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

This information is current as Jun Yan and Mark J. Mamula of October 2, 2021. J Immunol 2002; 168:3188-3194; ; doi: 10.4049/jimmunol.168.7.3188 http://www.jimmunol.org/content/168/7/3188 Downloaded from References This article cites 50 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/168/7/3188.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Autoreactive T Cells Revealed in the Normal Repertoire: Escape from Negative Selection and Peripheral Tolerance1

Jun Yan and Mark J. Mamula2

Self-reactive T cells are known to be eliminated by negative selection in the 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 synthesis in nonautoimmune anti-snRNP Tg mice. Transferred CD4 T cells migrate to regions of the spleen proximal to the follicles, suggesting that cognate B 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 Downloaded from B cell anergy in nonautoimmune mice may be reversed by appropriate T cell help. The Journal of Immunology, 2002, 168: 3188–3194.

cell development in the thymus undergoes both positive tors (8–17). In B cell-deficient autoimmune-prone MRL lpr/lpr and negative selective processes, in which they are pro- mice, populations of activated T cells are virtually eliminated as T grammed for self-MHC restriction and elimination of po- compared with wild-type (wt) MRL lpr/lpr mice (12), suggesting http://www.jimmunol.org/ tentially self-reactive cells (1–4). However, studies in animal mod- that B cells play a central role in the activation of autoreactive T els of T cell-mediated show that these processes in cells. In the present studies, we have used Ig Tg mice, in which B the thymus may not be sufficient for controlling self-reactive T cells have specificity for a target of lupus autoimmunity, the small cells. For example, analysis of self-reactive TCR transgenic (Tg)3 nuclear ribonucleoprotein particle (snRNP; Ref. 18). We find that mouse strains have established that as many as 25–40% of auto- autoreactive snRNP-specific T cells can be activated from normal reactive T cells escape even in the presence of the naive mice and MRL lpr/lpr mice. Anti-snRNP B cells tolerize T deleting ligand (5). Studies of organ-specific autoimmune diseases cells in Tg B10.A mice, but activate T cells in MRL lpr/lpr mice. such as multiple sclerosis and type-1 diabetes mellitus reveal that Most importantly, we find populations of autoreactive T cells in by guest on October 2, 2021 autoimmune and potentially pathogenic self-reactive T cells are the thymus of Tg B10.A mice while peripheral T cells are aner- present in the normal peripheral T cell repertoire (6, 7). Therefore, gized, suggesting that tolerance induction is mediated by B cells in these autoreactive T cells are not efficiently deleted during normal the periphery. Finally, autoreactive T cells from MRL lpr/lpr mice development in the thymus and/or periphery. However, one im- could provide a source of in vivo help for anti-snRNP B cells to portant implication of this concept is in whether the remaining make in Tg B10.A strains of mice. Thus, this sys- autoreactive T cells may be activated and functionally competent tem provides us the unique opportunity to examine how B cells can to create autoimmunity. In addressing this hypothesis, we investi- either activate or tolerize autoreactive T cells in the normal and gated the physiological status of autoreactive T cells in the normal autoimmune-prone repertoire. repertoire of naive mice. In essence, what is the clinical signifi- cance of self-reactive T cells in the normal repertoire, and to what Materials and Methods extent can these cells be activated and cause ? Mice The importance of B cells as autoantigen presenting cells has been extensively studied in our laboratory and by other investiga- 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 C␮ region gene segment (18). Tg mice have been backcrossed for Ͼ10 generations to C57BL/6, B10.A, or MRL lpr/lpr mice. The presence of the Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520 transgene was identified by PCR analysis of tail DNA using 2-12-specific primers 5Ј-GAGGTCCAGCTGCAGTCTGGA-3Ј in the first coding region Received for publication November 12, 2001. Accepted for publication Janurary of the V region, and 5Ј-CGCTCCACCAGACCTCTCTAGA-3Ј comple- 24, 2002. mentary to the XbaI site of JH4. Prior studies demonstrated that 92% of B The costs of publication of this article were defrayed in part by the payment of page cells possessed the transgene H chain (Ref. 18 and our unpublished obser- charges. This article must therefore be hereby marked advertisement in accordance vations). Animals are age- and sex-matched in all experiments, and are with 18 U.S.C. Section 1734 solely to indicate this fact. housed in a conventional facility at Yale University (New Haven, CT). 1 This study was supported by grants from the National Institutes of Health (AI36529 and AR41032; to M.J.M.). Purification of recombinant snRNP D protein (rSm-D) fusion 2 Address correspondence and reprint requests to Dr. Mark J. Mamula, Yale protein University School of Medicine, 333 Cedar Street, Laboratory of Clinical Inves- tigation 6, P.O. Box 208031, New Haven, CT 06520-8031. E-mail address: rSm-D expressed in Escherichia coli was used as stimulating Ag in T cell [email protected] proliferation assays (19). rSm-D was purified by anion and cation exchange 3 Abbreviations used in this paper: Tg, transgenic; snRNP, small nuclear ribonucle- chromatography as previously described (20), and was absorbed for po- oprotein particle; rSm-D, recombinant snRNP D protein; wt, wild type; HEL, hen egg tential mitogens by anti-LPS column chromatography using agarose beads lysosome. coated with polymyxin B (Pierce, Rockford, IL). In some experiments,

Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00 The Journal of Immunology 3189 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, 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 posi- tively selected by magnetic separation. B cells were purified by negative selection, using anti-mouse CD43 and anti-CD90 microbeads. The purity of CD4ϩ T cells or B cells was Ͼ94% as assessed by flow cytometry. Conventional T cell proliferation assays were performed with CD4ϩ T cells (2 ϫ 105/well) and irradiated purified B cells (500 rad) as APCs at 5 ϫ 105/well. All assays were performed with triplicate samples and in- cubated with or without Ag for 3 days. proliferation was assessed by [3H]thymidine incorporation (1.0 ␮Ci/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 liq- uid scintillation counter (Wallac, Gaithersburg, MD). Downloaded from In vitro studies or in vivo administration of CFSE-labeled CD4 T cells CD4 T cells purified from B10.A or MRL lpr/lpr (Ͼ16-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 ϫ FIGURE 1. Anti-snRNP Tg mice do not delete self-reactive B cells. 107/ml in prewarmed PBS containing a final concentration of 10 ␮M CFSE Spleen cells were prepared from anti-snRNP Tg or wt mice and costained and incubated for 30 min at 37°C. The cells were washed once in FCS and with anti-mouse IgM-FITC, anti-B220-Cy5, and biotinylated D protein of http://www.jimmunol.org/ twice in PBS before transfer. CD4 T cells (20 ϫ 106) were transferred i.v. the snRNP particle. Dot plots are gated on B220ϩ cells. 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 reduces the binding of biotin-rSm-D by Ͼ80%. Control recombi- incubation, cells were harvested for FACS analysis. nant fusion protein failed to block snRNP D-specific binding to B Tissue immunofluorescence cells. Thus, anti-snRNP Tg mice have significant numbers of Spleens were suspended in OCT, frozen in 2-methyl-butane cooled with snRNP D-specific B cells in the periphery, demonstrating that

dry ice, sectioned, and fixed with acetone. The sections were blocked using these cells are not actively deleted in either normal mice or in by guest on October 2, 2021 PBS/3% BSA, and then stained with anti-B220-PE overnight at 4°Cto autoimmune-prone MRL mice. detect follicular B cells. Following three washes with PBS/1% BSA, sec- tions were mounted with Fluoromount-G and then visualized by laser scan- Autoreactive T cells in normal naive mice and MRL lpr mice ning confocal fluorescence microscope (Zeiss Axiovert 100 M; Zeiss, are activated by 2-12 Tg B cells Oberkochen, Germany). It has been suggested that B cells could promote T cell activation Flow cytometry and infiltrative disease by several mechanisms. Because cognate Cells (1 ϫ 106) were surface-stained with various mAbs by conventional T-B interactions are crucial for the development of lupus autoim- methodology. The following Abs were used: anti-B220-cy5, anti- munity (23–27), we hypothesized that B cells may exert their ef- heat-stable Ag-FITC, anti-CD4-PE, anti-B7-1-FITC, anti-B7-2-PE, and anti- fects on CD4ϩ T cells through autoantigen presentation. Toward IgM-FITC (BD PharMingen, San Diego, CA). All samples were analyzed on a FACSCalibur flow cytometry (BD Biosciences, Mountain View, CA) this end, we used anti-snRNP B cells as APCs to examine the using CellQuest software. Between 10,000 and 20,000 events were col- status of autoreactive T cells inhabiting the repertoire of normal lected within a live lymphocyte gate set based on forward and side scatter. and autoimmune-prone mice. T cell proliferation was examined with B cell APCs from anti-snRNP Ig Tg animals. Results As illustrated in Fig. 2A, CD4 T cells from B10.A wt mice did Anti-snRNP 2-12 B cells are not deleted in either normal or not respond to rSm-D protein presented by autologous non-Tg autoimmune-prone mice APCs, but had a significant response to 2-12 Tg B cells either with The 2-12H transgene construct used to generate the Tg mice con- or without rSm-D protein. This observation indicated that self- sists of the 2-12 VDJ cloned upstream of the Igha C␮ reactive T cells inhabit the normal repertoire, and can be activated exon. An anti-snRNP B cell hybridoma, designated 2-12, was orig- by appropriate snRNP-presenting APCs. The ability of wt B10.A inally cloned from an MRL lpr/lpr mouse. Abs from the 2-12 T cells to respond to Tg B cells is likely due to specific Ag-pro- hybridoma are specific for the D protein of murine snRNPs and to cessing functions of these cells. We hypothesize that Tg B cells, by denatured DNA (22). To determine whether anti-snRNP autoreac- virtue of their surface receptor specificities, present a unique group tive B cells develop and reach the periphery, three-color immuno- of snRNP D peptides which T cells in the non-Tg mice have never fluorescence of spleen cells with anti-B220, anti-IgM, and biotin- contacted and which have never been deleted. CD4 T cells origi- ylated rSm-D was performed. The percentage of IgMϩ cells that nating from B10.A Tg mice failed to respond to peptide presented stain with rSm-D ranges from 15–30% in B10.A, C57BL/6, and by either non-Tg or Tg B10.A B cells, suggesting that these au- MRL lpr/lpr 2-12 Tg mice (Fig. 1), consistent with our previous toreactive T cell subsets have been anergized in Tg mice, presum- studies performed in C57BL/6 mice (18). The specificity of the Tg ably by Tg B cells. All T cell responses were inhibited by the B cells binding to snRNP D protein was confirmed with cold Ag presence of Abs to class II MHC (Ref. 19 and data not shown). It competition (data not shown). In such assays, unlabeled snRNP is unlikely that T cell responses in this study are due to autologous 3190 REGULATION OF T CELL TOLERANCE AND AUTOIMMUNITY

As seen in Fig. 2B, CD4 T cells from wt mice went through five rounds of division compared with no division of “tolerized” CD4 T cells from 2-12 Tg B10.A mice. Together, these results demon- strate that autoreactive T cells can be found in the normal reper- toire of naive mice by activation with Ag-specific APC. These observations were in contrast to similar experiments per- formed 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 signif- icantly 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 toler- ized 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 T cells from either Tg or wt B10.A mice responded significantly to snRNP peptide presented by Tg MRL lpr/lpr B cells, but not from MRL lpr/lpr wt B cells (Fig. 3B). This outcome implies that Downloaded from autoreactive T cells are not deleted in B10.A Tg mice and that “tolerance” can be overcome in vitro by Tg MRL B cells as APCs. Autoreactive T cells are tolerized in the periphery of 2-12 Tg normal mice We next investigated whether the tolerance induction of autoreac- tive T cells is mediated by 2-12 Tg B cells and the sites where http://www.jimmunol.org/ tolerance occurs. The expression of tolerance or autoimmunity was dependent on either the presence of anti-snRNP Tg B cells and/or FIGURE 2. The induction of activation/tolerance of autoreactive T cells ϩ the background, B10.A vs MRL. With these two variables in mind, 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 we investigated whether tolerance was mediated centrally in the purified 2-12 anti-snRNP B cells of B10.A mice with or without self-Ag thymus or in the periphery. CD4 T cells were purified from the rSm-D (5 ␮g/ml) for 72 h. Proliferation was quantified by uptake of thymus or spleen of individual mice and examined for their re- [3H]TdR (see Materials and Methods). CD4 T cells from same source were sponse to snRNP autoantigen. As illustrated in Fig. 4A, both thy- also CFSE-labeled and stimulated with 2-12 Tg B cells in vitro for 3 days. mic and splenic CD4 T cells from wt mice responded to D protein by guest on October 2, 2021 B, The CD4 T cells in two critical conditions as indicated were examined presented by Tg B cells. As found in Fig. 2, splenic CD4 T cells for cell division by FACS analysis. from Tg mice failed to respond to D proteins. However, thymic CD4 T cells from the same Tg mouse had a significant response (ϳ10-fold greater) to rSm-D protein presented by Tg B cells. This mixed lymphocyte reaction because the final data panel (T cells observation indicates that autoreactive T cells are not centrally and APCs from Tg B10.A mice) shows no detectable proliferative tolerized in the thymus, but instead are tolerized by Tg B cells in responses. We next labeled CD4 T cells with CFSE in cultures with 2-12 4 J. Yan, S. Shinde, M. Shlomchik, and M. Mamula. Differential regulation of T cell Tg B cells to further confirm that autoreactive T cells from wt mice tolerance and activation by B cells in normal or autoimmunity-prone mice. Submitted are able to divide in vitro from stimulation with 2-12 Tg B cells. for publication.

FIGURE 3. T cell autoimmunity in lupus-prone MRL lpr/lpr anti-snRNP Ig Tg mice. CD4ϩ T cells were purified from wt or Tg MRL lpr/lpr mice, as indicated. CD4ϩ T cells were cocultured with irradiated purified B cells with medium alone or with rSm-D (5 ␮g/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. The Journal of Immunology 3191

FIGURE 4. The induction of autoreactive T cell tolerance in B10.A Tg mice is mediated by Tg B cells in the periphery. CD4ϩ T cells purified from wt (A)orTg(B) B10. A thymus and spleen were cocul- tured with B10.A Tg B cells for 72 h. Proliferation was quantified by uptake of [3H]TdR.

the periphery. The level of TCR expression in all CD4 T cells is B10.A mice transferred into B10.A Tg mice failed to drive auto- similar (data not shown). production (data not shown). These latter observations demonstrate that minor MHC differences between donor and re- Downloaded from Dual TCR T cells contribute to the autoimmune repertoire to cipient mouse strains was not a basis for autoantibody production. snRNP D protein More importantly, this observation suggests that Ag-specific au- Studies using TCR Tg mice have demonstrated that several mech- toreactive T cells can deliver helper signals for autoreactive Tg B anisms could be involved in preventing autoreactive T cells from cells of nonautoimmune-prone mice. being deleted (28–31). For example, it has been found that dual TCR-bearing T cells expressing self-specific TCRs escape and are functionally competent to the “self” Ag in vivo. http://www.jimmunol.org/ 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 ␣Ϫ/ϩ␤Ϫ/ϩ mice as well as wt mice were cocultured with 2-12 Tg B cells in the presence of ␣Ϫ/ϩ␤Ϫ/ϩ

snRNP D protein. TCR mice are unable to generate two by guest on October 2, 2021 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 pro- tein as we described above (Fig. 2A). However, the response from TCR ␣Ϫ/ϩ␤Ϫ/ϩ mice is reduced by 50% compared with that from the wt. The decreased response is specific and comparable because wt and TCR ␣Ϫ/ϩ␤Ϫ/ϩ CD4 T cells respond identically to anti-CD3 stimulation (Fig. 2B). The reduced response of TCR ␣Ϫ/ϩ␤Ϫ/ϩ CD4 T cells suggests that dual TCR T cells constitute part of the self-reactive T cell repertoire in the periphery. How- ever, 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.

Anti-snRNP autoantibody production in B10.A 2-12 Tg mice reconstituted with MRL lpr/lpr anti-snRNP Tg mice CD4 T cells 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 trans- FIGURE 5. The ␣ϩ/Ϫ␤ϩ/Ϫ CD4 T cell repertoire contains reduced ferred into B10.A 2-12 Tg mice and wt littermates. MRL lpr/lpr autoreactive T cells against snRNP D protein compared with the wt re- animals with autoantibody production (16–30 wk of age) were pertoire. CD4 T cells purified from unimmunized C57BL/6 wt and used as the source of donor CD4 T cells. Anti-snRNP autoanti- TCR␣Ϫ/ϩ␤Ϫ/ϩ mice and cocultured with 2-12 Tg B cells. The proliferation bodies were detected in B10.A 2-12 Tg mice, but not in non-Tg was measured after a 3-day culture. As control, anti-CD3 Ab 2C11 was mice 1 mo after transfer of T cells from MRL lpr/lpr Tg mice (Fig. added with B6 B cells to demonstrate CD4 T cell proliferation is nearly 6). In contrast, donor CD4 T cells from B10.A Tg or non-Tg identical between mouse strains (B). 3192 REGULATION OF T CELL TOLERANCE AND AUTOIMMUNITY

FIGURE 6. Anti-snRNP autoantibodies are pro- duced in B10.A 2-12 Tg mice reconstituted with CD4ϩ T cells from MRL lpr/lpr Tg mice. Ten mil- lion purified CD4ϩ 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 rep- resents individual mouse serum before and after CD4ϩ T cell transfer. Downloaded from

Transferred CD4 T cells can migrate to regions of lymphoid 13, 36). There is an accumulating data suggesting that B cells are tissue proximal to follicles capable of presenting autoantigen in activating autoreactive T cells T cell-dependent Ab responses typically require cognate interac- (12, 15). Direct evidence comes from studies of B cell-deficient tion between Ag-presenting B cells and Ag-specific T cells. Ag- MRL lpr/lpr mice which fail to generate spontaneously activated T http://www.jimmunol.org/ mediated cross-linking of the B cell receptor promotes B cell lo- cell populations that are normally found in wt MRL lpr/lpr mice calization 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 by guest on October 2, 2021 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 res- ident B cells. It also provides rationale for why MRL T cells drive autoantibody production through cognate B-T interactions in recipient mice.

Discussion Self-reactive T cells are conventionally regarded to be eliminated by negative selection in the thymus or by the induction of tolerance in the periphery (4, 35). The results described here demonstrate that snRNP-reactive T cells can be detected and activated in nor- mal naive mice by autoantigen-specific B cells as APCs. However, these autoreactive T cells are normally anergic because they can- not proliferate in vitro by coculturing with autologous nonspecific B cells and self peptides. In contrast, autoreactive T cells are spon- taneously activated in lupus-prone MRL lpr/lpr mice. In addition, 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 spontaneously activated autoreactive T cells can provide help to ϩ autoreactive B cells from normal mice (anti-snRNP Tg B10.A confocal microscopy of splenic sections of CFSE-labeled CD4 T cells transferred into recipient mice (donor and recipient mice as indicated). strains) for autoantibody production. Splenic tissues were isolated and examined 24 h after cell transfer. CFSE- Autoreactive T cells inhabit the naive repertoire of normal mice 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 The importance of B cells as autoantigen presenting cells has been representative of three mice for each transfer group. F, follicle; T, T cell emphasized by previous studies in our laboratory and others (8, 10, zone. Original magnification: ϫ100. The Journal of Immunology 3193

(12, 13). In the present study, we made use of Ig Tg mice in which It is also possible that the frequency of self-reactive T cells are B cells have specificity for the snRNP D protein (18). Our results decreased in the single TCR repertoire compared with dual TCR- indicate that naive CD4 T cells respond to peptides presented by bearing T cells. Enumerating snRNP-reactive T cells from both Tg B cells even without exogenously added Ag (Fig. 1). Two single and dual TCR T cells is presently underway and should important concepts have emerged from this simple experiment. resolve the latter question. First, autoreactive B cells bearing surface Ig specific for snRNP T cell help drives autoantibody production in encounter a source self-Ag in vivo, and subsequently bind and nonautoimmune-prone mice present snRNP peptides to T cells. We do not yet know the phys- iologic site of snRNP autoantigen that is captured and presented by snRNP-specific, autoreactive T cells are actively tolerized in non- Tg B cells, whether from natural cell death or apoptotic cells. autoimmune Tg mice, but not in autoimmune-prone MRL mice. Second, autoreactive T cells inhabit the naive repertoire of normal Furthermore, the transfer of CD4 T cells from MRL lpr/lpr Tg mice and can be activated by autoantigen-specific B cells as APCs. mice into B10.A Tg mice initiates an anti-snRNP autoantibody Autoreactive T cells constitute the normal T cell peripheral rep- response. This observation demonstrates that Ag-specific T cells ertoire as found here and by studies showing that a high frequency can provide help for anti-snRNP B cell of nonautoimmune origin of proteolipid protein139–151-reactive T cells are found in naive to produce autoantibodies in vivo. We do not yet understand the mice without immunization (6). Additionally, studies from TCR factors that explain a lack of adequate CD4 T cell helper functions Tg mice have demonstrated that most self-reactive T cells are not of wt B10.A mice when transferred into B10.A Tg mice. However, completely deleted by negative selection in the thymus. Low-avid- we presume that a high frequency of snRNP-specific T cells exist ity T cells escape thymic deletion and are retained in the mature in Tg MRL mice because they are easily activated with Ag in vitro. Downloaded from population (37–39). Not surprisingly, even CTLs spe- This premise is also supported by recent studies demonstrating that ␤ cific to ubiquitous proteins such as 2-microglobulin, hemoglobin, MRL T cells are activated by lower thresholds of Ag stimulation or kallikrein persist in the peripheral repertoire and can be acti- as compared with normal background T cells (43). vated under appropriate conditions (40, 41). Moreover, autoantibody production requires autoreactive T-B Splenic CD4 T cells from Tg B10.A mice encountering Ag pre- cell collaboration, and is supported by the colocalization of T cells sented by Tg B cells in vivo become anergic. This provides one and B cells in the follicles as presented here. The trafficking of explanation why Tg B cells of normal (nonautoimmune) origin are transferred cells within secondary lymphoid tissues of the host was http://www.jimmunol.org/ not able to spontaneously produce autoantibodies. This observa- performed to help explain biology of T cell help for autoantibody tion is also consistent with previous studies demonstrating the pro- production (44–47). CD4 T cells from MRL lpr/lpr Tg mice mi- liferative response of T cells to hen egg lysozyme (HEL) “autoan- grated to within the margins of B cell zones in the spleen. In tigen” delivered by anti-HEL Tg B cells or by Ag delivered to contrast, transferred CD4 T cells from B10.A Tg or non-Tg mice other APCs by B cells (14). However, unlike these studies, we do failed to migrate to the B cell follicles consistent with their inabil- not have evidence for Fas-mediated B cell death after T cells are ity to drive autoantibody production under these conditions. Pre- activated in Tg B10.A mice. vious studies have demonstrated that Ag-specific Tg T and B cells

Our data clearly show that T cells are not deleted in 2-12 Tg initially remain localized without contact in their respective zones. by guest on October 2, 2021 model and can be partially activated by 2-12 MRL lpr/lpr Tg B Following Ag stimulation, these cells move in a synchronous fash- cells in vitro. Recent studies using HEL Ab/Ag double Tg mice ion out of their respective sites for cognate interactions at the in- which have shown that HEL-specific T cells are tolerized in HEL terface of these zones (34, 48, 49). Ag single Tg mice (50). However, T cells from Ag/Ab double Tg In conclusion, these studies illustrate the presence of autoreac- mice escape central deletion and experience a partial breakdown of tive T cells both in normal naive mouse and MRL lpr/lpr mouse, peripheral tolerance presumably due to the presentation of HEL a murine model of human systemic lupus erythematosus. The in- Ag by HEL-specific B cells in double Tg mice (50). We found that duction of autoimmunity relies on the presence and cognate inter- thymic T cells from 2-12 Tg mice are activated by 2-12 Tg B cells actions of specific T cell-helper functions with B cells. Perhaps while peripheral CD4 T cells from the same mouse are anergic. more importantly, these studies illustrate that B cells as APCs play This observation suggests that autoreactive T cells in the 2-12 Tg an important role both in the activation and tolerance induction of B10.A mice are not deleted in the thymus, but are actively aner- autoreactive T cells, depending on the background. These obser- gized by Tg B cells in the periphery. Autoreactive T cells in the vations have implications for the understanding of autoreactive T thymus of 2-12 Tg mice emigrate into periphery-encountering Tg cell tolerance or autoimmunity and the initiation of autoantibody B cells, and result in T cell tolerance via cognate interactions. We responses in normal individuals in which aberrant autoreactive T believe this to be the mechanism by which 2-12 Tg mice in the cell activation may arise. normal background maintain self-tolerance. T cell anergy in the periphery can be overcome by the transfer of nontolerant MRL Acknowledgments lpr/lpr T cells that provide help for autoantibody production. The We thank Drs. Stephen H. Clarke and Sandra Santulli-Marotto for the activation of B cells and autoantibody in the B10.A Tg mice also development of the 2-12 Tg mice and for their helpful discussion. We also leads to Ab deposition and cellular infiltration into the kidney.4 thank Dr. Mark Shlomchik for his constructive comments of this manu- script. We thank Dr. Linda Bockenstedt for developing the technology of Our results also indicate that dual TCR-bearing T cells may purifying cloned recombinant snRNP-D protein. partially contribute to the autoreactive T cell repertoire. Although the physiological roles of dual TCR T cells still are not clear, References previous work has demonstrated that dual TCR T cells may rescue 1. Bevan, M. J. 1977. In a radiation chimaera, host H-2 determine immune autoreactive T cells from negative selection in the thymus (30). responsiveness of donor cytotoxic cells. Nature 269:417. However, our data imply that autoreactive T cells are also found in 2. Kappler, J. W., N. Roehm, and P. Marrack. 1987. T cell tolerance by clonal elimination in the thymus. Cell 49:273. the single TCR repertoire. This latter notion is supported by the 3. Singer, A., R. Bosselut, and A. Bhandoola. 1999. Signals involved in CD4/CD8 work from the Allen laboratory (42) which demonstrates the bind- lineage commitment: current concepts and potential mechanisms. Semin. Immu- nol. 11:273. ing of two separate ligands, a self-peptide (arthritic peptide), and a 4. von Boehmer, H. 1990. Developmental biology of T cells in T cell-receptor foreign on distinct MHC by T cells bearing a single TCR. transgenic mice. Annu. Rev. Immunol. 8:531. 3194 REGULATION OF T CELL TOLERANCE AND AUTOIMMUNITY

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