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The Journal of Immunology, 2004, 172: 5900-5907.
Copyright © 2004 by The American Association of Immunologists

Generation of Anergic and Regulatory T Cells following Prolonged Exposure to a Harmless Antigen 1

Tse-Ching Chen2, Stephen P. Cobbold, Paul J. Fairchild and Herman Waldmann3

Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Regulatory CD4+ T cells are known to develop during the induction of donor-specific peripheral tolerance to transplanted tissues; it is proposed that such tolerance is a consequence of persistent, danger-free stimulation by Ag. To test this hypothesis, male RAG-1–/– mice were recolonized with small numbers of monospecific CD4+ T cells specific for the male H-2Ek-restricted Ag Dby. After 6 wk in the male environment, the monospecific CD4+ T cells, having recolonized the host, had become anergic to stimulation in vitro and had acquired a regulatory capacity. CD4+ T cells in these mice expressed higher levels of CTLA-4 and glucocorticoid-induced TNF-related receptor than naive CD4+ T cells, but only 3% of the recolonizing cells were CD25+ and did not express significant foxP3 mRNA. In vivo, these tolerant T cells could censor accumulation of, and IFN-{gamma} production by, naive T cells, with only a slight inhibition of proliferation. This suppressive effect was not reversed by the addition of fresh bone marrow-derived male dendritic cells. These results suggest that persistent exposure to Ag in conditions that fail to evoke proinflammatory stimuli leads to the development of T cells that are both anergic and regulatory.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A major therapeutic goal has been to understand and harness processes associated with immunological tolerance so that short-term inductive therapy may lead to long-term benefits with reduced maintenance immunosuppression. Short courses of nondepleting CD4 Abs producing coreceptor blockade can harness such processes in mice (1, 2, 3, 4), and since then many other biological agents have been shown capable of doing the same (3, 5). A mechanism has been identified that is dominant, regulatory, and mediated by Ag-reactive CD4+ regulatory T cells that emerge following the therapy (2, 3).

Simultaneously, studies in autoimmune disease and chronic gut immunopathology have shown that naturally occurring CD4+CD25+/CD4+CD45RBlow regulatory T cells prevent self-tissue aggression (6, 7, 8). The Ag specificity of "natural" regulatory T cells is uncertain, and thus the role of Ag in recruiting their function cannot be reliably studied. Some evidence suggests that such cells arise within the thymus during development (9), usually before exposure to any foreign alloantigen.

In contrast, the regulatory T cells generated from coreceptor blockade and from therapy with other agents that modulate T cell stimulation by APC clearly display Ag specificity (2, 3). We have sought to identify common features following therapy that might guide T cells exposed to Ag to become regulatory. It is known that the development of tolerance and regulation takes some weeks to be established (2, 10), that regulatory T cells are primed to donor Ags indirectly processed by host T cells (11), and that tolerance is maintained as long as Ag persists (10, 12, 13). A testable hypothesis would be that tolerogenic therapies might prevent graft rejection, allowing the graft to heal and function. This healed graft would then be a constant source of shed Ags available to host APC. Such APC, it is proposed, would not receive maturation (danger) signals to drive T cell aggression, but would, instead, be able to signal T cells toward anergy and the capacity to regulate.

We examined whether a set of monospecific naive CD4+ T cells could acquire the ability to regulate and prevent graft rejection if persistently exposed to Ag in the absence of danger signals. A small number of monospecific anti-male CD4+ T cells from A1(M).RAG-1–/– (CBA/Ca background) mice were adoptively transferred into normal (nonirradiated) male CBA/Ca RAG-1–/– mice (hereafter referred to as F>M mice) or control female mice (hereafter referred to as F>F mice) and allowed to colonize the recipients. After 6 wk, colonization had indeed occurred, but with no evidence of graft-versus-host disease (GvHD). 4 Monospecific T cells persistently exposed to the nominal male Ag showed classical anergy, i.e., the inability to proliferate to Ag in vitro unless exogenous IL-2 was added. Acquired F>M regulatory T cells were also capable of regulating naive T cell function both in vitro and in vivo. These data suggest that prolonged exposure to Ag can, in the absence of full activating signals, lead to selection of T cells with regulatory properties.


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

CBA/Ca mice (H-2k), RAG-1–/–, A1(M).RAG-1–/–, (A1(M) x Thy1.1)F1 mice, and B10.BR (H-2k) mice were bred and maintained in the specific pathogen-free facility of the Sir William Dunn School of Pathology, University of Oxford (Oxford, U.K.). A1(M).RAG-1 –/– mice bear a transgenic TCR specific for the male Ag Dby in the context of H-2E k on a RAG-1 –/– background. CBA/Ca and B10.BR share the same H-2k haplotype but differ for multiple minor Ags. Mice ages 6–16 wk were used in the present study. All procedures were performed in accordance with the Home Office Animals (Scientific Procedures) Act of 1986.

Cell transfers

For the induction of CD4+ regulatory T cells, 1 x 106 female A1(M).RAG-1–/– splenocytes were adoptively transferred into male and female RAG-1–/– mice via the lateral tail vein. The recipients were monitored every day and body weight was measured every 3 days during the first 6 wk.

Histology

Tissues were fixed in 10% buffered Formalin and embedded in paraffin. Four-micrometer sections of tissues were stained with H&E for histopathologic examination.

Synthetic peptides

The Dby-Ek peptide (REEALHQFRSGRKPI) was synthesized by the Medical Research Council Peptide Synthesis Unit, Imperial College School of Medicine (London, U.K.). Purity was >95% by HPLC.

Abs and flow cytometry

Four-color flow cytometric analysis was performed on a FACSCalibur with CellQuest 3.1 software (BD Biosciences, San Jose, CA). mAb to CD4 (H129.19; rat IgG2a), CD8 (53-6.7; rat IgG2a), CD25 (PC61; rat IgG1), CD44 (IM7; rat IgG2b), CD69 (H1.2F3; hamster IgG), CTLA-4 (UC10-4F10-11; hamster IgG), and Thy1.1 (OX-7; mouse IgG1) were purchased from BD PharMingen (San Diego, CA). Biotinylated polyclonal goat glucocorticoid-induced TNF-related receptor (GITR) Ab was purchased from R&D Systems (Abingdon, U.K.). Allophycocyanin-streptavidin was used as secondary reagent. mAbs to CD4 (YTA3.1.2), CD62L (Mel/14), and CD3 (KT3) were prepared in our own laboratory. For intracellular CTLA-4 staining, cells were fixed in a final concentration of 2% Formalin at room temperature for 20 min after surface staining. Subsequently, cells were washed, then permeabilized with 0.5% saponin buffer (Sigma-Aldrich, St. Louis, MO). PE anti-CTLA-4 (BD PharMingen) diluted in 0.5% saponin buffer was used to stain for 30 min.

CFSE labeling

CFSE (Molecular Probes, Leiden, The Netherlands) was added into cell suspensions at a final concentration of 8 µM and incubated at room temperature for 10 min. The labeling process was terminated with 10% FCS at 4°C. Cells were washed three times before use.

Cell separation

Spleens were harvested and a single-cell suspension was obtained by passing the splenocytes through a 70-µm cell strainer (BD Biosciences) and erythrocytes were lysed by 0.15 M Tris-buffered ammonium chloride. For negative selection of CD4+ T cells, cell suspensions were incubated with the CD4+ T cell isolation kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. Cells were sorted by using an AutoMACS "possels" program. Typical purity was >80%.

Measurement of proliferation in vitro

Unfractioned splenocytes from naive A1(M).RAG-1–/–, F>M, and F>F mice (containing 2 x 104 CD4+ T cells) were stimulated with 8 x 104 T cell-depleted CBA/Ca splenocytes as APCs and 10 µg/ml anti-CD3 mAb (2C11) for 72 h. Sorted CD4+ T cells (5–10 x 103) were stimulated with mitomycin C- treated T cell-depleted splenocytes (2–4 x 104) from CBA/Ca mice and cultured for 96 h in the presence of the Dby-Ek peptide. To determine the suppressive effect in vitro, cocultures of 5 x 103 sorted naive A1(M).RAG-1–/– CD4+ T cells and titrated numbers of CD4 sorted test T cells were stimulated with 100 nM Dby-Ek peptide and APC for 96 h. [3H]Thymidine (0.5 µCi/well; Amersham Pharmacia Biotech, Piscataway, NJ) was added to the culture for another 18 h. Plates were harvested onto glass fibers and [3H]thymidine incorporation was counted in a beta-plate scintillation counter (LKB/Pharmacia). All cultures were tested in triplicate. Depletion of the total splenic T lymphocyte population was achieved by i.p. injection of 1 mg each of depleting anti-CD8 mAbs (YTS 156.7 and YTS 169.4) and anti-CD4 mAbs (YTA 3.1.2 and YTS 191.2).

ELISA

Supernatants were collected on day 3 and IL-2, IFN-{gamma}, IL-4, or IL-10 were measured by ELISA using JES6-1A12, R46A2, 11B11, and JES5 as capture Abs, respectively, and biotinylated anti-mouse IL-2 mAb (JES6-5H4), IFN-{gamma} mAb (XMG 1.2), anti-IL-4 mAb (BVD6-24G2), or anti-IL-10 mAb (SXC-1) as the detection Abs (all mAbs from BD PharMingen).

Measurement of T cell proliferation in vivo

CFSE-labeled female (A1(M) x Thy1.1)F1 splenocytes (5 x 106) were injected into test mice. On day 4, spleens were harvested. To measure the proliferation of injected T cells, the intensity of CFSE fluorescence on Thy-1.1+ gated cells was analyzed. The average number of divisions was calculated using the following equation: average number of divisions = log2(A/B), where A represents the mean intensity of a mitomycin C-treated aliquot of the CFSE-labeled CD4+ T cells injected into female A1(M).RAG-1–/– mice and B represents the mean intensity of untreated CFSE-labeled CD4+ T cells injected into experimental mice. Values of p for differences between the mean values for groups of mice were estimated using the Student’s t test.

Preparation of bone marrow-derived dendritic cells (DC)

Bone marrow cells from male CBA/Ca mice were cultured in RPMI 1640 plus 10% FCS supplemented with GM-CSF. On days 3 and 6, nonadherent cells were removed and remaining cells were cultured in fresh medium supplemented with GM-CSF and harvested for use on day 7.

Intracellular cytokine staining

Splenocytes from test mice were stimulated ex vivo for 6 h with 5 µM Dby-Ek peptide at 37°C. Brefeldin A (Sigma-Aldrich) was added at a final concentration of 10 µg/ml for the last 3 h. Thereafter, cells were washed three times and stained with PE-anti-Thy1.1 (BD PharMingen) and 7-aminoactinomycin D (7-AAD; Sigma-Aldrich). Cells were permeabilized with 0.5% saponin (Sigma-Aldrich) and stained with allophycocyanin-anti-IFN-{gamma}, allophycocyanin-anti-IL-4, or allophycocyanin-anti-IL-10 (all from BD PharMingen). Four-color FACSCalibur analysis was performed using CellQuest 3.1 software and dot plots were gated on Thy1.1+7-AAD– cells.

Real-time quantitative RT-PCR

RNA was prepared using the SV Total RNA isolation system (Promega, Madison, WI) that includes DNase I treatment. Reverse transcription used the ProStar kit with random hexamers (Stratagene, La Jolla, CA). Forward and reverse primer sequences for foxp3 are 5'-CCCAGGAAAGACAGCAACCTT-3' and 5'-TTCTCACAACCAGGCCTCTTG-3'. Primer sequences for CD3{gamma} are 5'-TTACAGAATGTGTGAAAACTGCATTG-3' and 5'-CACCAAGAGCAAGGAAGAAGATG-3'. TaqMan probes of foxp3 and CD3{gamma} are FAM-5'-CTACCCACTGCTGGCAAATGGAGTC3'-TAMRA, and VIC-5'-ACATAGGCACCATATCCGGCTTTATCTTCG-3'-TAMRA, respectively. Multiplex PCR was performed with gene-specific primers, fluorogenic probes and the Universal MasterMix kit (PE Applied Biosystems, Foster City, CA) in triplicate with primers at a concentration of 300 nM and the probe at 200 nM. A hot start, two-step PCR (15 s at 95°C and 60 s at 60°C) was applied for 40 cycles. PCR and TaqMan analysis were performed using the 7700 Sequence Detector System (PE Applied Biosystems). Standard curves of appropriate cDNAs were used to calibrate the amounts of cDNAs in each test sample.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Colonization of male RAG-1–/– mice with monospecific CD4+ anti-male T cells without GvHD

One million female A1(M).RAG-1–/– splenocytes were adoptively transferred by i.v. injection into male RAG-1–/– mice. As controls the same number of A1(M).RAG-1–/– splenocytes was injected into female RAG-1–/– mice. Most male recipients showed no >5% weight loss up to the second week, and after that time their weight increased in parallel with controls (Fig. 1a). Absolute CD4+ T cell counts in the spleens of the F>M mice were 2- to 3-fold higher (Fig. 1b) than in the F>F mice (p = 0.008) and had therefore not undergone deletion en mass. However, the numbers of CD4+ T cells in both of the F>M and F>F mice were less than in the unmanipulated naive female A1(M).RAG-1–/– mice (p < 0.0001).



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  		FIGURE 1. Female A1(M).RAG-1–/– CD4+T cells reconstitute male RAG-1–/– mice. Male and female RAG-1–/– mice were adoptively transferred i.v. with 1 x 106 female A1(M).RAG-1–/– splenocytes on the same day. Weights (a) were monitored every 3 days. Mice were killed 6 wk later and absolute CD4+ T cells (b) were enumerated in recipients’ spleens.

 
Histology of liver and colon (Fig. 2) excluded GvHD in the F>M mice (14). Although lymphocytes reconstituted the colon, there was no significant infiltration into the lamina propria, no goblet cell depletion, and no epithelial apoptosis in the F>M and F>F mice as compared with naive female A1(M).RAG-1–/– mice. In the liver, minimal or mild infiltration of lymphocytes was seen in the portal tract of both F>M and F>F mice, but there was no bile duct damage, hepatocyte apoptosis, nor endothelialitis.



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  		FIGURE 2. Both male and female recipients show no evidence of GvHD. H&E sections of liver and colon from naive A1(M).RAG-1–/– mice and both male and female RAG-1–/– recipients (n = 4) show minimal infiltrations of small lymphocytes.

 
Development of anergy in monospecific T cells colonizing male RAG-1–/– mice

The ability of F>M and F>F CD4+ T cells to mount proliferative responses after stimulation by anti-CD3 mAb or by Dby-Ek peptide was assessed. Unfractionated F>M spleen cells were hyporesponsive to anti-CD3 stimulation as compared with F>F and naive monospecific T cells (data not shown), and a similar result was observed in experiments where 5–10 x 103 sorted F>M and F>F CD4+ T cells were stimulated with 2–4 x 104 APC in the presence of varying amounts of Dby-Ek peptide for 72 h (Fig. 3a). To assess cytokine production, supernatants were collected on day 3 and IL-2, IFN-{gamma}, IL-4, and IL-10 were measured by ELISA. Naive monospecific T cells and F>F CD4+ T cells were able to produce IL-2, yet none could be detected in cultures of F>M CD4+ T cells (Fig. 3b). IFN-{gamma}, IL-4, and IL-10 could not be detected in any of the groups (data not shown).



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  		FIGURE 3. A1(M).RAG-1–/– T cells colonizing male RAG-1–/– mice become anergic. CD4+ T cells sorted from F>M mice fail to proliferate (a) and secrete IL-2 (b) in response to Dby-Ek peptide. Results shown are representative of six independent experiments. CFSE-labeled F>M CD4+ T cells are uniformly anergic to Dby-Ek stimulation compared with A1(M).RAG-1–/– CD4+ T cells. Results shown are typical of three independent experiments (c). The anergy of F>M CD4+ T cells is partially reversed by exogenous IL-2 (d). Results shown are representative of six independent experiments.

 
To determine whether the F>M CD4+ T cells responded uniformly to the male peptide, we assessed the proliferative response of CFSE-labeled cells (15). Indeed. F>M CD4+ T cells were completely anergic to 100 nM Dby-Ek peptide stimulation (Fig. 3c) by comparison with F>F CD4+ T cells, which themselves were slightly less active than naive monospecific CD4+ T cells. The anergic state of F>M T cells could be rescued, at least in part, by the addition of 100U/ml IL-2 (Fig. 3d).

Anergic T cells can suppress the function of naive monospecific CD4+ T cells

Several reports have shown that anergic T cells can suppress naive CD4+ T cells in vitro (16, 17, 18). The suppressive activity of F>M or F>F CD4+ T cells was assessed by a coculture of 5–10 x 103 sorted naive monospecific CD4+ T cells and a serial titration of F>M or F>F CD4+ T cells with 4-fold excess of APCs and 100 nM Dby-Ek peptide for 72 h. The F>M T cells suppressed naive monospecific T cell proliferation by up to 85% at a ratio of 1:1 and around 50% at a ratio of 0.25 (Fig. 4a). In contrast, F>F CD4+ T cells did not suppress naive CD4+ T cell proliferation at all. F>M CD4+ T cells not only suppressed naive T cell proliferation but also completely inhibited IL-2 secretion at the ratio of 1:1 (Fig. 4b). The inhibitory effect was marked even at a ratio of 0.125. F>F cells showed no such suppression.



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  		FIGURE 4. F>M CD4+ T cells can suppress naive T cell proliferation and IL-2 secretion in vitro. CD4+ T cells sorted from F>M mice suppress naive A1(M).RAG-1–/– CD4+ T cell proliferation (a) and IL-2 secretion (b) in response to 100 nM Dby-Ek peptide. Values plotted are means ± SD of triplicate cultures. Results shown are typical of six independent experiments.

 
Phenotypic characterization of the F>M and F>F CD4+ T cells

The surface phenotype of CD4+ T cells emerging in the F>M and F>F CD4+ populations was analyzed. CD25 has been widely accepted as a marker for naturally occurring regulatory T cells, while CTLA-4 and GITR have also been shown to be particularly prominent in these cells (6, 19, 20). Only 3% of the F>M CD4+ T cells were CD25+ (Fig. 5a), while, in contrast, ~ 15% of control F>F CD4+ T cells had become CD25+.



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  		FIGURE 5. The phenotype of F>M and F>F CD4+ T cells. Splenocytes from female A1(M).RAG-1–/–, F>M, and F>F mice were gated on the CD4+ population, permitting analysis of the expression of CD25 (a) GITR, CTLA-4, CD69, and CD4 expression (b). Results shown are typical of at least three independent experiments. Control female A1(M) RAG-1–/–CD4+ T cells are indicated by the thin line and F>M and F>F CD4+ T cells are indicated by the thick line. Total splenocytes from F>M, F>F, and female A1(M).RAG-1–/– mice were assessed for foxp3 mRNA using real-time quantitative RT-PCR (c). Foxp3 mRNA expression was normalized against CD3{gamma} mRNA expression. Sorted CD4+CD25+ T cells and CD4+CD25– T cells and unfractionated spleen cells from a pool of naive CBA/Ca mice (29 ) were included as controls (purity >90%). Data from F>M, F>F, and A1(M).RAG-1–/– mice are shown as geometric means + SDs of three individual mice per group.

 
Three markers were selectively up-regulated in F>M CD4+ T cells when compared with F>F and naive controls. These were: GITR (median fluorescence intensity (MFI) = 57.03), while F>F CD4+T cells (MFI = 18.84) expressed this marker at a level similar to that of naive monospecific T cells (MFI = 13.93; Fig. 5b); CD69, which was also up-regulated in the F>M CD4+ T cells (MFI = 26.47), by comparison with F>F CD4+ and naive T cells (MFI = 16.29 and MFI = 10.39, respectively); and, finally, CD4 that was up-regulated in F>M T cells (MFI = 1152.72), compared with F>F T cells (MFI = 821.46), was similar to naive T cells. Both F>M (MFI = 9.33) and F>F (MFI = 10.28) CD4+ T cells had up-regulated CTLA-4 compared with naive T cells (MFI = 3.16). Both F>M and F>F CD4+ T cells expressed an activated phenotype, CD44high and CD62Llow, while naive CD4+ T cells did not (data not shown).

Anergic F>M CD4+ T cells do not express foxP3 mRNA

It has recently been shown that the forkhead transcription factor foxP3 is specifically expressed in, and required for, the development of CD4+CD25+ regulatory T cells (21, 22, 23). In light of this, we assessed the expression of foxP3 mRNA in the F>M, F>F, and A1(M).RAG-1–/– T cells by using quantitative real-time PCR analysis. CD4+CD25+ and CD4+CD25– T cells and unfractionated normal spleen from CBA/Ca mice were included as controls. FoxP3 mRNA expression was normalized against CD3{gamma} mRNA expression. Control CBA/Ca CD4+CD25+ T cells and total spleen showed significant expression of foxP3 mRNA, yet little, if any, significant expression was observed in mRNA samples from splenic CD4+CD25– T cells of CBA/Ca mice, or unfractionated spleen from F>M, F>F, and A1(M).RAG-1–/– mice (Fig. 5c).

Anergic F>M CD4+ T cells can suppress CD4+ T cell expansion and differentiation in vivo

CFSE-labeled (A1(M) x Thy-1.1)F1 spleen cells (5 x 106) were transferred into F>M, F>F, female RAG-1–/–, and male RAG-1–/– mice. Mice were killed on day 4. Splenocytes were stimulated ex vivo with 5 µM Dby peptide for 6 h and brefeldin A was added for the last 3 h. Cells were then stained with fluorochrome-labeled anti-IFN-{gamma}, anti-IL-4, and anti-IL-10 Abs and analyzed by flow cytometry. Dot plots were gated on Thy1.1+7-AAD– cells (Fig. 6a). It was found that 3–6% of Thy-1.1-marked T cells from control male RAG-1–/– recipients produced IFN-{gamma}, and these cells showed evidence of being able to undergo at least six cycles of proliferation. In the F>M mice, proliferation was only marginally reduced, but no obvious IFN-{gamma}-secreting cells were detected. As expected, none was seen in the control F>F mice and female RAG-1–/– mice. Nor was there evidence for further immune deviation in the F>M mice, as neither IL-4- nor IL-10-secreting cells could be found (data not shown). No obvious difference in proliferation was seen between the Thy-1.1-marked T cell proliferation in the F>F mice compared with the female RAG-1–/– mice (p > 0.05). The absolute number of Thy-1.1-marked T cells that accumulated in the spleens of F>M mice was substantially (15-fold) lower compared with male RAG-1–/– mice (p = 0.0006; Fig. 6b) while there was no significant difference in accumulation in F>F mice and female RAG-1–/– mice (p > 0.05). The conclusion is, therefore, that F>M CD4+ T cells can suppress naive CD4+ T cell expansion and IFN-{gamma} production in vivo.



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  		FIGURE 6. F>M CD4+ T cells can suppress naive CD4+ T cell expansion and IFN-{gamma} production in vivo. CFSE-labeled (Thy1.1 x A1)F1 splenocytes (5 x 106) were adoptively transferred into female RAG-1–/–, male RAG-1–/–, F>F mice, or F>M mice. On day 4, spleens were harvested for flow cytometric analysis. Thy1.1+CD4+ T cells in F>M mice contained far less IFN-{gamma}-secreting cells compared with those in male RAG-1–/– mice when given a brief stimulation (6 h) with 5 µM Dby-Ek ex vivo (a). Thy1.1+CD4+ T cells had undergone fewer cell divisions in F>M mice compared with those in male RAG-1–/– mice, as indicated in each plot. Naive Thy1.1+CD4+ T cell accumulation (b) showed no significant difference between F>F mice and female RAG-1–/– mice, whereas naive Thy1.1+CD4+ T cells accumulated in male RAG-1–/– recipients but not in F>M mice.

 
Challenge with male DC does not overcome in vivo regulation

It was possible that host APC had been decommissioned in F>M mice and that this might explain the results (24, 25). We therefore asked whether function could be restored by the adoptive transfer of fresh male APC. We adoptively transferred 5 x 106 CFSE-labeled Thy-1.1-marked T cells into F>M, F>F, female RAG-1–/–, and male RAG-1–/– mice. The next day, 1 x 106 bone marrow-derived DC were injected into the test groups. On day 4, spleen cells were enumerated and stained as before. The Thy-1.1-marked monospecific T cells contained IFN-{gamma}-secreting cells among the proliferating T cells of the control F>F and naive female mice, therefore, validating the ability of the male bone marrow-derived DC to stimulate T cells. There were, however, no detectable IFN-{gamma}-secreting cells identified in the F>M mice injected with or without male DC (Fig. 7a), even though those cells had been able to undergo several rounds of proliferation. No obvious difference in proliferation was observed between F>M CD4+ T cells with or without male DC stimulation (p > 0.05). However, proliferation was enhanced in the F>F mice and female RAG-1–/– mice challenged by male DC as compared with those without DC challenge (p = 0.0004). The absolute number of Thy-1.1-marked CD4+ T cells that accumulated in the spleens of F>M mice with or without DC challenge was not substantially different (p > 0.05; Fig. 7b). There was, however, a significant increase in Thy1-marked monospecific T cell accumulation in the F>F mice following male DC challenge (p < 0.05). Therefore, normal male DC could not restore the capacity for naive T cells to expand and produce IFN-{gamma} in vivo in the F>M mice, ruling out the possibility that the defect was solely at the level of host APC.



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  		FIGURE 7. Fresh male DC were not able to promote naive CD4 proliferation and IFN-{gamma} production. CFSE-labeled (Thy1.1 x A1)F1 splenocytes (5 x 106) were adoptively transferred into female RAG-1–/–, male RAG-1–/–, F>F, and F>M mice. Some of these mice were then challenged i.v. with 1 x 106 male bone marrow-derived DC the following day, and spleen cells were taken on day 4 for analysis. The challenge of male bone marrow DC failed to increase IFN-{gamma}-secreting cells in F>M mice when stimulated for 6 h with 5 µM Dby-Ek peptide ex vivo (a), whereas it did result in Thy1.1+ T cells producing IFN-{gamma} in the F>F and female RAG-1–/– mice. Results were pooled from five individual mice. Naive Thy1.1+CD4+ T cell proliferation, indicated in each plot, and net accumulation (b) show no significant differences between F>M mice with and without the challenge of male DC, whereas male bone marrow DC promoted Thy1.1+CD4+ cell proliferation and accumulation in F>F mice.

 
Abs to IL-10R and TGF-{beta} fail to abrogate the suppressive effect of F>M regulatory T cells

F>M mice were treated with either 2 mg anti-TGF-{beta} mAb (1D11), or anti-IL-10R mAb (1B1 · 2), or isotype control (YCATE 55) i.p. on days –1, 0, and 2. CFSE-labeled (A1(M) x Thy-1.1)F1 splenocytes (5 x 106) (Thy-1.1-marked T cells) were adoptively transferred into F>M mice the following day. Mice were killed on day 4 and spleen cells were stained and analyzed by flow cytometry. No differences between the Thy 1.1-marked T cell proliferation, absolute cell numbers, or IFN-{gamma} secretion were observed in the F>M recipients given the neutralizing Abs compared with the isotype control (data not shown). The suppressive effect of F>M regulatory T cells does not, therefore, seem to involve IL-10 or TGF-{beta}.

Anergic regulatory T cells are able to impair rejection directed to third-party Ags

To determine whether F>M regulatory T cells engaging their cognate Ag could regulate responses to third-party alloantigens, we examined their ability to suppress the rejection of third-party (B10.BR) cells by naive CBA/Ca T cells. One x 107 genetically (human (h) CD52) marked CBA/Ca spleen cells were injected into F>M and F>F mice. Three days later, mice were challenged with CFSE-labeled B10.BR T cell-depleted male and female splenocytes. The percentages of CFSE+ B10.BR cells in the peripheral blood were analyzed by tail bleeding at weekly intervals. Female B10.BR cells were cleared in most of the F>F mice by day 14. In contrast, male B10.BR cells were eliminated by day 7 in F>F mice, the faster rate undoubtedly caused by the contribution of host monospecific anti-male T cells. However, both male and female B10.BR cells exhibited longer survival in F>M mice than in F>F mice (p < 0.05; Fig. 8), but were not significantly different from each other. It would seem therefore that in this monospecific T cell system, the effects of regulation could be extended to Ags unrelated to and dissociated from those eliciting the regulation.



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  		FIGURE 8. Anergic regulatory T cells can impede rejection of third-party Ags. One x 107 female CP-1 (hCD52 transgenic) spleen cells were injected into F>M and F>F mice. Three days later, mice were challenged with either CFSE-labeled, in vivo T cell-depleted (4 ), female or male B10.BR splenocytes. The percentages of hCD52–CFSE+ (i.e., B10.BR) lymphocytes in the peripheral blood were analyzed by tail bleeding at weekly intervals and flow cytometry. Male and female B10.BR cells were cleared in most of the F>F mice by days 7 and 14, respectively. However, both male and female B10.BR cells exhibited longer survival in F>M mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We started with a monospecific CD4+ T cell system lacking naturally occurring CD4+CD25+ T cells. The fate of mature monospecific anti-male CD4+ T cells was monitored following persistent Ag stimulation after adoptive transfer into male RAG-1–/– mice. These anti-male (A1(M).RAG-1–/–) monospecific CD4+ T cells colonized their hosts and did not cause GvHD as evidenced by the unremarkable histology of liver and colon and rapid reversal of weight loss. When examined 42 days after initial exposure to male Ag, they behaved both as anergic and regulatory, exhibiting the ability to suppress naive T cell CD4+ proliferation and IL-2 secretion in vitro and naive CD4+ T cell expansion in vivo and IFN-{gamma} production ex vivo.

Nondepleting Abs to CD4 have been shown to induce transplantation tolerance by promoting the development of CD4+ regulatory T cells (2, 4, 26, 27). More recently, Abs to the costimulatory molecule (CD40L) (28, 29, 30) and TCR complex (CD3) (31) have also been used to induce regulatory T cells in vivo. Although the parameters involved in the induction of Ag-specific regulatory T cells are poorly defined, we have proposed that grafts, once accepted, may be able to provide persistent, danger-free Ag stimulation which will, in time, lead to the generation of regulatory T cells (32). The present study was undertaken to test this hypothesis by exposing naive monospecific T cells to a harmless source of persisting Ag and examining whether regulatory T cells were indeed generated.

A critical control for these recolonization studies was the behavior of the F>F group. Previous studies have identified some changes in CD4+ T cells reconstituting T cell-deprived mice. Tanchot et al. (33, 34) reported that naive CD4+ T cells acquired a stable memory-like phenotype in the absence of Ag stimulation in the CD3{epsilon}-deficient and RAG-2–/– recipients. Proliferative responses and cytokine release were also impaired and the injected cells did not completely recolonize the naive CD4+ T cell pool. These results are not dissimilar to those with the F>F CD4+ T cells, which expressed CD44, down-regulated CD62L, and showed a slightly impaired proliferative response. Importantly, these cells did not show regulatory activity by the various criteria measured. The phenotypic changes they exhibited might be secondary to the homeostatic expansion that occurs when small numbers of T cells recolonize a lymphocyte-deficient environment (33, 34).

Adoptive transfer of CD4+CD25+ regulatory T cells can prevent autoimmune disease (6), graft rejection (29), and GvHD. However, the F>M CD4+ T cells containing 3% CD4+CD25+ T cells were far more efficient at regulation than the F>F population that had expanded CD4+CD25+ T cells to 15%. One possibility to explain this is the loss of CD25 expression by regulatory T cells after expansion (35) in F>M recipients. As we could not detect significant foxP3 in the unfractionated spleens of these mice (although control, unfractionated CBA spleen cells were clearly positive), we might conclude that any putative CD4+CD25+ populations that had emerged were unlike natural regulatory T cells (36, 37, 38). Previous evidence for CD4+ CD25– regulatory T cells comes from studies in mice rendered tolerant of allogeneic skin (29) and mice expressing an agonist Ag on the cortical and medullary thymic epithelium and B cells (39). Anti-male regulatory T cells in the F>M mice expressed abundant CTLA-4 and GITR (40) as do natural regulatory T cells (19, 41, 42). Whereas both CTLA-4 and GITR are also expressed by recently activated T cells, it was GITR that remained exclusive to F>M rather than F>F cells.

In vitro suppression of proliferation of naive cells has been widely used to indicate the presence of regulatory T cells (18, 43). However, it is not clear that this readout adequately reflects the physiological role of regulatory T cells in vivo. In the present study, F>M CD4+ T cells could suppress naive CD4+ T cell proliferation in a dose-dependent manner in vitro. However, these T cells had only a marginal effect on naive CD4+ T cell proliferation in vivo yet greatly inhibited naive T cell accumulation and effector cytokine production. These results support a similar finding in a model of dominant transplantation tolerance where the regulatory T cells did not suppress alloreactive CD8+ T cell proliferation yet disarmed their capacity for effector function (44). Part of the suppressive activity of regulatory T cells in vivo may relate to the creation of conditions that do not support survival of Ag-activated T cells and/or their expression of effector functions (45, 46).

Natural CD4+CD25+ T cells, once activated through their TCR, show a capacity for Ag-nonspecific suppression in vitro (18, 47). The literature may be unresolved on this point in being unable to distinguish the induction and effector phases of regulation. In the present study, in a TCR-transgenic recipient, the regulation was seen to extend to third-party Ags, with little added benefit coming from linkage to the cognate male Ag. In these rather special circumstances host monospecific T cells would be in continuous contact with Ag, and a substantive proportion should be permanently activated. Even if each were to regulate in a local microenvironment, it might be difficult, given the frequencies of cells involved, for third-party T cells to escape from being regulated as bystanders. Such a situation would not be easily achieved in a mouse with a conventional polyclonal repertoire of T cells, where local or "linked" suppression might be the likely outcome. It is also possible that although the major population of regulatory CD4+ T cells in this system is inherently Ag specific, they are able to act through other regulatory components of the immune system that do not have such specificity.

The mechanism of action of regulatory T cells derived here remains to be defined. Some models of regulation have reported a necessary role for TGF-{beta} or IL-10 (48, 49, 50), while others have failed to show a need for these cytokines (29, 51). Our own evaluation of this TCR monospecific system seems to exclude an absolute need for IL-10 and TGF-{beta} for this form of regulation to be expressed.

We showed that restoration of fresh male DC failed to overcome suppression. Although this excludes the possibility that failure to respond was not solely due to a lack of host DC, it does not rule out the possibility that regulatory T cells have the ability to neutralize any Ag-bearing DC that might enter the system. In other words, it cannot rule out microenvironment-modifying models of regulation (52).

In summary, sustained exposure to Ag can result in the generation of anergic T cells with regulatory capacity that are predominantly CD25–GITR+CTLA-4+/–, even in the absence of tutoring by any pre-existing regulatory T cells. This form of regulation would not seem to require a role for foxP3. These data support the notion of a form of peripheral tolerance compatible with the "Civil Service Model" (53) expounded over a decade ago, where anergic T cells can compete out emerging naive responding cells that then default to tolerance themselves. By implication, drugs or biological modulators acting to curtail danger/activation signals in T cell and APC interactions may enhance the attainment of tolerance by regulation.


   Footnotes
 
1 This work was supported by a program grant from the Medical Research Council, U.K.. Back

2 Current address: Department of Pathology, Chang Gung Memorial Hospital, Chang Gung University, Tao-Yuan, Taiwan. Back

3 Address correspondence and reprint requests to Prof. Herman Waldmann, Sir William Dunn School of Pathology, South Parks Road, Oxford, OX1 3RE, U.K. E-mail address: herman.waldmann{at}path.ox.ac.uk Back

4 Abbreviations used in this paper: GvHD, graft-versus-host disease; GITR, glucocorticoid-induced TNF-related receptor; DC, dendritic cell; L, ligand; 7-AAD, 7-aminoactinomycin D; MFI, median fluorescence intensity; hCD52, human CD52. Back

Received for publication January 5, 2004. Accepted for publication March 1, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cobbold, S. P., A. Jayasuriya, A. Nash, T. D. Prospero, H. Waldmann. 1984. Therapy with monoclonal antibodies by elimination of T-cell subsets in vivo. Nature 312:548.[Medline]
  2. Qin, S., S. P. Cobbold, H. Pope, J. Elliott, D. Kioussis, J. Davies, H. Waldmann. 1993. "Infectious" transplantation tolerance. Science 259:974.[Abstract]
  3. Waldmann, H., S. Cobbold. 1998. How do monoclonal antibodies induce tolerance? A role for infectious tolerance?. Annu. Rev. Immunol. 16:619.[Medline]
  4. Qin, S., S. Cobbold, H. Tighe, R. Benjamin, H. Waldmann. 1987. CD4 monoclonal antibody pairs for immunosuppression and tolerance induction. Eur. J. Immunol. 17:1159.[Medline]
  5. Graca, L., K. Honey, E. Adams, S. P. Cobbold, H. Waldmann. 2000. Cutting edge: anti-CD154 therapeutic antibodies induce infectious transplantation tolerance. J. Immunol. 165:4783.[Abstract/Free Full Text]
  6. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, M. Toda. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor {alpha}-chains (CD25): breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155:1151.[Abstract]
  7. Powrie, F., M. W. Leach, S. Mauze, L. B. Caddle, R. L. Coffman. 1993. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in CB-17 scid mice. Int. Immunol. 5:1461.[Abstract/Free Full Text]
  8. Powrie, F., R. Correa-Oliveira, S. Mauze, R. L. Coffman. 1994. Regulatory interactions between CD45RBhigh and CD45RBlow CD4+ T cells are important for the balance between protective and pathogenic cell-mediated immunity. J. Exp. Med. 179:589.[Abstract/Free Full Text]
  9. Itoh, M., T. Takahashi, N. Sakaguchi, Y. Kuniyasu, J. Shimizu, F. Otsuka, S. Sakaguchi. 1999. Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J. Immunol. 162:5317.[Abstract/Free Full Text]
  10. Scully, R., S. Qin, S. Cobbold, H. Waldmann. 1994. Mechanisms in CD4 antibody-mediated transplantation tolerance: kinetics of induction, antigen dependency and role of regulatory T cells. Eur. J. Immunol. 24:2383.[Medline]
  11. Wise, M. P., F. Bemelman, S. P. Cobbold, H. Waldmann. 1998. Linked suppression of skin graft rejection can operate through indirect recognition. J. Immunol. 161:5813.[Abstract/Free Full Text]
  12. Marshall, S. E., S. P. Cobbold, J. D. Davies, G. M. Martin, J. M. Phillips, H. Waldmann. 1996. Tolerance and suppression in a primed immune system. Transplantation 62:1614.[Medline]
  13. Chen, Z. K., S. P. Cobbold, H. Waldmann, S. Metcalfe. 1996. Amplification of natural regulatory immune mechanisms for transplantation tolerance. Transplantation 62:1200.[Medline]
  14. Hattori, K., T. Hirano, H. Miyajima, N. Yamakawa, M. Tateno, K. Oshimi, N. Kayagaki, H. Yagita, K. Okumura. 1998. Differential effects of anti-Fas ligand and anti-tumor necrosis factor {alpha} antibodies on acute graft-versus-host disease pathologies. Blood 91:4051.[Abstract/Free Full Text]
  15. Lyons, A. B., C. R. Parish. 1994. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171:131.[Medline]
  16. Lechler, R., J. G. Chai, F. Marelli-Berg, G. Lombardi. 2001. T-cell anergy and peripheral T-cell tolerance. Philos. Trans. R, Soc. London B 356:625.[Abstract/Free Full Text]
  17. Jordan, M. S., M. P. Riley, H. von Boehmer, A. J. Caton. 2000. Anergy and suppression regulate CD4+ T cell responses to a self peptide. Eur. J. Immunol. 30:136.[Medline]
  18. Takahashi, T., Y. Kuniyasu, M. Toda, N. Sakaguchi, M. Itoh, M. Iwata, J. Shimizu, S. Sakaguchi. 1998. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int. Immunol. 10:1969.[Abstract/Free Full Text]
  19. Takahashi, T., T. Tagami, S. Yamazaki, T. Uede, J. Shimizu, N. Sakaguchi, T. W. Mak, S. Sakaguchi. 2000. Immunologic self-tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J. Exp. Med. 192:303.[Abstract/Free Full Text]
  20. Zelenika, D., E. Adams, S. Humm, C. Y. Lin, H. Waldmann, S. P. Cobbold. 2001. The role of CD4+ T-cell subsets in determining transplantation rejection or tolerance. Immunol. Rev. 182:164.[Medline]
  21. Khattri, R., T. Cox, S. A. Yasayko, F. Ramsdell. 2003. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 4:337.[Medline]
  22. Fontenot, J. D., M. A. Gavin, A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4:330.[Medline]
  23. Hori, S., T. Nomura, S. Sakaguchi. 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science 299:1057.[Abstract/Free Full Text]
  24. Fairchild, P. J., H. Waldmann. 2000. Dendritic cells and prospects for transplantation tolerance. Curr. Opin. Immunol. 12:528.[Medline]
  25. Vendetti, S., J. G. Chai, J. Dyson, E. Simpson, G. Lombardi, R. Lechler. 2000. Anergic T cells inhibit the antigen-presenting function of dendritic cells. J. Immunol. 165:1175.[Abstract/Free Full Text]
  26. Qin, S. X., S. Cobbold, R. Benjamin, H. Waldmann. 1989. Induction of classical transplantation tolerance in the adult. J. Exp. Med. 169:779.[Abstract/Free Full Text]
  27. Qin, S. X., M. Wise, S. P. Cobbold, L. Leong, Y. C. Kong, J. R. Parnes, H. Waldmann. 1990. Induction of tolerance in peripheral T cells with monoclonal antibodies. Eur. J. Immunol. 20:2737.[Medline]
  28. Honey, K., S. P. Cobbold, H. Waldmann. 2000. Dominant tolerance and linked suppression induced by therapeutic antibodies do not depend on Fas-FasL interactions. Transplantation 69:1683.[Medline]
  29. Graca, L., S. Thompson, C. Y. Lin, E. Adams, S. P. Cobbold, H. Waldmann. 2002. Both CD4+CD25+ and CD4+CD25– regulatory cells mediate dominant transplantation tolerance. J. Immunol. 168:5558.[Abstract/Free Full Text]
  30. Honey, K., S. P. Cobbold, H. Waldmann. 1999. Dominant regulation: a common mechanism of monoclonal antibody induced tolerance?. Immunol. Res. 20:1.[Medline]
  31. Chatenoud, L.. 2003. CD3-specific antibody-induced active tolerance: from bench to bedside. Nat. Rev. Immunol. 3:123.[Medline]
  32. Waldmann, H.. 2002. Reprogramming the immune system. Immunol. Rev. 185:227.[Medline]
  33. Tanchot, C., A. Le Campion, S. Leaument, N. Dautigny, B. Lucas. 2001. Naive CD4+ lymphocytes convert to anergic or memory-like cells in T cell-deprived recipients. Eur. J. Immunol. 31:2256.[Medline]
  34. Tanchot, C., A. Le Campion, B. Martin, S. Leaument, N. Dautigny, B. Lucas. 2002. Conversion of naive T cells to a memory-like phenotype in lymphopenic hosts is not related to a homeostatic mechanism that fills the peripheral naive T cell pool. J. Immunol. 168:5042.[Abstract/Free Full Text]
  35. Gavin, M. A., S. R. Clarke, E. Negrou, A. Gallegos, A. Rudensky. 2002. Homeostasis and anergy of CD4+CD25+ suppressor T cells in vivo. Nat. Immunol. 3:33.[Medline]
  36. Bennett, C. L., J. Christie, F. Ramsdell, M. E. Brunkow, P. J. Ferguson, L. Whitesell, T. E. Kelly, F. T. Saulsbury, P. F. Chance, H. D. Ochs. 2001. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat. Genet. 27:20.[Medline]
  37. Wildin, R. S., F. Ramsdell, J. Peake, F. Faravelli, J. L. Casanova, N. Buist, E. Levy-Lahad, M. Mazzella, O. Goulet, L. Perroni, et al 2001. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat. Genet. 27:18.[Medline]
  38. Brunkow, M. E., E. W. Jeffery, K. A. Hjerrild, B. Paeper, L. B. Clark, S. A. Yasayko, J. E. Wilkinson, D. Galas, S. F. Ziegler, F. Ramsdell. 2001. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat. Genet. 27:68.[Medline]
  39. Apostolou, I., A. Sarukhan, L. Klein, H. von Boehmer. 2002. Origin of regulatory T cells with known specificity for antigen. Nat. Immunol. 3:756.[Medline]
  40. Zelenika, D., E. Adams, S. Humm, L. Graca, S. Thompson, S. P. Cobbold, H. Waldmann. 2002. Regulatory T cells overexpress a subset of Th2 gene transcripts. J. Immunol. 168:1069.[Abstract/Free Full Text]
  41. Shimizu, J., S. Yamazaki, T. Takahashi, Y. Ishida, S. Sakaguchi. 2002. Stimulation of CD25+CD4+ regulatory T cells through GITR breaks immunological self-tolerance. Nat. Immunol. 3:135.[Medline]
  42. McHugh, R. S., M. J. Whitters, C. A. Piccirillo, D. A. Young, E. M. Shevach, M. Collins, M. C. Byrne. 2002. CD4+CD25+ immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16:311.[Medline]
  43. Thornton, A. M., E. M. Shevach. 1998. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188:287.[Abstract/Free Full Text]
  44. Lin, C. Y., L. Graca, S. P. Cobbold, H. Waldmann. 2002. Dominant transplantation tolerance impairs CD8+ T cell function but not expansion. Nat. Immunol. 3:1208.[Medline]
  45. Madakamutil, L. T., I. Maricic, E. Sercarz, V. Kumar. 2003. Regulatory T cells control autoimmunity in vivo by inducing apoptotic depletion of activated pathogenic lymphocytes. J. Immunol. 170:2985.[Abstract/Free Full Text]
  46. Curtsinger, J. M., D. C. Lins, M. F. Mescher. 2003. Signal 3 determines tolerance versus full activation of naive CD8 T cells: dissociating proliferation and development of effector function. J. Exp. Med. 197:1141.[Abstract/Free Full Text]
  47. Thornton, A. M., E. M. Shevach. 2000. Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific. J. Immunol. 164:183.[Abstract/Free Full Text]
  48. Hara, M., C. I. Kingsley, M. Niimi, S. Read, S. E. Turvey, A. R. Bushell, P. J. Morris, F. Powrie, K. J. Wood. 2001. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J. Immunol. 166:3789.[Abstract/Free Full Text]
  49. Davies, J. D., G. Martin, J. Phillips, S. E. Marshall, S. P. Cobbold, H. Waldmann. 1996. T cell regulation in adult transplantation tolerance. J. Immunol. 157:529.[Abstract]
  50. Onodera, K., W. W. Hancock, E. Graser, M. Lehmann, M. H. Sayegh, T. B. Strom, H. D. Volk, J. W. Kupiec-Weglinski. 1997. Type 2 helper T cell-type cytokines and the development of "infectious" tolerance in rat cardiac allograft recipients. J. Immunol. 158:1572.[Abstract]
  51. Piccirillo, C. A., J. J. Letterio, A. M. Thornton, R. S. McHugh, M. Mamura, H. Mizuhara, E. M. Shevach. 2002. CD4+CD25+ regulatory T cells can mediate suppressor function in the absence of transforming growth factor {beta}1 production and responsiveness. J. Exp. Med. 196:237.[Abstract/Free Full Text]
  52. Mellor, A. L., D. H. Munn. 2003. Tryptophan catabolism and regulation of adaptive immunity. J. Immunol. 170:5809.[Free Full Text]
  53. Waldmann, H., S. Qin, S. Cobbold. 1992. Monoclonal antibodies as agents to reinduce tolerance in autoimmunity. J. Autoimmun. 5:(Suppl. A):93.

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P. Yu, R. K. Gregg, J. J. Bell, J. S. Ellis, R. Divekar, H.-H. Lee, R. Jain, H. Waldner, J. C. Hardaway, M. Collins, et al.
Specific T Regulatory Cells Display Broad Suppressive Functions against Experimental Allergic Encephalomyelitis upon Activation with Cognate Antigen
J. Immunol., June 1, 2005; 174(11): 6772 - 6780.
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M. A. Curotto de Lafaille, A. C. Lino, N. Kutchukhidze, and J. J. Lafaille
CD25- T Cells Generate CD25+Foxp3+ Regulatory T Cells by Peripheral Expansion
J. Immunol., December 15, 2004; 173(12): 7259 - 7268.
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