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The Journal of Immunology, 2004, 173: 2942-2951.
Copyright © 2004 by The American Association of Immunologists

CD4+CD25+ Cells Controlling a Pathogenic CD4 Response Inhibit Cytokine Differentiation, CXCR-3 Expression, and Tissue Invasion1

Nadia Sarween*, Anna Chodos{dagger}, Chandra Raykundalia*, Mahmood Khan*, Abul K. Abbas{dagger} and Lucy S. K. Walker2,*

* Medical Research Council Center for Immune Regulation, University of Birmingham Medical School, Birmingham, United Kingdom; and {dagger} Department of Pathology, University of California, San Francisco, CA 94143


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It is well established that CD4+CD25+ regulatory T cells (Tregs) inhibit autoimmune pathology. However, precisely how the behavior of disease-inducing T cells is altered by Tregs remains unclear. In this study we use a TCR transgenic model of diabetes to pinpoint how pathogenic CD4 T cells are modified by Tregs in vivo. We show that although Tregs only modestly inhibit CD4 cell expansion, they potently suppress tissue infiltration. This is associated with a failure of CD4 cells to differentiate into effector cells and to up-regulate the IFN-{gamma}-dependent chemokine receptor CXCR-3, which confers the ability to respond to pancreatic islet-derived CXCL10. Our data support a model in which Tregs permit T cell activation, yet prohibit T cell differentiation and migration into Ag-bearing tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ability of the CD4+CD25+ subset of regulatory T cells (Tregs)3 to inhibit autoimmunity has attracted much attention (1, 2, 3, 4, 5). Despite this, it is still unclear which components of a pathogenic immune response are influenced by regulatory T cells in vivo. In vitro studies have suggested that CD4+CD25+ cells impact the earliest stages of T cell activation, inhibiting activation marker expression, IL-2 production, proliferation, and even the increase in cell size associated with activation (6, 7). However, in vitro studies bypass the anatomical constraints on APC, Th, and Treg localization that exist in vivo, and subsets of cells that suppress equivalently in vitro do not necessarily show equivalent regulatory function in vivo (8).

Multiple animal models have convincingly demonstrated the regulatory activity of CD4+CD25+ cells in vivo (2, 5, 8, 9, 10, 11). One factor that often limits such studies is the inability to identify the Ag-specific T cells responsible for disease induction and to directly track their response to regulation. We have developed a TCR transgenic model of diabetes that enables us to dissect how the phenotype and behavior of the Ag-specific T cells that mediate tissue injury is impacted by CD4+CD25+ regulatory T cells. In this model, activation of CD4 T cells of a single specificity (OVA-specific DO11 cells) is sufficient to trigger diabetes, and this can be prevented by the cotransfer of CD4+CD25+ cells. Using the clonotypic anti-TCR Ab to track the DO11 cells in both regulated (plus CD4+CD25+) and unregulated (plus CD4+CD25–) contexts, we have been able to define which parameters of a pathogenic CD4 cell response are modulated in vivo by CD4+CD25+ cells. We show that although CD4+CD25+ cells allow Ag-specific activation and accumulation of pathogenic T cells, they potently inhibit the ability of such T cells to produce IFN-{gamma} and infiltrate tissue.

Regulation of the nature and extent of pancreatic islet infiltration is known to be a critical checkpoint in the control of diabetes (12). In many cases, insulitis can be tolerated for long periods of time without perturbation of blood glucose homeostasis. Examples include models involving the transgenic expression of certain cytokines in pancreatic {beta} cells (13, 14, 15, 16) or where T cell specificity is transgenically directed against an islet Ag, either natural (17) or engineered (18, 19). In such cases the infiltrating lymphocytes are frequently restricted to the periphery of the pancreatic islets (termed peri-insulitis) rather than penetrating the {beta} cell mass. The genetic pathways that control the progression of benign peri-insulitis to invasive destructive insulitis are clearly of critical importance in the regulation of autoimmune pathology.

Interestingly, some studies implicate IFN-{gamma} in controlling this particular checkpoint. The amelioration of diabetes observed in the context of IFN-{gamma} blockade (20, 21, 22) or genetic deficiency of IFN-{gamma} (23, 24) or its receptor (25) is not merely a reflection of reduced {beta} cell-directed cytotoxicity, but instead presents as a defect at the level of pancreatic islet infiltration. This suggests that T cell effector cytokine production may be important in the transition from benign to invasive insulitis. Using our TCR transgenic model we show that the control of diabetes by CD4+CD25+ cells is associated with a striking reduction in IFN-{gamma} production from Ag-specific CD4 cells and a concomitant inhibition of pancreatic islet infiltration. Expression of the IFN-{gamma}-dependent chemokine receptor CXCR3, which has been implicated in pancreatic islet infiltration (26), is reduced in the presence of CD4+CD25+ cells, providing a mechanism by which regulatory T cells can curb pathogenicity through altered T cell trafficking.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

DO11.10 TCR transgenic and BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). RAG2–/– mice were purchased from Taconic Laboratories (Germantown, NY). Rat insulin promoter (RIP)-mOVA mice on a BALB/c background expressing a membrane-bound form of OVA under the control of the rat insulin promoter (fromline 296-1B) were a gift from W. Heath (WEHI, Melbourne, Australia). DO11.10 mice and RIP-mOVA mice were bred to a RAG2–/– background (designated DO11/rag and RIP-mOVA/rag, respectively). DO11.10 mice and RIP-mOVA were crossed as previously described (27). Mice were housed at the University of California San Francisco animal facility and the University of Birmingham Biomedical Services Unit and were used according to institutional guidelines. Mice were genotyped using PCR and flow cytometry and were between 6 and 10 wk of age at the start of each experiment.

T cell transfers

Combined lymph node (LN; axillary, inguinal, brachial, popliteal, and mesenteric) cells from DO11/rag mice were stained with the clonotypic Ab KJ126-allophycocyanin (Caltag Laboratories, Burlingame, CA) to assess the proportion of DO11 cells (routinely >85%), and the indicated number of DO11 cells was injected into RIP-mOVA/rag recipient mice i.v. in the tail vein. CD4 cells were purified from the combined LN of normal or CD28–/– BALB/c mice by negative selection using CD8-FITC and CD19-FITC, followed by anti-FITC magnetic beads (Qiagen, Valencia, CA). CD4+CD25+ and CD4+CD25– cells (from BALB/c LN) or KJ126+CD25+ and KJ126+CD25– cells (from LN of DO11xRIP-mOVA mice) were purified by high speed cell sorting (MoFlo; DakoCytomation, Fort Collins, CO). Where indicated, cells were incubated before transfer with 1 µM CASE (Molecular Probes, Eugene, OR) for 10 min at room temperature, followed by two washes with RPMI 1640 supplemented with 1 mM L-glutamine, penicillin, streptomycin, nonessential amino acids, sodium pyruvate, HEPES (all from Invitrogen Life Technologies, Grand Island, NY), 5 x 10–5 M 2-ME, and 10% FBS (Sigma-Aldrich, St. Louis, MO).

Immunization

OVA protein (Sigma-Aldrich) was prepared emulsified in IFA (Difco, Detroit, MI), and 200 µg was administered s.c. in the flank where indicated. Anti-IFN-{gamma} (clone R46A2) was prepared by protein G purification of hybridoma culture supernatant. Rat IgG was purchased from Zymed Laboratories (South San Francisco, CA). One milligram of the indicated Ab in PBS was injected i.p. on days 0 and 3 (relative to OVA/IFA administration) where indicated.

Flow cytometry

Cells were stained with KJ126-allophycocyanin, CD25-PE (PC61), CD62L-FITC (Mel-14), CD69-PE (H1.2F3), CD4-PerCP (L3T4), IFN-{gamma}-PE (XMG1.2), IL-4-PE (11B11), and IL-2-PE (JES6-544). CXCR-3 was detected with a rabbit polyclonal Ab (ZCM3; Zymed Laboratories) and visualized using anti-rabbit PE (Jackson ImmunoResearch Laboratories, West Grove, PA), anti-rabbit FITC (Southern Biotechnology Associates, Birmingham, AL), or anti-rabbit biotin (Southern Biotechnology Associates) followed by streptavidin-allophycocyanin or streptavidin-PE. Equivalent data were obtained using each method of CXCR-3 detection, although the overall intensity of staining varied slightly depending on the flurochrome used. All Abs were purchased from BD Pharmingen (San Diego, CA) unless otherwise indicated. For intracellular cytokine staining, cells were restimulated for 4 h with 1 µg/ml OVA peptide in the presence of 10 µg/ml brefeldin A for the final 2 h. Cells were fixed for 10 min with 4% paraformaldehyde after surface staining, then permeabilized with 0.5% saponin (Sigma-Aldrich) and stained with Abs against intracellular markers for 15 min at room temperature. Stained cells were washed once with 0.5% saponin and once with 1% FBS in PBS before analysis. Gates were set using isotype-matched control Abs.

Immunohistology

Acetone-fixed frozen pancreas sections were stained with KJ126 mAb that was detected with alkaline phosphate-conjugated anti-mouse IgG2a and rabbit polyclonal anti-mouse insulin (Santa Cruz Biotechnology, Santa Cruz, CA) that was detected with HRP-conjugated goat anti-rabbit IgG (Southern Biotechnology Associates). Pancreatic islets were scored according to the degree of infiltration, with individual islets being classified as having no infiltrate, peri-islet infiltrate (KJ126+ cells present, but confined to the periphery of islets), or invasive infiltrate (KJ126+ cells penetrating {beta} cell mass). A minimum of 15 islets were scored per pancreas section.

PCR

RNA was extracted from pancreas sections taken from the indicated mice using RNAzol B (Biogenesis, Poole, U.K.) and reverse transcribed to cDNA. PCR was performed using the following primers: {beta}-actin forward, AGCGGGAAATCGTGCGTG; {beta}-actin reverse, CAGGGTACATGGTGGTGCC; CXCL10 forward, ATCCTGCTGGGTCTGAG; and CXCL10 reverse, TAGCAGCTGATGTGACC.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Diabetes can be induced in RIP-mOVA mice in a system devoid of Tregs

To study how CD25+ Tregs modify pathogenic CD4 cells, we have established an adoptive transfer-based model of diabetes in which the availability of the CD25+ subset can be experimentally manipulated. The starting point for this project was to establish a disease model characterized by the complete absence of CD25+ Tregs, so that the effects of selectively restoring this population could be studied. The donor CD4 T cells in this system are derived from DO11 mice on a RAG2–/– background that do not contain detectable CD4+CD25+ cells (28) (Fig. 1A). The recipient mice express a membrane-bound form of OVA on pancreatic {beta} cells and have been bred to a RAG2–/– background to preclude the development of T and B cells (RIP-mOVA/rag mice). Despite the absence of Tregs, RIP-mOVA/rag mice injected with DO11 cells remained normoglycemic for at least 26 days after transfer (Fig. 1B and data not shown). However, activation of the transferred DO11 cells by peripheral immunization with OVA in IFA led to rapid diabetes induction (Fig. 1B).



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  		FIGURE 1. Diabetes induction in RIP-mOVA/rag mice is dependent on immunization and is inhibited CD4+CD25+ cells. A, FACS profile of donor cells from the LN of DO11/rag mice stained for CD4 and the clonotypic Ab KJ126. CD25 expression within the gated CD4+KJ126+ population is shown. B, DO11/rag cells (2 x 106) were adoptively transferred into RIP-mOVA/rag recipients that were either left untreated or immunized s.c. with 200 µg of OVA protein emulsified in IFA 24 h later. Blood glucose readings for three mice per group are shown. Data show one representative experiment of seven. C, DO11/rag cells (1–2 x 106) were transferred into RIP-mOVA/rag mice that had been injected 24 h previously with the CD4 cells indicated. All mice were immunized with 200 µg of OVA 24 h after transfer of DO11/rag cells. RIP-mOVA/rag mice received no CD4 cells, 107 wild-type CD4 cells, or 107 CD28–/– CD4 cells (panel i) or received 106 CD4+CD25+ cells or 106 CD4+CD25– cells (panel ii). The CD4+ populations were purified from BALB/c (or CD28–/– BALB/c) peripheral LN. Blood glucose readings on days 14–20 are shown. Data were compiled from five separate experiments.

 
CD4+CD25+ cells are sufficient to inhibit diabetes in RIP-mOVA/rag mice

To test the ability of CD4 T cells to control disease induction, the effect of reconstituting RIP-mOVA/rag mice with purified CD4 T cells from a wild-type mouse was examined. In the presence of CD4 cells, the injection of DO11 cells followed by peripheral immunization failed to induce diabetes in RIP-mOVA/rag recipients (Fig. 1C). These animals maintained normal blood glucose levels for >80 days after transfer (data not shown). Consistent with a role for CD4+CD25+ Tregs in disease prevention, CD4 cells from CD28–/– mice, which are deficient in CD4+CD25+ regulatory T cells (5) (data not shown), failed to protect from diabetes (Fig. 1C). To address whether the CD25+ population of CD4 cells was sufficient to inhibit disease, the effect of transferring purified CD4+CD25+ cells vs CD4+CD25– cells was assessed. The transfer of CD4+CD25+ cells was sufficient to confer long term protection from diabetes (Fig. 1C; and up to 60 days (data not shown)). In contrast, purified CD4+CD25– cells were far less effective at controlling disease, with the majority of recipients exhibiting high blood glucose by day 14 (Fig. 1C). Therefore, introduction of purified CD4 cells or the CD25+ subset therein was sufficient to prevent diabetes induction in RIP-mOVA/rag mice.

Effects of Tregs on pancreatic islet infiltration

The above model provided an experimental system in which the presence of CD25+ Tregs could be precisely manipulated in a manner that had functional consequences for disease onset. This presented a novel opportunity to pinpoint how pathogenic DO11 cells are modified in vivo by CD4+CD25+ cells. We first examined the effect of CD4+CD25+ cells on pancreatic islet infiltration. DO11 cells were injected into RIP-mOVA/rag recipients that had either received CD4+CD25+ cells (which protect from disease; see Fig. 1C) or control CD4+CD25– cells. Recipient mice were immunized 24 h later with OVA/IFA and were killed on day 6 for analysis. This time point preceded the onset of hyperglycemia, such that all mice had normal blood glucose levels at the time of harvest (data not shown). DO11 T cells could be detected immunohistologically in a proportion of the pancreatic islets in both groups of mice. However, the extent and nature of the pancreatic infiltrate were clearly different. As shown in Fig. 2A, recipients of CD4+CD25+ cells had fewer islets exhibiting invasive infiltration of DO11 cells (on the average, 9.28% of islets showed invasive infiltration in the presence of CD4+CD25+ cells compared with 70.05% in the presence of CD4+CD25– cells; p = 0.04). Indeed, the majority of islets in the recipients of CD4+CD25+ cells remained free of infiltration, and when infiltrate was present, it was frequently peri-islet rather than invasive (Fig. 2B). Examination of islet infiltration at later time points (days 10 and 17) showed a similarly low level of infiltration of DO11 cells in recipients of CD4+CD25+ cells, suggesting that infiltration was not simply delayed. Control mice that received CD4+CD25– or CD4+CD25+ cells alone or received DO11 cells but were not immunized did not exhibit detectable pancreatic islet infiltration at the time points examined (data not shown). Therefore, the ability of CD4+CD25+ cells to inhibit diabetes induction was associated with an early capacity to control T cell infiltration of pancreatic islets.



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  		FIGURE 2. Characterization of pancreatic islet infiltration in the presence of CD4+CD25+ cells or CD4+CD25– cells. DO11/rag cells (106) were transferred into RIP-mOVA/rag mice that had been injected with 106 CD4+CD25+ cells or CD4+CD25– cells 24 h previously. Mice were immunized s.c. with 200 µg of OVA/IFA 24 h after injection of DO11/rag cells and were harvested 6 days later. Frozen pancreas sections were stained for the presence of DO11 T cells and insulin, and islets were scored for degree of infiltration. A, Percentage of islets exhibiting invasive infiltration of KJ126+ cells. B, Proportion of islets with invasive, peri-islet, or no infiltration. Each bar indicates one animal and shows scoring from a minimum of 15 islets. Data show one representative experiment of two.

 
Effects of Tregs on DO11 T cell number and phenotype

We next wished to assess how the altered islet infiltration related to the number and phenotype of Ag-specific T cells in the presence or the absence of Tregs. We first quantified the number of KJ126-positive cells in both pancreatic LN (draining the site of tissue Ag) and peripheral LN (draining the site of immunization). The DO11 cells clearly expanded in response to immunization in both mice bearing CD4+CD25+ cells and mice bearing CD4+CD25– cells (Fig. 3). At the site of immunization, the expansion of DO11 cells was moderately reduced in recipients of CD4+CD25+ cells compared with that in recipients of CD4+CD25– cells (on the order of 2- to 3-fold; Fig. 3, top panel). A similar pattern was observed in the spleen (Fig. 3, bottom panel). In the pancreatic LN, the number of DO11 cells was comparable between mice that received CD4+CD25– and those that received CD4+CD25+ cells on both day 3 and day 6 (Fig. 3, middle panel). At later time points, the number of DO11 cells in the pancreatic LN tended to be lower in recipients of CD4+CD25+ cells (0.014 x 106± 0.005 in the presence of CD4+CD25+ cells compared with 0.05 x 106 ± 0.01 in the presence of CD4+CD25– cells on day 16; n = 3). Therefore, although the accumulation of DO11 cells in the pancreatic LN could eventually be inhibited by CD4+CD25+ cells, this effect manifested itself relatively late. In mice that did not receive CD4+CD25+ cells or CD4+CD25– cells, DO11 cell expansion was typically equivalent to or slightly greater than that seen with CD4+CD25– cotransfers (data not shown). Therefore, the presence of Tregs was associated with a modest reduction in DO11 T cell number in peripheral LN, but not pancreatic LN.



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  		FIGURE 3. Effect of CD4+CD25+ cells on DO11 T cell numbers. DO11/rag cells (106) were transferred into RIP-mOVA/rag mice that had been injected with 106 purified CD4+CD25+ cells or CD4+CD25– cells 24 h previously. Recipient mice were immunized s.c. with 200 µg of OVA/IFA 24 h after transfer of DO11/rag cells where indicated. Crosses depict cell numbers in mice that received DO11/rag cells alone in the absence of immunization. On days 3 and 6 postimmunization, peripheral LN cells (draining the site of immunization), pancreatic LN cells, and spleen cells were isolated and analyzed by flow cytometry. The number of DO11 T cells (CD4+KJ126+) in each location is shown. The average value for seven mice per group is shown with the SD.

 
To probe the phenotype of the Ag-specific T cells, we examined cytokine expression by intracellular staining in both the peripheral LN draining the site of immunization and the pancreatic LN draining the site of tissue Ag. In the absence of immunization, <1% of the DO11 cells transferred to RIP-mOVA/rag mice produced IFN-{gamma} (data not shown). Three days after immunization, ~30% of the DO11 cells in mice bearing control CD4+CD25– cells had differentiated to become IFN-{gamma} secretors in the peripheral LN of RIP-mOVA/rag recipients (Fig. 4A). However, in mice bearing CD4+CD25+ cells, the production of IFN-{gamma} in response to immunization was strikingly inhibited, with <3% of the transferred DO11 cells staining positively for this cytokine (Fig. 4A). A similar trend was evident in the pancreatic LN on day 3, but was more obvious on day 6 (day 6 shown, Fig. 4A). Despite poor IFN-{gamma} production, the DO11 T cells in mice that received Tregs showed high levels of CD69 and partially down-regulated CD62L indicative of activation (data not shown). The modulation of cytokine secretion by CD4+CD25+ cells in four separate experiments (Fig. 4B) revealed that the number of IFN-{gamma}-secreting DO11 cells was reduced, on the average, 75% in the presence of CD4+CD25+ cells compared with in the presence of CD4+CD25– cells. Interestingly, the number of IL-2-secreting DO11 cells was only modestly reduced (average, 33% inhibition) in the presence of CD4+CD25+ cells (Fig. 4). The number of DO11 cells producing IL-4 in these experiments was consistently low in both groups of mice (Fig. 4). T cell transfers that did not result in protection from diabetes, such as those involving CD28–/– CD4 cells, did not inhibit DO11 cell IFN-{gamma} expression (data not shown). Thus, although DO11 T cells were numerically broadly similar in recipients of CD4+CD25+ cells vs CD4+CD25– cells, clear qualitative differences were evident with respect to effector cytokine differentiation.



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  		FIGURE 4. Effect of CD4+CD25+ cells on cytokine production. DO11/rag cells (106) were transferred into RIP-mOVA/rag mice that had been injected with 106 purified CD4+CD25+ cells or CD4+CD25– cells 24 h previously. Mice were immunized s.c. with 200 µg of OVA/IFA 24 h after transfer of DO11/rag cells. Peripheral LN cells and pancreatic LN cells were isolated 3 and 6 days after immunization and were restimulated in vitro with OVA323–339 for 4 h, then analyzed for intracellular cytokine expression. A, Plots are gated on the CD4+KJ126+ population and show pooled samples comprising three (for peripheral LN) or six (for pancreatic LN) mice per group. Peripheral LN are shown on day 3 and pancreatic LN on day 6. B, Four separate experiments were conducted as described above, and the percentage of KJ126+ cells expressing IFN-{gamma}, IL-4, or IL-2 in peripheral LN on day 3 was assessed. Each bar shows pooled cells from two mice.

 
Control of islet infiltration by IFN-{gamma} production

As CD4+CD25+ Tregs appeared to control both IFN-{gamma} production and pancreatic islet infiltration, we sought to determine whether these two phenomena were linked. We therefore tested the role of IFN-{gamma} in promoting pancreatic islet invasion in the transgenic model by examining the effect of administering blocking anti-IFN-{gamma} Abs. RIP-mOVA/rag mice were adoptively transferred with DO11 cells, then immunized with OVA/IFA as described above, and anti-IFN-{gamma} or control rat IgG was injected i.p. on days 0 and 3. Mice were killed on day 6, and pancreas sections were scored for infiltration of DO11 cells. Blockade of IFN-{gamma} decreased the number of islets exhibiting invasive infiltration (from 70.36% with control Ab to 16.8% with anti-IFN-{gamma}; Fig. 5, A and C). This was reminiscent of the effect seen after transfer of CD4+CD25+ cells (see Fig. 3B). The pattern of lymphocytic accumulation at the periphery of affected islets in anti-IFN-{gamma}-treated mice also closely resembled that observed in mice injected with CD4+CD25+ cells (Fig. 5A). Thus, the presence of CD4+CD25+ cells or blockade of IFN-{gamma} caused a similar modulation of pancreatic islet infiltration.



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  		FIGURE 5. Effect of IFN-{gamma} blockade on pancreatic islet infiltration. RIP-mOVA/rag mice were injected with DO11/rag cells (106) and immunized s.c. with 200 µg of OVA/IFA 24 h later (day 0). One milligram of anti-IFN-{gamma} (R46A2) or control rat IgG was injected i.p. on days 0 and 3, and mice were killed on day 6. A, Frozen pancreas sections were stained for DO11 T cells (blue) and insulin (brown). Original magnification, x10. Upper panel, Pancreas sections from mice that received CD4+CD25– or CD4+CD25+ cells (as described in Fig. 2); lower panel, mice that received rat IgG or anti-IFN-{gamma}. B, Percentage of pancreatic islets exhibiting invasive infiltration of KJ126+ cells after injection of anti-IFN-{gamma} or control rat IgG. C, Proportion of islets with invasive, peri-islet, or no infiltration after injection of anti-IFN-{gamma} or control rat IgG. Islets were scored as described in Fig. 2. Data are representative of two independent experiments.

 
Reduced T cell CXCR-3 in the presence of Tregs

The above data suggested that Tregs controlled pancreatic islet infiltration by inhibiting IFN-{gamma} production. This implied a critical role for IFN-{gamma} signaling in orchestrating tissue infiltration by pathogenic lymphocytes. IFN-{gamma} can trigger the production of a number of chemokines from microcapillary endothelial cells, including RANTES (29), CXCL10/inducing protein 10, and CXCL9/monokine induced by IFN-{gamma}. Recently, it has been shown that pancreatic {beta} cells themselves can produce CXCL10 and CXCL9 in response to inflammation (26). The receptor that binds these chemokines, CXCR-3, is preferentially expressed on Th1 cells (30, 31), and intriguingly, its expression is dramatically inhibited by IFN-{gamma} blockade or gene ablation (32). We therefore investigated whether the presence of CD4+CD25+ cells altered the expression of CXCR-3 on DO11 T cells. As shown in Fig. 6A, DO11 cells isolated from unimmunized hosts were largely devoid of CXCR-3 expression. In mice that had been immunized, CXCR-3 was induced on the DO11 cells, but this was clearly inhibited in the presence of CD4+CD25+ cells compared with control CD4+CD25– cells (Fig. 6A). Similar data were obtained when DO11 cells isolated from the pancreatic LN were examined (Fig. 6B). Blockade of IFN-{gamma} in our adoptive transfer system led to a marked reduction in the level of CXCR-3 expression on DO11 cells (Fig. 6C). Analysis of mRNA from whole pancreas sections of recipient mice confirmed the presence of the chemokine CXCL10, a ligand for CXCR-3 (Fig. 6D). Consistent with a critical role for CXCR-3 expression in pancreatic islet infiltration, CXCR-3 knockout mice have recently been shown to exhibit dramatically impaired insulitis in a virus-induced diabetes model (26). Thus, the failure of DO11 cells to invade pancreatic islets in the presence of CD4+CD25+ cells was associated with reduced expression of the IFN-{gamma}-dependent CXCR-3.



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  		FIGURE 6. A and B, Effect of CD4+CD25+ cells on CXCR-3 expression. DO11/rag cells (106) were transferred into RIP-mOVA/rag mice that had been injected with 106 purified CD4+CD25+ cells or CD4+CD25– cells 24 h previously. Mice were immunized s.c. with 200 µg of OVA/IFA 24 h after transfer of DO11/rag cells. Plots show CXCR-3 expression on gated CD4+KJ126+ cells from the LN draining the site of immunization on day 3 (A) or from the pancreatic LN on day 6 (B). C, Effect of IFN-{gamma} blockade on CXCR-3 expression. DO11/rag cells (106) were transferred into RIP-mOVA/rag mice, which were immunized s.c. with 200 µg of OVA/IFA 24 h later. One milligram of anti-IFN-{gamma} or control rat IgG was administered i.p. on days 0 and 2, and CXCR-3 expression is shown on day 3 on gated CD4+KJ126+ cells isolated from the LN draining the site of immunization. D, Expression of the chemokine CXCL10 in the pancreas of adoptive transfer recipients (lane 2). DO11/rag cells (106) were transferred into RIP-mOVA/rag mice, which were immunized s.c. 24 h later with 200 µg of OVA/IFA. Six days later, mice were killed; cDNA was prepared from frozen pancreas sections, and PCR was performed for CXCL10 and {beta}-actin. Normal pancreas is shown as a control (lane 1).

 
Tregs inhibit IFN-{gamma} production and CXCR-3 expression in nonlymphopenic mice

As the above experiments documented the activities of CD4+CD25+ Tregs in rag gene-deficient mice, it was important to confirm that similar modulation occurred in a nonlymphopenic environment. To address this issue, we exploited the TCR transgenic Tregs (KJ+CD25+ cells) that we have recently demonstrated arise in double-transgenic mice bearing the DO11 TCR and its respective Ag (RIP-mOVA) (27). KJ+CD25+ cells have regulatory function in vitro and in vivo (27) and prevent diabetes induction in the adoptive transfer system described in this study (Fig. 7A). The advantage of having access to a large number of OVA-specific Tregs is that they enable us to study Treg function in normal lymphocyte-competent hosts. The phenotype of adoptively transferred naive DO11 cells responding to immunization in BALB/c recipients in the presence of KJ+CD25+ cells (or control KJ+CD25– cells) was therefore examined. CFSE labeling was used to distinguish cotransferred populations. Less than 1% of the adoptively transferred DO11 cells produced IFN-{gamma} in the absence of immunization (data not shown). The number of DO11 cells that produced IFN-{gamma} after immunization was lower than that observed in rag-deficient animals (compare Fig. 7B with Fig. 4A), probably reflecting the actions of endogenous Tregs within the BALB/c hosts. Nonetheless, mice in which the endogenous Treg population had been supplemented by injection of KJ+CD25+ cells showed a clear reduction in the number of IFN-{gamma}-positive cells (Fig. 7B). Mirroring our findings in the RIP-mOVA/rag system, DO11 cells responding to immunization in the presence of Ag-specific Tregs showed markedly lower levels of CXCR-3 (Fig. 7C). Thus, the ability of CD25+ Tregs to inhibit IFN-{gamma} production and CXCR-3 expression could be demonstrated in both RIP-mOVA/rag mice and lymphocyte-intact BALB/c recipients. The targeting of Ag to the pancreas in the former hosts allowed us to reveal the consequences of such regulation for the control of tissue invasion and injury.



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  		FIGURE 7. Inhibition of IFN-{gamma} and CXCR-3 by TCR-transgenic CD4+CD25+ cells in BALB/c hosts. A, TCR-transgenic CD4+CD25+ cells (KJ+CD25+) inhibit diabetes in the adoptive transfer model. DO11/rag cells (106) were transferred into RIP-mOVA/rag mice that had been injected with 106 purified KJ+CD25+ cells or KJ+CD25– cells 24 h previously. Blood glucose is shown on day 16. B, Inhibition of IFN-{gamma} by TCR transgenic Tregs. DO11/rag cells (106) were adoptively transferred into BALB/c mice that had been injected 24 h previously with CFSE-labeled KJ126+CD25+ cells (106) or KJ126+CD25– cells (106) from the LN of DO11xRIP-mOVA double-transgenic mice. One day later, mice were immunized s.c. with 200 µg of OVA/IFA. Three days after immunization, draining LN cells were isolated and restimulated in vitro with OVA323–339 for 4 h, then analyzed for intracellular cytokine expression. IFN-{gamma} levels for gated CD4+KJ126+CFSE– cells are shown. C, Inhibition of CXCR-3 by TCR-transgenic Tregs. BALB/c mice were subjected to adoptive transfer and immunization as described above. Three days after immunization, draining LN cells were isolated and stained for CXCR-3 levels. Plots are gated on CD4+KJ126+CFSE– cells. Data are representative of at least three experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Despite overwhelming data to support the importance of CD4+CD25+ Tregs in controlling immune responses, a detailed understanding of which aspects of T cell activation are modulated in vivo by Tregs has been lacking. Analysis of the mechanisms underlying Treg suppression has produced conflicting results, with a clear role for TGF-{beta} (33, 34) and/or IL-10 (35, 36, 37) in some models, but not others (38, 39, 40). Regardless of the molecular mechanisms, uncovering the functional consequences of regulation for pathogenic T cells promises to provide useful insight into Treg activity while potentially providing scope to mimic their downstream effects therapeutically. The lack of consensus regarding multiple aspects of Treg biology probably reflects the existence of several distinct populations of cells with regulatory activity that have divergent phenotypes and properties. In fact, IL-10 production from CD25– cells can prevent diabetes in NOD mice (41), and IL-10-producing B cells can also inhibit autoimmunity (42). Because regulation can also involve cooperation between different cell subsets (38, 43, 44), this adds further complexity to the picture.

The above issues highlight the need for well-defined experimental systems in which distinct aspects of peripheral regulation can be dissected. To this end, we have developed a novel transgenic model in which regulatory and pathogenic T cells populations can be independently manipulated. Two features of our model make it particularly amenable for this analysis. The first is that T cells bearing a single specificity are sufficient to instigate tissue destruction in the absence of CD8 cells or B cells. The second is that the disease-inducing T cells can be distinguished from cotransferred regulatory populations with a clonotypic Ab. This experimental setup has allowed us to track how disease-inducing CD4 T cells targeting a known tissue Ag are modulated by CD4+CD25+ cells. Our study reveals several novel aspects relating to the actions of CD25+ Tregs in vivo. We show that CD4 cells primed in the presence of Tregs show only modestly impaired expansion and IL-2 production, yet exhibit a striking failure to express IFN-{gamma}. This suggests that Tregs target effector cytokine production rather than preventing T cell activation wholesale. Our study also reveals that T cells experiencing regulation fail to up-regulate high levels of the IFN-{gamma}-dependent CXCR-3 and are inefficient at infiltrating the Ag-bearing tissue. Collectively, these data indicate that the ability of Tregs to control the pathogenicity of CD4 cells reflects their capacity to coordinately inhibit cytokine production and tissue infiltration.

To define the suppressive effects of CD4+CD25+ cells in isolation, without the possibility of participation by additional regulatory subsets that are CD25–, we have developed a model in which the entire T cell population is removed (by rag gene deficiency) and is then selectively replaced at will by adoptive transfer. In general, removal of T (or T and B) cells has the effect of exacerbating disease in several transgenic autoimmune models (43, 45). Because autoreactive T cells are likely to undergo lymphopenia-induced proliferation in such hosts, this may contribute to the heightened aggressiveness of disease observed. In our model, lymphopenia-induced proliferation of adoptively transferred DO11/rag T cells in RIP-mOVA/rag recipients is not sufficient to induce diabetes, and immunization with the cognate Ag is required. The effects of regulation in this model are unlikely to merely reflect inhibition of lymphopenia-induced proliferation, because it has previously been demonstrated that CD4+CD25+ cells do not control the lymphopenia-induced proliferation of CD4+CD25– cells during the first 2 wk after adoptive transfer into RAG–/– recipients (46, 47). Moreover, the consequences of regulation in our study are measured at time points that precede the onset of lymphopenia-induced proliferation, and the data can be reproduced in lymphocyte-sufficient hosts.

The link between IFN-{gamma} and diabetes pathogenesis is somewhat controversial. Increasing levels of IFN-{gamma} have been shown to correlate with progression to diabetes in both NOD (48) and TCR-HAxRIP-hemagglutinin double-transgenic mice (19), and transgenic expression of IFN-{gamma} in pancreatic islets is sufficient to provoke {beta} cell destruction (49). NOD mice genetically deficient in IFN-{gamma}R{alpha}–/– are strikingly resistant to diabetes (25), but this is now thought to reflect the role of a closely linked gene on chromosome 10 that was carried over from the 129 background (50, 51). Nevertheless, ablation of the IFN-{gamma}R {beta}-chain (on chromosome 16) slightly delays diabetes onset, whereas ablation of IFN-{gamma} itself results in a similar delay (24, 52) or even prevention (23) of diabetes. Furthermore, in vivo blockade of IFN-{gamma}, using Ab or soluble forms of the IFN-{gamma}R, clearly inhibits diabetes induction (20, 21, 22) (53). Consistent with the latter, preliminary analysis of a small number of mice suggests that IFN-{gamma} blockade prevents, or at least delays, diabetes in our adoptive transfer model system (data not shown).

The idea that CD4+CD25+ cells regulate cytokine production receives support from a recent study in which CD25 depletion enhanced IFN-{gamma} production from T cells responding to Ag-pulsed mature DCs (54). These authors also documented a reduction in IL-4 after CD25 depletion. The latter observation suggests that CD4+CD25+ cells can also enhance Th2 differentiation under certain conditions, consistent with findings obtained in an experimental autoimmune encephalomyelitis model (11). Our data show that the ability of CD25+ Treg to inhibit IFN-{gamma} production does not require the production of Th2 cytokines, because the decrease in IFN-{gamma} observed in the presence of Tregs was not associated with a reciprocal increase in IL-4.

IFN-{gamma} can clearly modulate multiple aspects of immunity, even playing an anti-inflammatory role under certain circumstances (55, 56). Our studies highlight a poorly appreciated role of IFN-{gamma} in regulating tissue infiltration (20, 23, 24, 25). Inhibition of IFN-{gamma} in our TCR transgenic model, by CD4+CD25+ cells or by Ab blockade, is sufficient to prevent the conversion of peri-insulitis to invasive insulitis. IFN-{gamma} is known to play a role in the induction of chemokines from endothelial cells of the microcapillaries, with potential candidates including CXCL10/inducing protein 10, CXCL9/monokine induced by IFN-{gamma} (57), CCL20 (58), and RANTES (29). IFN-{gamma} can also induce endothelial cell expression of selectins, including E-selectin (59, 60), which has recently been implicated in the local transmigration of T cells at the site of injection of Ag in adjuvant (61). Consistent with a role for IFN-{gamma} in promoting transmigration, pancreatic expression of this cytokine greatly accelerates the homing of T cells to pancreatic islets (62). As Ag presentation by endothelial cells can also promote T cell transmigration (63, 64), the ability of IFN-{gamma} to up-regulate MHC class II expression and Ag processing may contribute to its role in potentiating tissue invasion.

We have found that the expression of CXCR3, which is implicated in pancreatic islet infiltration, is reduced in the presence of CD4+CD25+ cells. The role of CXCR3 in tissue infiltration was recently highlighted in a virus-induced diabetes model in which CXCR3-knockout lymphocytes exhibited defective islet infiltration (26). Intriguingly, in the few islets that did become infiltrated, the T cells were exclusively localized to the rim of the islets, reminiscent of our findings in the presence of CD4+CD25+ Tregs or after IFN-{gamma} blockade. Blockade of CXCL10, a CXCR-3 ligand, is sufficient to abrogate virus-induced diabetes, supporting a key role for this pathway in destructive tissue inflammation (65). High CXCR-3 expression has recently been linked to several autoimmune disorders, including Graves’ disease (66) and, in particular, multiple sclerosis (67), in which CXCR-3 up-regulation correlates with relapse (68). Together with the findings presented in this study, this suggests that mimicking the effect of Tregs by blockade of CXCR-3 may present a broadly applicable strategy for inhibiting T cell recruitment to inflammatory sites (69).


   Acknowledgments
 
We are grateful to S. Jiang, C. McArthur, and R. Bird for Moflo sorting, and to C. Benitez and the University of Birmingham Biomedical Services Unit staff for animal husbandry. We thank J. Cyster, M. Krummel, D. Sansom, and D. Adams for helpful comments on the manuscript.


   Footnotes
 
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.

1 This work was supported by the Wellcome Trust (to L.S.K.W.) and National Institutes of Health Grants PO1AI35297 and R37AI25022 (to A.K.A.). L.S.K.W. is a Medical Research Council Career Development Fellow. Back

2 Address correspondence and reprint requests to Dr. Lucy Walker, Medical Research Council Center for Immune Regulation, University of Birmingham Medical School, Vincent Drive, Birmingham, U.K. B15 2TT. E-mail address: l.s.walker{at}bham.ac.uk Back

3 Abbreviations used in this paper: Treg, regulatory T cell; LN, lymph node; RIP, rat insulin promoter. Back

Received for publication February 23, 2004. Accepted for publication June 14, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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