To study the role of CD25+ regulatory T cells (Tregs) in peripheral B cell tolerance, we generated transgenic rat insulin promoter RIP-OVA/HEL mice expressing the model Ags OVA and HEL in pancreatic islet β cells (where RIP is rat insulin promoter and HEL is hen egg lysozyme). Adoptively transferred transgenic OVA-specific CD4+ and CD8+ T cells proliferated only in the autoantigen-draining pancreatic lymph node (PLN), demonstrating pancreas-specific Ag expression. Transferred HEL-specific transgenic B cells (IgHEL cells) disappeared within 3 wk from transgenic but not from nontransgenic mice immunized with autoantigen. Depletion of CD25+ FoxP3+ cells completely restored IgHEL cell numbers. Treg exerted an analogous suppressive effect on endogenous HEL-specific autoreactive B cells. Tregs acted by inhibiting the proliferation of IgHEL cells in the spleen and PLN and by systemic induction of their apoptosis. Furthermore, they reduced BCR and MHC II surface expression on IgHEL cells in the PLN. These findings demonstrate that autoreactive B cells specific for a nonlymphoid tissue autoantigen are controlled by Tregs.
Autoreactive B cells are controlled by several checkpoints that operate in the bone marrow by receptor editing or central deletion (1, 2, 3, 4) and in the periphery by inducing deletion or anergy (4, 5). Anergic B cells fail to enter the long-lived mature B cell pool and are arrested in a transitional developmental stage that is characterized by low surface IgM, reduced BCR signaling, and Ab secretion (4, 6, 7). In the periphery, autoreactive B cells are deleted after continuous BCR signaling (8) or controlled by limiting T cell help (9). Recently, regulatory T cells (Tregs)4 have been proposed to also suppress B cells based on the observations that Treg depletion aggravated disease in some autoantibody-mediated disease models (10, 11, 12) and that Tregs induced B cell apoptosis in vitro (13, 14, 15). Suppression of autoreactive T cells by Tregs has been shown to occur in autoantigen (auto-Ag)-draining lymph nodes (LNs) by inhibiting their proliferation (16). The in vivo role of Tregs in the suppression of B cells against nonlymphoid tissue-restricted auto-Ags has not yet been studied. In the present work, we addressed this question by using a novel transgenic (tg) model that allowed following the fate of naive B cells specific for a tg pancreatic auto-Ag after adoptive transfer.
Materials and Methods
Animals and reagents
C57/BL6, OT-I.Rag−/−, OT-II, and IgHEL (B cells expressing a tg BCR for hen egg lysozyme (HEL)) mice were bred under specific pathogen-free conditions and used at 8–16 wk of age in accordance with German animal experimentation guidelines. To generate tg mice expressing OVA and HEL under control of the rat insulin promoter (RIP) (ROHhigh), HEL cDNA amplified from the plasmid plExV3-HEL (17) was introduced into the pBlueRIP/Tfr-Ova plasmid that had been used for generating RIP-membrane bound OVA (mOVA) mice (18, 19). A 3.47-kb fragment containing RIP, the human transferrin receptor membrane domain, the genes for OVA (aa 161–407) and HEL, and an SV40 poly(A) tail was excised and injected into pronuclei of fertilized C57/BL6 oocytes. All reagents, if not specified otherwise, were from Sigma-Aldrich. CD25+ cells were depleted by injecting 300 μg of PC61.5 Ab purified from a hybridoma supernatant.
Cell preparation and adoptive transfer
IgHEL cells were isolated from spleens of IgHEL mice, depleted of erythrocytes by buffered NH4Cl, and enriched by magnetic separation using a B cell separation kit (Miltenyi Biotec). OT-I and OT-II cells were isolated and labeled with CFSE as described (19).
ELISPOT and Western blotting
Cell suspensions were incubated for 4 h on ELISPOT plates (Millipore) coated with HEL. Bound Ab was detected with biotinylated goat anti-mouse IgG (Dianova) or rat anti-mouse IgMa Ab (BD Biosciences). Spots were developed with a 3-amino-9-ethycarboazole substrate and analyzed with a Bioreader 200 and Bioreader 2006 software (Bio-Sys). Western blotting was performed according to standard procedures using rabbit anti-OVA antiserum or rabbit anti-actin Ab, goat anti-rabbit-HRP, and an ECL Western blotting substance kit (Pierce, Bonn Germany).
Flow cytometry and statistics
Ab were from BD Biosciences except for anti-B220 and anti-Foxp3 from eBioscience. Soluble HEL was conjugated to the Alexa Fluor 647 fluorochrome with a commercial kit (Invitrogen) and used at 2.5 μg/ml to stain specific B cells. Apoptosis was detected with a fluorescent dye specific for activated caspase 3/7 (Immunochemistry Technologies), and dead cells were detected by Hoechst 33342 dye. Statistical t test analysis was done with Prism 4.0 (GraphPad). p < 0.05 was considered statistically significant.
Results and Discussion
Activation of autoreactive T cells in the pancreatic LN of ROHhigh mice
We generated transgenic ROHhigh mice expressing the membrane-bound model Ags OVA and HEL as a fusion protein in pancreatic islet β cells (construct shown in Fig. 1,A). These animals allowed the use of tg OVA-specific T cells (19) and HEL-specific B cells of the MD4 line (6) as surrogate autoreactive T and B cells, respectively. Western blot analysis showed transgene expression only in the pancreas (Fig. 1,B). Site-specific presentation of the tg autoantigen was determined using CFSE-labeled OVA-specific CD8+ T cells (OT-I cells) that represent sensitive in vivo probes for OVA presentation (19). Three days after adoptive transfer, OT-I cells had proliferated only in the pancreatic LN (PLN) of tg mice, whereas few proliferated cells were seen in other LNs or in non-tg mice (Fig. 1,C). OVA-specific CD4+ T cells (OT-II cells) also proliferated only in the PLN of ROHhigh mice, albeit at a very low rate detectable on day 5 (Fig. 1 D), consistent with previous studies reporting weak responses of these cells against tg auto-Ag (19, 20).
CD25+ cells induce peripheral tolerance of B cells specific for pancreatic auto-Ag in the spleen and pancreatic LN
To study peripheral B cell tolerance against pancreatic auto-Ag, we transferred autoreactive B cells into ROHhigh mice. As B cells of a given specificity are extremely rare in a normal repertoire, we used transgenic IgHEL cells from the well-characterized MD4 line as surrogate autoreactive B cells, with the constraint that their tg IgM BCR cannot switch to other subclasses (6). Adoptive transfer avoids central tolerance, which cannot be excluded if IgHEL mice are crossed with HEL-expressing mice. Such transfer studies had been instrumental in dissecting the mechanisms of peripheral CD8+ T cell cross-tolerance against pancreatic auto-Ag in the transgenic RIP-mOVA system (19).
After the transfer of IgHEL cells, ROHhigh mice were challenged with HEL/alum (protocol in Fig. 2,A). After 3 wk, the spleens were examined by ELISPOT for B cells forming HEL-specific spots of the IgMa allotype characteristic of IgHEL cells. Splenocytes from ROHhigh mice produced 80% fewer spots than non-tg controls (Fig. 2,B), demonstrating Ag-specific peripheral tolerance of IgHEL cells. To investigate whether this depended on Tregs, we transferred IgHEL cells into mice treated with PC61.5 Ab. This treatment eliminated >98% of CD25+FoxP3+CD4+ T cells in the blood (data not shown). Depletion of CD25+ cells restored the functionality of splenic IgHEL cells, whereas no changes were observed in non-tg mice (Fig. 2,B). Also, endogenous autoreactive B cells, which could be detected by measuring HEL-specific IgG+ spots, were less numerous in the spleens of ROHhigh mice (Fig. 2,C). Depleting CD25+ cells also restored their numbers to those in non-tg mice, whereas no changes were seen in non-tg mice (Fig. 2 C). Thus, CD25+ cells were required for Ag-specific peripheral tolerization of IgHEL cells in the spleen.
Few Ab-forming sports were found in LNs by ELISPOT (Fig. 2,B and data not shown), with one exception. In the tiny PLN, autoreactive B cells of ROHhigh mice depleted of CD25+ cells were increased ∼5-fold over other LNs (Fig. 2,B). To verify this observation with a different technique that does not rely on Ab secretion, we enumerated IgHEL cells in the pooled PLNs of four mice by flow cytometrical detection of HEL-binding IgMa+ B220+ cells. This analysis confirmed that IgHEL cells were less abundant in the PLNs of ROHhigh mice than in those of non-tg controls (Fig. 2,D). Depletion of CD25+ cells restored IgHEL cell numbers to levels even higher than in non-tg mice (Fig. 2 D). Thus, CD25+ cells suppressed IgHEL cells in the spleen and PLN of ROHhigh mice. This interpretation was further confirmed by in vitro culture studies where cells from these two organs, but not those from a nondraining control LN, suppressed Ab production by cocultured IgHEL cells challenged with Ag, albeit this was statistically significant only for PLN cells (supplemental. Fig. 1).5 No suppression was observed when these cells were separated from IgHEL cells in Transwell chambers (data not shown).
The CD25+ cell-dependent B cell loss from the spleen may be related to the ability of this organ to concentrate circulating Ags, such as HEL used for immunization, by means of a conduit system that renders this organ more efficient at presenting such Ags than LNs (21, 22). The conduit system of LNs is more efficient at gathering Ag arriving through afferent lymphatics (23), which in our system was the PLN. Thus, the loss of autoreactive B cells from the PLN may have been due to the presentation of an islet-derived auto-Ag there (Fig. 1). Such presentation has been reported to induce CD25+FoxP3+ Tregs in the PLN of related tg RIP-mOVA mice (24).
CD25+ cells prevent proliferation of autoreactive B cells and induce their apoptosis
The CD25+ cell-dependent loss of IgHEL cells theoretically may be due to reduced proliferation in response to auto-Ag, increased apoptosis, or both. To examine proliferation, we transferred CFSE-labeled IgHEL cells into ROHhigh mice (Fig. 3,A). Without immunization, IgHEL cells did not proliferate in any site, including the PLN, even after depletion of CD25+ cells (data not shown). After HEL/alum immunization, IgHEL cells proliferated in the spleens of tg and non-tg mice, and this was substantially higher in ROHhigh mice depleted of CD25+ cells after 3 (Fig. 3,B) or 5 days (supplemental. Fig. 2). Also in the PLN, significant IgHEL cell proliferation was detected in CD25+ cell-depleted ROHhigh mice as opposed to other LNs (Fig. 3,B). This effect on B cell proliferation may explain why IgHEL cells were more frequent in the PLNs of PC61.5-treated ROHhigh mice than in those of non-tg controls (Fig. 2 C).
To examine apoptosis, we determined caspase-3/7 activity. Strikingly, in the PLNs and spleens of ROHhigh mice >90% of the transferred IgHEL cells were caspase-3/7-positive and in nondraining LNs the number was ∼75% (data not shown), whereas in non-tg controls only 20–30% were apoptotic (Fig. 3,C). Depletion of CD25+ cells reduced apoptosis in ROHhigh mice to the levels in non-tg mice, while in these controls again no changes were seen (Fig. 3,C). Thus, CD25+ cells not only impaired proliferation but also induced apoptosis of autoreactive B cells in an Ag-specific manner. This may be important to incapacitate IgHEL cells in ROHhigh mice, because their proliferation could only be partially suppressed by CD25+ cells in the spleen (Fig. 3 B). This manner of peripheral B cell tolerance resembles cross-tolerance of CD8+ T cells, where deletion was also preceded by proliferation (19). Although auto-Ag in our transgenic systems was confined to a nonlymphatic organ, B cell apoptosis was seen also in the spleen and nondraining LNs. This may be due to B cell apoptosis induction in these sites, e.g., by recirculated Tregs or to B cell recirculation after apoptosis induction elsewhere, e.g., in the PLN. This may be important to incapacitate autoreactive B cells also in sites that do not drain auto-Ag.
Theoretically, apoptosis of autoreactive B cells may have been mediated by auto-Ag-specific CD25+ CD8+ CTLs. In this scenario, PC61.5 treatment might have caused depletion of CD25+ CTLs and thereby prevented B cell apoptosis (Fig. 3 C). To rule out this possibility, we immunized PC61.5-treated ROHhigh mice with OVA/alum and determined OVA-specific cytotoxicity. We detected hardly any in non-tg controls and none in ROHhigh mice, even after CD25+ cell depletion (supplemental Fig. 3), arguing against CTLs as inducers of B cell apoptosis. Furthermore, ROHhigh mice did not develop diabetes after PC61.5 treatment and auto-Ag challenge (data not shown), which would be expected if significant cytotoxicity had been induced.
We next examined whether the BCR was down-regulated, which is known to occur during peripheral B cell tolerization (6, 25). Indeed, IgHEL cells in the PLN of ROHhigh mice showed nearly 50% BCR reduction (Fig. 3,D) and even 80% reduction of MHC II (Fig. 3,E), which were again restored after CD25+ cell depletion (Fig. 3, D and E). Interestingly, these effects were not observed in the spleen (Fig. 3, D and E). CD40 and CD86 expression were not markedly changed (supplemental Fig. 4).
This phenotype was partially reminiscent of anergic IgHEL cells in double transgenic mice coexpressing HEL as systemic self-Ag, because these also lacked proliferation and showed reduced surface IgM expression (1, 4, 7, 25). In contrast, IgHEL cells in our system were not irreversibly arrested in transitional development state 2 but instead could proceed to activated mature B cells (supplemental Fig. 5) and produce IgMa autoantibodies after antigenic challenge once CD25+ cells were depleted (Fig. 2 B). The down-regulation of MHC II by tolerized B cells, which has not been reported to date, may further contribute to suppression by reducing the effectiveness of T cell help.
In summary, we showed that CD25+ cells can suppress the response of autoreactive B cells to challenge with self-Ag by preventing their proliferation and by inducing their apoptosis in the spleen and auto-Ag-draining LN. Apoptosis induction is consistent with the previous demonstration that CD25+ T cells can kill B cells in vitro by Fas-Fas ligand (13) or by granzyme/perforin in an in vivo system with a systemic auto-Ag (14). In the PLN intrinsic functions were also impaired, as evidenced by reduced BCR and MHC II expression. An interesting mechanistic question for future studies is whether CD25+ cells tolerize autoreactive B cells by direct interactions or indirectly, e.g., by suppressing Th cells. Evidence for both mechanisms has been reported in other systems (13, 14, 15), and it is conceivable that both synergized in our model. Experimental separation between direct and indirect suppression is difficult, as B cell responses in Th-depleted animals are generally weak, hampering the assessment of further suppression by CD25+ cells. An additional question for future studies is whether B cell suppression involves also BCR editing, which cannot be studied with MD4 IgHEL cells. The ability to tolerize B cells specific for nonlymphoid tissue auto-Ags represents a further mechanism by which Tregs can control autoimmunity. Thus, Tregs may also be attractive therapeutic targets in diseases involving autoreactive B cells.
We thank Booshini Fernando for technical assistance, Stefan Thiel for providing the plExV3-HEL plasmid, Sjef Verbeek for microinjections, and David Tarlinton for helpful comments. We acknowledge technical support by the flow cytometry core facility and by the animal facilities of the University Clinic of Bonn (House for Experimental Therapy).
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
I.L.-P. was supported by fellowship Lu 1387/1-1 of the Deutsche Forschungsgemeinschaft and C.K. by a career development grant of the government of the German state of Nordrhein-Westfalen.
Abbreviations used in this paper: Treg, regulatory T cell; auto-Ag, autoantigen; HEL, hen egg lysozyme; IgHEL, B cell expressing tg Ig receptor for HEL; LN, lymph node; PLN, pancreatic lymph node; RIP, rat insulin promoter; ROHhigh mice, tg mice expressing OVA and HEL under control of RIP; tg, transgenic.
The online version of this article contains supplemental material.