumably by Tg B cells. All T cell responses were inhibited by the presence of Abs to class II MHC (Ref. 19 and data not shown). It is unlikely that T cell responses in this study are due to autologous mixed lymphocyte reaction because the final data panel (T cells and APCs from Tg B10.A mice) shows no detectable proliferative responses.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. The induction of activation/tolerance of autoreactive T cells by 2-12 anti-snRNP B cells in normal background mice. A, CD4+ T cells were purified from wt or Tg B10.A mice and cocultured with irradiated purified 2-12 anti-snRNP B cells of B10.A mice with or without self-Ag rSm-D (5 µg/ml) for 72 h. Proliferation was quantified by uptake of [3H]TdR (see Materials and Methods). CD4 T cells from same source were also CFSE-labeled and stimulated with 2-12 Tg B cells in vitro for 3 days. B, The CD4 T cells in two critical conditions as indicated were examined for cell division by FACS analysis.

These observations were in contrast to similar experiments performed with the autoimmune-prone MRL lpr/lpr mice. As in B10.A mice, CD4 T cells from MRL lpr/lpr mice or Tg mice failed to respond to snRNP protein presented by B10.A wt APCs (Fig. 3A). However, T cells from MRL lpr/lpr mice or Tg mice significantly responded to peptide presented by B10.A Tg B cells (Fig. 3A). This observation is in sharp contrast to T cell responses of B10.A Tg mice, indicating autoreactive CD4 T cells are not tolerized or deleted by Tg B cells in MRL lpr/lpr Tg mice. This notion is supported by the analysis of T cell clones from 2-12 Tg MRL mice that respond to individual snRNP D peptides.⁴

View larger version (17K): [in this window] [in a new window]   FIGURE 3. T cell autoimmunity in lupus-prone MRL lpr/lpr anti-snRNP Ig Tg mice. CD4+ T cells were purified from wt or Tg MRL lpr/lpr mice, as indicated. CD4+ T cells were cocultured with irradiated purified B cells with medium alone or with rSm-D (5 µg/ml) for 72 h. A, CD4 T cells of MRL lpr/lpr or 2-12 Tg mice cocultured with a B cell APC source identical to that found in Fig. 2A. B, CD4 T cells of B10.A Tg or wt mice with a B cell source from MRL lpr/lpr Tg or wt origin.

Autoreactive T cells are tolerized in the periphery of 2-12 Tg normal mice

We next investigated whether the tolerance induction of autoreactive T cells is mediated by 2-12 Tg B cells and the sites where tolerance occurs. The expression of tolerance or autoimmunity was dependent on either the presence of anti-snRNP Tg B cells and/or the background, B10.A vs MRL. With these two variables in mind, we investigated whether tolerance was mediated centrally in the thymus or in the periphery. CD4 T cells were purified from the thymus or spleen of individual mice and examined for their response to snRNP autoantigen. As illustrated in Fig. 4A, both thymic and splenic CD4 T cells from wt mice responded to D protein presented by Tg B cells. As found in Fig. 2, splenic CD4 T cells from Tg mice failed to respond to D proteins. However, thymic CD4 T cells from the same Tg mouse had a significant response (10-fold greater) to rSm-D protein presented by Tg B cells. This observation indicates that autoreactive T cells are not centrally tolerized in the thymus, but instead are tolerized by Tg B cells in the periphery. The level of TCR expression in all CD4 T cells is similar (data not shown).

View larger version (13K): [in this window] [in a new window]   FIGURE 4. The induction of autoreactive T cell tolerance in B10.A Tg mice is mediated by Tg B cells in the periphery. CD4+ T cells purified from wt (A) or Tg (B) B10. A thymus and spleen were cocultured with B10.A Tg B cells for 72 h. Proliferation was quantified by uptake of [3H]TdR.

Studies using TCR Tg mice have demonstrated that several mechanisms could be involved in preventing autoreactive T cells from being deleted (28, 29, 30, 31). For example, it has been found that dual TCR-bearing T cells expressing self-specific TCRs escape central tolerance and are functionally competent to the "self" Ag in vivo. These studies suggest that dual TCR T cells with one autoreactive TCR may survive negative selection through the second, nonself, TCR (29, 30, 32, 33). It is still not clear whether dual TCR T cells constitute autoreactive T cell repertoire in normal naive mice.

To examine the contribution of dual TCR T cells to self-reactive T cell repertoire, CD4 T cells from TCR ^-/+-/+ mice as well as wt mice were cocultured with 2-12 Tg B cells in the presence of snRNP D protein. TCR ^-/+-/+ mice are unable to generate two productively rearranged TCR loci or two loci; therefore, they have only single-specificity TCR T cells. As shown in Fig. 5, CD4 T cells from wt mice develop a significant response to the D protein as we described above (Fig. 2A). However, the response from TCR ^-/+-/+ mice is reduced by 50% compared with that from the wt. The decreased response is specific and comparable because wt and TCR ^-/+-/+ CD4 T cells respond identically to anti-CD3 stimulation (Fig. 2B). The reduced response of TCR ^-/+-/+ CD4 T cells suggests that dual TCR T cells constitute part of the self-reactive T cell repertoire in the periphery. However, we have yet to clone and identify the peptide specificity of snRNP-reactive T cells from hemizygous mice to quantify the fraction of self-reactive T cells that may still inhabit this repertoire.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. The +/-+/- CD4 T cell repertoire contains reduced autoreactive T cells against snRNP D protein compared with the wt repertoire. CD4 T cells purified from unimmunized C57BL/6 wt and TCR-/+-/+ mice and cocultured with 2-12 Tg B cells. The proliferation was measured after a 3-day culture. As control, anti-CD3 Ab 2C11 was added with B6 B cells to demonstrate CD4 T cell proliferation is nearly identical between mouse strains (B).

Thus far, we have demonstrated T cell tolerance induction and no autoantibody production in 2-12 Tg B10.A mice. In contrast, anti-snRNP T cells are readily observed in Tg MRL lpr/lpr mice. Next, we examined whether T cell help from MRL lpr/lpr mice could drive autoantibody production in MHC class II-matched 2-12 Tg B10.A mice.

Toward this end, purified CD4 T cells from age-matched wt MRL lpr/lpr or 2-12 Tg MRL lpr/lpr mice were adoptively transferred into B10.A 2-12 Tg mice and wt littermates. MRL lpr/lpr animals with autoantibody production (16–30 wk of age) were used as the source of donor CD4 T cells. Anti-snRNP autoantibodies were detected in B10.A 2-12 Tg mice, but not in non-Tg mice 1 mo after transfer of T cells from MRL lpr/lpr Tg mice (Fig. 6). In contrast, donor CD4 T cells from B10.A Tg or non-TgB10.A mice transferred into B10.A Tg mice failed to drive autoantibody production (data not shown). These latter observations demonstrate that minor MHC differences between donor and recipient mouse strains was not a basis for autoantibody production. More importantly, this observation suggests that Ag-specific autoreactive T cells can deliver helper signals for autoreactive Tg B cells of nonautoimmune-prone mice.

View larger version (22K): [in this window] [in a new window]   FIGURE 6. Anti-snRNP autoantibodies are produced in B10.A 2-12 Tg mice reconstituted with CD4+ T cells from MRL lpr/lpr Tg mice. Ten million purified CD4+ T cells from MRL lpr/lpr 2-12 Tg mice were adoptively transferred into B10.A 2-12 Tg or wt mice. After 1 mo, sera were collected, and anti-snRNP reactivity was measured. Experiments were performed in triplicate with three mice in each group with average data represented. Each bar represents individual mouse serum before and after CD4+ T cell transfer.

T cell-dependent Ab responses typically require cognate interaction between Ag-presenting B cells and Ag-specific T cells. Ag-mediated cross-linking of the B cell receptor promotes B cell localization to the boundary between the T cell zones and follicles, promoting encounters between Ag-bearing B cells and Ag-specific T cells (34). We labeled purified CD4 T cells of different origins with CFSE for adoptive transfer into B10.A 2-12 Tg or wt mice. After 24 h, we defined the localization of the transferred cells by immunostaining frozen sections of the spleen. In striking contrast to the CD4 T cells either from B10.A 2-12 Tg or wt, transferred CD4 T cells from MRL 2-12 Tg mice migrated into the B cell follicles (Fig. 7, A and B). Transferred CD4 T cells from B10.A 2-12 Tg and wt mice were found to localize within the T cell zone, with only occasional cells migrating near follicles (Fig. 7, C and D). Similar patterns were seen at 2, 3, or 5 days after transfer. However, no localization pattern difference occurred in B10.A 2-12 Tg and non-Tg mice. These observations suggest that T cells from 2-12 MRL Tg mice can acquire the intrinsic ability to migrate to B cell follicles, and that this process is independent of the resident B cells. It also provides rationale for why MRL T cells drive autoantibody production through cognate B-T interactions in recipient mice.

View larger version (46K): [in this window] [in a new window]   FIGURE 7. Transferred CD4 T cells of MRL lpr/lpr Tg mice migrate toward splenic B cell follicles in recipient B10.A Tg mice. Panels represent confocal microscopy of splenic sections of CFSE-labeled CD4+ T cells transferred into recipient mice (donor and recipient mice as indicated). Splenic tissues were isolated and examined 24 h after cell transfer. CFSE-labeled cells are indicated in green, and B cell follicles are stained in red with the use of mAbs to B220 (see Materials and Methods). Results are representative of three mice for each transfer group. F, follicle; T, T cell zone. Original magnification: x100.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Autoreactive T cells inhabit the naive repertoire of normal mice

The importance of B cells as autoantigen presenting cells has been emphasized by previous studies in our laboratory and others (8, 10, 13, 36). There is an accumulating data suggesting that B cells are capable of presenting autoantigen in activating autoreactive T cells (12, 15). Direct evidence comes from studies of B cell-deficient MRL lpr/lpr mice which fail to generate spontaneously activated T cell populations that are normally found in wt MRL lpr/lpr mice (12, 13). In the present study, we made use of Ig Tg mice in which B cells have specificity for the snRNP D protein (18). Our results indicate that naive CD4 T cells respond to peptides presented by Tg B cells even without exogenously added Ag (Fig. 1). Two important concepts have emerged from this simple experiment. First, autoreactive B cells bearing surface Ig specific for snRNP encounter a source self-Ag in vivo, and subsequently bind and present snRNP peptides to T cells. We do not yet know the physiologic site of snRNP autoantigen that is captured and presented by Tg B cells, whether from natural cell death or apoptotic cells. Second, autoreactive T cells inhabit the naive repertoire of normal mice and can be activated by autoantigen-specific B cells as APCs.

Autoreactive T cells constitute the normal T cell peripheral repertoire as found here and by studies showing that a high frequency of proteolipid protein139–151-reactive T cells are found in naive mice without immunization (6). Additionally, studies from TCR Tg mice have demonstrated that most self-reactive T cells are not completely deleted by negative selection in the thymus. Low-avidity T cells escape thymic deletion and are retained in the mature naive T cell population (37, 38, 39). Not surprisingly, even CTLs specific to ubiquitous proteins such as ₂-microglobulin, hemoglobin, or kallikrein persist in the peripheral repertoire and can be activated under appropriate conditions (40, 41).

Splenic CD4 T cells from Tg B10.A mice encountering Ag presented by Tg B cells in vivo become anergic. This provides one explanation why Tg B cells of normal (nonautoimmune) origin are not able to spontaneously produce autoantibodies. This observation is also consistent with previous studies demonstrating the proliferative response of T cells to hen egg lysozyme (HEL) "autoantigen" delivered by anti-HEL Tg B cells or by Ag delivered to other APCs by B cells (14). However, unlike these studies, we do not have evidence for Fas-mediated B cell death after T cells are activated in Tg B10.A mice.

Our data clearly show that T cells are not deleted in 2-12 Tg model and can be partially activated by 2-12 MRL lpr/lpr Tg B cells in vitro. Recent studies using HEL Ab/Ag double Tg mice which have shown that HEL-specific T cells are tolerized in HEL Ag single Tg mice (50). However, T cells from Ag/Ab double Tg mice escape central deletion and experience a partial breakdown of peripheral tolerance presumably due to the presentation of HEL Ag by HEL-specific B cells in double Tg mice (50). We found that thymic T cells from 2-12 Tg mice are activated by 2-12 Tg B cells while peripheral CD4 T cells from the same mouse are anergic. This observation suggests that autoreactive T cells in the 2-12 Tg B10.A mice are not deleted in the thymus, but are actively anergized by Tg B cells in the periphery. Autoreactive T cells in the thymus of 2-12 Tg mice emigrate into periphery-encountering Tg B cells, and result in T cell tolerance via cognate interactions. We believe this to be the mechanism by which 2-12 Tg mice in the normal background maintain self-tolerance. T cell anergy in the periphery can be overcome by the transfer of nontolerant MRL lpr/lpr T cells that provide help for autoantibody production. The activation of B cells and autoantibody in the B10.A Tg mice also leads to Ab deposition and cellular infiltration into the kidney.⁴

Our results also indicate that dual TCR-bearing T cells may partially contribute to the autoreactive T cell repertoire. Although the physiological roles of dual TCR T cells still are not clear, previous work has demonstrated that dual TCR T cells may rescue autoreactive T cells from negative selection in the thymus (30). However, our data imply that autoreactive T cells are also found in the single TCR repertoire. This latter notion is supported by the work from the Allen laboratory (42) which demonstrates the binding of two separate ligands, a self-peptide (arthritic peptide), and a foreign epitope on distinct MHC by T cells bearing a single TCR. It is also possible that the frequency of self-reactive T cells are decreased in the single TCR repertoire compared with dual TCR-bearing T cells. Enumerating snRNP-reactive T cells from both single and dual TCR T cells is presently underway and should resolve the latter question.

T cell help drives autoantibody production in nonautoimmune-prone mice

snRNP-specific, autoreactive T cells are actively tolerized in nonautoimmune Tg mice, but not in autoimmune-prone MRL mice. Furthermore, the transfer of CD4 T cells from MRL lpr/lpr Tg mice into B10.A Tg mice initiates an anti-snRNP autoantibody response. This observation demonstrates that Ag-specific T cells can provide help for anti-snRNP B cell of nonautoimmune origin to produce autoantibodies in vivo. We do not yet understand the factors that explain a lack of adequate CD4 T cell helper functions of wt B10.A mice when transferred into B10.A Tg mice. However, we presume that a high frequency of snRNP-specific T cells exist in Tg MRL mice because they are easily activated with Ag in vitro. This premise is also supported by recent studies demonstrating that MRL T cells are activated by lower thresholds of Ag stimulation as compared with normal background T cells (43).

Moreover, autoantibody production requires autoreactive T-B cell collaboration, and is supported by the colocalization of T cells and B cells in the follicles as presented here. The trafficking of transferred cells within secondary lymphoid tissues of the host was performed to help explain biology of T cell help for autoantibody production (44, 45, 46, 47). CD4 T cells from MRL lpr/lpr Tg mice migrated to within the margins of B cell zones in the spleen. In contrast, transferred CD4 T cells from B10.A Tg or non-Tg mice failed to migrate to the B cell follicles consistent with their inability to drive autoantibody production under these conditions. Previous studies have demonstrated that Ag-specific Tg T and B cells initially remain localized without contact in their respective zones. Following Ag stimulation, these cells move in a synchronous fashion out of their respective sites for cognate interactions at the interface of these zones (34, 48, 49).

In conclusion, these studies illustrate the presence of autoreactive T cells both in normal naive mouse and MRL lpr/lpr mouse, a murine model of human systemic lupus erythematosus. The induction of autoimmunity relies on the presence and cognate interactions of specific T cell-helper functions with B cells. Perhaps more importantly, these studies illustrate that B cells as APCs play an important role both in the activation and tolerance induction of autoreactive T cells, depending on the background. These observations have implications for the understanding of autoreactive T cell tolerance or autoimmunity and the initiation of autoantibody responses in normal individuals in which aberrant autoreactive T cell activation may arise.

	Acknowledgments

 
We thank Drs. Stephen H. Clarke and Sandra Santulli-Marotto for the development of the 2-12 Tg mice and for their helpful discussion. We also thank Dr. Mark Shlomchik for his constructive comments of this manuscript. We thank Dr. Linda Bockenstedt for developing the technology of purifying cloned recombinant snRNP-D protein.

	Footnotes

 
¹ This study was supported by grants from the National Institutes of Health (AI36529 and AR41032; to M.J.M.).

² Address correspondence and reprint requests to Dr. Mark J. Mamula, Yale University School of Medicine, 333 Cedar Street, Laboratory of Clinical Investigation 6, P.O. Box 208031, New Haven, CT 06520-8031. E-mail address: mark.mamula{at}yale.edu

³ Abbreviations used in this paper: Tg, transgenic; snRNP, small nuclear ribonucleoprotein particle; rSm-D, recombinant snRNP D protein; wt, wild type; HEL, hen egg lysosome.

⁴ J. Yan, S. Shinde, M. Shlomchik, and M. Mamula. Differential regulation of T cell tolerance and activation by B cells in normal or autoimmunity-prone mice. Submitted for publication.

Received for publication November 12, 2001. Accepted for publication January 24, 2002.

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