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Sir William Dunn School of Pathology, Oxford, U.K.
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Abstract |
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. The nondepleting anti-CD4 mAb was also able to induce tolerance in vivo in such monospecific TCR-transgenic mice, and this too was dependent on TGF-. As in conventional mice, acquired tolerance was dominant, such that naive monospecific T cells were not able to override tolerance. Splenic T cells from tolerant mice proliferated normally in response to Ag, and secreted IFN-
and some IL-4, similar to control mice undergoing primary or secondary graft rejection. High levels of foxP3 mRNA, and glucocorticoid-induced TNFR superfamily member 18 (GITR)+ CD25+ T cells were found within the tolerated skin grafts of long-term tolerant recipients. These data suggest that regulatory T cells maintaining transplantation tolerance after CD4 Ab blockade can be induced de novo through a TGF--dependent mechanism, and come to accumulate in tolerated grafts. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Natural CD4+CD25+ regulatory T cells are thought to play an important role in controlling responses to self Ags, nonpathogenic organisms within the gut, lymphocyte homeostasis, and the innate response, particularly in lymphopenic conditions (12). They are generated within the thymus, possibly via high avidity recognition of self peptides (13), and have recently been shown to express the transcription factor foxP3. Ectopic expression of foxp3 can also convert naive CD4+ T cells to a regulatory phenotype (14, 15, 16). The mechanisms by which natural CD4+CD25+ regulatory T cells act in the different autoimmune disease models seem to involve a discretionary requirement for the cytokines IL-10 and TGF- (17), and may depend on signals through CTLA-4 (18, 19, 20), as well as regulation coordinated by glucocorticoid-induced TNFR superfamily member 18 (GITR)5 (21, 22). A role for both CD4+CD25+ and CD4+CD25– cell populations has been demonstrated in transplantation tolerance after adoptive transfer to lymphopenic mice, but involvement of IL-10 or CTLA-4 remains controversial in that context (23). Regulatory T cells have also been described within the tolerated graft itself, their presence detected following transfer and expansion within recombination-activating gene-1–/– (RAG-1–/–) recipient mice, although most expanded cells exhibited a CD4+CD25– phenotype (24).
An experimentally induced population of cytokine-polarized T cells, known as Tr1 cells, are also able to suppress autoimmune diseases after adoptive transfer in vivo (25, 26). Tr1 cells are thought to be dependent on IL-10 for both their generation and suppressive activity, but it is still unclear how, or if, they may relate to CD4+CD25+ regulatory populations. Gene expression analyses have shown that CTLA-4, GITR, CD25, CD103, and OX40 are all constitutively expressed on both Tr1-like clones and CD4+CD25+ cells from the spleen, but there are many other differences to suggest the populations are quite distinct (27). Tr1-like cells seem to be related to Th2 cells, with the former expressing very few unique genes, but rather expressing only a subset of the Th2-associated transcripts (28). We have found that neither Tr1-like nor Th2 clones express foxP3 by RT-PCR, even after activation with CD3 cross-linking, although activated Tr1-like cells overexpress the repressor of GATA (ROG) (28).
It has previously been shown that Th2 (and Th1) clones with identical specificity can cause rapid, and Ag-specific, rejection of skin grafts after adoptive transfer to lymphocyte-deficient mice (29). Two different Tr1-like clones from such mice were unable to reject male skin, yet were instead able to suppress the subsequent rejection of fresh male grafts by either Th1 or Th2 cells from the same source (26, 27). Although such experiments confirm that Tr1-like cells are capable of suppressing graft rejection in vivo, it also remains a critical issue whether dominant tolerance is a result of the selection of a pre-existing population of natural regulatory T cells that cross-react with graft alloantigens, or the differentiation or polarization of graft-specific peripheral T cells toward a regulatory function.
In this study, we use female transgenic mice expressing a single TCR specific for the male transplantation Ag (Dby presented by H-2Ek) on a RAG-1–/– background (29) that have no detectable pre-existing CD4+CD25+foxP3+ regulatory T cells in the thymus or periphery. We show that Ag stimulation in the presence of saturating nondepleting CD4 Abs induces foxP3 expression in a TGF--dependent manner, both in vitro and in vivo, and that this correlates with lifelong acceptance of male skin grafts. This long-term acceptance is shown to be tolerance as such mice accepted further male skin grafts at any time thereafter, while mice receiving Ab without the tolerizing graft rejected subsequent male skin grafts. Evidence for the presence of induced regulatory T cells resides in the property of tolerant mice to resist the rejection by infused naive, male-specific, monospecific T cells and the simultaneous expression of foxP3, GITR, and CD25 within tolerated skin grafts. This suggests that foxP3+ regulatory T cells can be induced de novo in the periphery by appropriate Ag presentation in the presence of TGF-.
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Materials and Methods |
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A1(M).RAG-1–/– TCR-transgenic mice (29), CBA/Ca.RAG-1–/–, C57/B10, and CBA/Ca mice were bred and maintained in specific pathogen-free conditions at the Sir William Dunn School of Pathology (Oxford, U.K.). Skin grafting was conducted by grafting full thickness tail skin (1 x 1 cm) on the lateral flank as previously described (1). Grafts were observed on alternate days after the removal of the bandage at day 8 and considered rejected when no viable donor skin was present. Statistical analysis of graft survival was made by the log rank method. Tolerance to H-Y was induced at the time of grafting female A1(M).RAG-1–/– mice with male CBA.RAG-1–/– skin by a single injection on day 0 of 1 mg of anti-CD4 mAb (YTS 177.9 (1)). Tolerance was induced in CBA/Ca mice at the time of grafting C57/B10 skin by three doses of 1 mg of the anti-CD4 (YTS 177.9), anti-CD8 (YTS 105.18), and anti-CD154 (MR1 (30)) nondepleting mAbs over 1 wk. In some experiments, neutralizing Abs to TGF- (1D11 (31)) or IL-10R (1B1.2 (32)) were given at doses indicated. All procedures were conducted in accordance with the Home Office Animals (Scientific Procedures) Act of 1986.
Immunofluorescence analysis and Abs
Spleen cells were either labeled directly or after stimulation with PMA (50 ng/ml) plus ionomycin (500 ng/ml) for 4 h at 37°C in the presence of brefeldin A. FACS staining: CD3-FITC (clone KT3; produced in-house), CD25-PE or CD25-allophycocyanin (clone PC61; BD PharMingen, San Diego, CA), CD4-CyChrome (CD4-PerCP; BD PharMingen), and biotin conjugated anti-GITR mAb (YGITR 765 (33)) were used to label live T cells in PBS containing 0.1% NaN3, 1% BSA, 10 µg/ml Fc Block (BD PharMingen), and 5% heat-inactivated normal rabbit serum at 4°C. When required, the cells were then washed, fixed in 2% paraformaldehyde, and permeabilized in PBS containing 0.5% saponin and stained with anti-IFN--PE (BD PharMingen) in the dark at 4°C. Streptavidin-allophycocyanin (BD PharMingen) was also added to detect biotinylated first-step Abs. Four-color analysis was performed using a FACSCalibur (BD Biosciences, Oxford, U.K.) with dual laser (488 nm and 633 nm) excitation. The analysis gate was set on the forward and side scatters to eliminate cell debris and dead cells.
Regulatory T cells from tolerated skin grafts
T cells were prepared from tolerated skin grafts by cutting them into small pieces, digesting with trypsin at 37°C for 1 h, and removing dead cells on nylon wool. Viable lymphocytes were enriched by Histopaque-1083 centrifugation followed by AutoMACS (Miltenyi Biotec, Auburn, CA) positive selection of CD4+ T cells according to the manufacturer’s instructions. These T cells were tested either by real-time RT-PCR or immunofluorescence and FACS analysis.
Real-time quantitative RT-PCR
DNase-I-treated total RNA from tissues was prepared using the SV Total RNA isolation system (Promega, Madison, WI). Reverse transcription was performed using the proStar kit with random hexamers (Stratagene, Cedar Creek, TX). Real-time quantification was performed using gene-specific, fluorogenic probes and the Universal MasterMix kit (PE Applied Biosystems, Warrington, U.K.) in a final volume of 25 µl. The reaction mixture contains all primers at 300 nM and the probe at 200 nM. The enzyme was heat activated 10 min at 95°C. A two-step PCR procedure of 15 s at 95°C and 60 s at 60°C was applied for 50 cycles. PCR and TaqMan analysis were performed using the ABI/PRISM 7700 sequence detector system (PE Applied Biosystems). Multiplex PCRs were performed using appropriate gene-specific primers and VIC-labeled CD3 or hypoxanthine phosphoribosyltransferase (HPRT) probes and FAM-labeled test probes as shown in Table I. Standard curves of cDNAs from normal spleen or Tr1D1 clone were used to calibrate threshold cycles to amounts of test and normalizing cDNAs on each 96-well plate run. Normalized values for mRNA expression were calculated as test mean/normalizer mean. All samples were run in triplicate.
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The production and maintenance of the male-specific Th1, Th2, Tr1D1, and SKA T cell clones from A1(M).RAG-1–/– TCR female transgenic mice has been described elsewhere (26, 29). The clones were harvested by Histopaque-1083 centrifugation and in some cases then stimulated with anti-CD3 (100 ng/ml; 145.2C11 absorbed to 24-well plates) at 37°C overnight.
Proliferation assays
To assess T cell responses from A1(M).RAG-1–/– groups, spleen cell suspensions were depleted of RBC by water lysis, washed, and set up at 5 x 104 cells/200 µl well in RPMI 1640 medium containing 10% FCS. Stimulator cells were mitomycin C-treated (30 min at 37°C) male or female CBA/Ca spleen cells (2 x 105/well), or male or female bone marrow-derived dendritic cells (bmDCs; 1 x 104/well) generated by 7 days culture in recombinant mouse GM-CSF (34). Where required, H-Y peptide (Dby-Ek: REEALHQFRSGRKPI) was added at 10 or 100 nM as specific Ag. After 72 h culture at 37°C, 100 µl of supernatants were taken from each well for cytokine ELISAs, and the volume replaced with medium containing 20 µCi/ml [3H]thymidine for further culture overnight. Plates were harvested onto glass fiber filters and [3H]thymidine incorporation was counted in a Betaplate (Pharmacia, Gaithersburg, MD) scintillation counter. All cultures were tested in triplicate, presented as geometric means and SDs, and significance was calculated by Student’s t test.
Alternatively, CD4+ T cells were enriched from responder spleen cell suspensions using the "CD4 untouched" MACS kit (Miltenyi Biotec) according to manufacturer’s instructions, and labeled with 5 µM CFSE (Molecular Probes, Eugene, OR) at room temperature for 10 min followed by washing in FCS-containing medium. The CFSE-labeled CD4+ T cells (105/well) were incubated for 96 h with female CBA/Ca bmDCs (105/well) alone or with 10 nM DBY-Ek peptide, in duplicate. Cultures were harvested, stained with anti-CD4-biotin, anti-CD25-PE, and 100 µM 7-aminoactinomycin D (7-AAD) to label dead cells, followed by streptavidin-allophycocyanin (BD PharMingen). Cells were fixed in 1% formaldehyde and analyzed using a FACSCalibur (BD Biosciences).
Cytokine detection in culture supernatants
Specific cytokines were assayed by ELISA according to the manufacturer’s instructions (BD PharMingen) with the following Abs: IL-4 was captured by 11B11 and detected by biotin-BVD6.24G2, IL-10 was captured by JESS2A5 and detected by biotin-JESS16E3, and IFN- was captured by R4-6A2 and detected by biotin-XMG1.2, and in each case the assay developed by streptavidin peroxidase followed by o-phenylenediamine substrate. Each ELISA was calibrated against a standard dilution series of the appropriate recombinant mouse cytokine.
Serum CD4 Ab levels
Sera from Ab-treated mice were taken at –1, 63, 94, and 132 days from the time of grafting and Ab injection, heat inactivated at 56°C for 30 min, and incubated with CBA/Ca thymocytes for 1 h at 4°C at 1:2 or 1:10 dilutions. After washing, bound CD4 Ab was detected using FITC anti-rat IgG (Sigma-Aldrich, St. Louis, MO) and flow cytometry (FACSCalibur; BD Biosciences), and calibrated against a standard dilution series of YTS 177.9 in heat inactivated normal mouse serum. The limit of detection of this assay was equivalent to 100 ng of YTS 177.9 per milliliter of neat serum, while saturation was achieved above 2 µg/ml.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dependent manner
With the recent discovery that the transcription factor foxP3 programs T cells to develop regulatory functions (14, 15, 16, 35), it has become possible to use foxP3 induction as a surrogate marker to identify conditions that may favor regulatory T cell differentiation. We have found that stimulation of naive T cells from female A1(M).RAG-1–/– (monospecific) mice, when stimulated in vitro with their cognate Ag (Dby-Ek peptide; 100 nM) in the presence of the nondepleting anti-CD4 mAb (YTS177.9), were not only inhibited in their proliferation (Fig. 1a), but also started to express foxP3 mRNA by day 3 (data not shown). By day 6 (Fig. 1b), there were levels of foxP3 transcripts similar to that of CD4+CD25+ regulatory T cells from normal mice (i.e., 10–100 U of foxP3/CD3). This induction of foxP3 was dependent on TGF-, as it was completely inhibited by including the neutralizing Ab 1D11 in the culture (Fig. 1b). The proliferation block at 100 µg/ml anti-CD4 was also partially reversed by anti-TGF-, but only to levels similar to 10 µg/ml anti-CD4 alone where considerable foxP3 mRNA was still induced (Fig. 1a). An isotype-matched anti-IL-10 receptor Ab had no effect on either proliferation or foxP3 induction (data not shown).
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We next wished to test whether the anti-CD4 mAb could induce dominant tolerance in mice with no endogenous CD4+CD25+ regulatory T cells or foxP3 expression. Female A1(M).RAG-1–/– TCR-transgenic mice normally reject male skin grafts acutely, within 15–20 days (Fig. 2). A single injection of the nondepleting anti-CD4 Ab was able to prolong skin graft survival reliably and indefinitely, at doses of 0.5 mg (data not shown) and 1 mg (Fig. 2). Anti-CD4 Ab was still detectable in the serum (up to 3 µg/ml) at day 63 after grafting, but had been eliminated from the circulation (<100 ng/ml) by day 94 (data not shown). Treated mice carrying male skin grafts for >100 days were able to accept fresh second male grafts indefinitely, demonstrating immune tolerance, while the group initially given Ab without a tolerizing graft rejected their male grafts (Fig. 2). Mice that had initially been allowed to reject a first male graft without Ab cover also rejected fresh second grafts at a similar rate to the first skin.
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Tolerance to graft Ags can be achieved by a number of mechanisms, including clonal elimination, immune regulation, or ignorance of the graft Ag. To determine which of these mechanisms might play a role in the TCR-transgenic system, where all the T cells have the potential to recognize donor Ag, we could determine the number and phenotype of the T cells in each animal. First, it was possible to rule out T cell clonal elimination, as the absolute numbers of CD4+ T cells in the spleen (Fig. 3a), blood, and lymph nodes (data not shown) were not significantly different between mice that were naive, anti-CD4-treated, skin grafted without treatment, or tolerized with a graft plus anti-CD4, even after second graft challenges.
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after a brief activation in vitro with PMA plus ionomycin (Fig. 3b). Furthermore, although the proportions of cells staining was quite variable between individual tolerant mice, they were generally very similar to primed (rather than naive) mice in the expression of a large number of immune markers (including CD152, CD45RB, CD69, CD103, IL-4, IL-10, CD62L, CD154, CD30, and LAG-3; data not shown). CD25 is considered a marker for both activated T cells as well as for CD4+ regulatory T cells. Although CD25 was highly expressed 7 days after a graft challenge in all groups, indicating recent T cell activation, there was evidence of only a small excess of CD4+CD25+ T cells in the spleens of tolerant mice at >100 days after the second graft challenge (Fig. 3c).
Tolerant mice still make a proliferative T cell response to male Ag
In vitro assays of T cell responsiveness to donor Ag after the induction of peripheral tolerance in normal mice with a full T cell repertoire have not generally shown any consistent correlation with the immune status of the recipient (36). We have now observed a similar outcome in the H-Y Ag monospecific TCR-transgenic system consistent with no substantive deletion to achieve this form of tolerance. All the groups, whether tolerant or immune to male skin grafts, were able to make similar dose-dependent, proliferative (Fig. 4a), and cytokine (>100 ng/ml IFN-, 0.5–5 ng/ml IL-4, <1 ng/ml IL-10; data not shown) responses in vitro when female spleen cells or bmDCs were used to present varying concentrations (1–1000 nM) of the Dby-Ek peptide. However, the tolerant group did seem to respond poorly to native male APCs (that in naive mice is equivalent to 10 nM Dby-Ek peptide), raising the possibility that tolerant T cells may in some way down-regulate presentation or processing of the endogenous Dby Ag. Prior removal of the small numbers of CD4+CD25+ T cells from the tolerant spleen cell populations had no effect on the proliferation of any population in vitro, whether stimulated with Dby-Ek peptide, native male Ag, or anti-CD3 (data not shown).
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Tolerance is associated with foxP3+, GITR+CD25+ T cells in the graft
We have previously found that two of the major regulatory T cell populations, the natural CD4+CD25+ cells and the "induced" Tr1-like cells, are associated with the expression of two different transcription factors, respectively foxP3 (14, 15, 16) and ROG (28, 37). As expected for TCR-transgenic mice on a RAG-1–/– background (38), there was no significant expression of mRNA by RT-PCR for either transcript in the thymus or spleens of naive male or female animals (Fig. 5a). High levels of foxP3 were found in the original, tolerizing, and secondary challenge skin grafts of the tolerized groups, but not in rejecting or syngeneic (i.e., female) skin grafts, or normal skin from any of the groups, at >100 days from first grafting. The level of foxP3 mRNA detected was similar to that seen in purified CD4+CD25+ T cells from normal CBA/Ca mice, and to that seen in the accepted skin grafts of C57/B10-tolerized nontransgenic CBA/Ca mice, when normalized to the amount of CD3 mRNA in each sample (Fig. 5a). Analysis of tissues from anti-CD4-treated female A1(M).RAG-1–/– mice at various times after grafting indicated that foxP3 message could first be detected in male, but not female, skin grafts at day 21, and at this time there was also a weak signal for foxP3 in the spleens of male tolerized mice that disappeared by day 28 (Fig. 5b). foxP3 has not been detected in any effector Th1, Th2, or Tr1-like cell clones from female A1(M).RAG-1–/– mice, whether resting or activated (28), suggesting that a regulatory T cell phenotype may have been induced specifically in those T cells colonizing a tolerated skin graft. In contrast, ROG was detected, but was not differentially expressed, in tolerated and rejecting skin grafts (Fig. 5a).
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50% coexpressed CD3+CD4+GITR+CD25+ (Fig. 6b), supporting a regulatory phenotype for these cells (14, 15, 16, 22). These T cells must have been derived from the naive CD3+CD4+GITR–CD25–foxP3– population of the monospecific mice, as the male skin grafts were derived from T cell-deficient RAG-1–/– donors.
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The presence of regulatory T cells and peripheral tolerance has generally been associated with a resistance to the rejection of established or fresh grafts by the infusion of immunocompetent lymphocytes (39). The male tolerized, monospecific mice also showed evidence of such regulatory activity, as the infusion of sufficient naive male-specific T cells to cause rapid graft rejection in empty RAG-1–/– recipients was unable to break the tolerant state (Fig. 7a). Giving sufficient Ag-primed cells, in this case a male-specific Th1 clone (29), was able to override tolerance in monospecific mice in a fashion similar to CD4 mAb-induced tolerance in normal CBA/Ca mice (1), leading to graft rejection, albeit with a small, but statistically significant, delay (Fig. 7b).
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Having demonstrated that anti-CD4 treatment was able to induce tolerance and regulatory T cells associated with the expression of foxP3, we wished to determine whether this would be TGF- dependent. We found that coadministration of a neutralizing anti-TGF- mAb (1D11) blocked the ability of the anti-CD4 to induce tolerance in the majority of female A1(M).RAG-1–/– mice (Fig. 8), while an Ab of the same isotype against the IL-10 receptor (1B1.2) had no effect, with all but one of the grafts surviving indefinitely.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The natural CD4+CD25+ regulatory T cell population is thought to be primarily specific for self Ags and to originate from selection events during T cell development within the thymus (13, 40, 41, 42, 43). Regulatory T cells that control mucosal inflammation are also thought to have some specificity for Ags expressed by commensal flora (44, 45), although it is not clear whether these are selected by cross-reaction from the thymic self-reactive population, whether they are generated in the periphery de novo (46), or whether they act primarily to control innate mechanisms of inflammation (47) and T cell homeostasis (48, 49).
The monospecific mice used in this study were found not to contain measurable foxP3+, CD4+CD25+ natural regulatory T cells in the thymus or periphery, as expected (9, 38), and all their T cells have identical specificity for the male (Dby) transplantation Ag (29) and are therefore very unlikely to have any significant cross-reaction with self Ags or normal gut flora. When taken together, the induction of tolerance, expression of foxP3, GITR, and CD25 on T cells within the tolerated graft, and evidence of regulatory activity as shown by the resistance to breaking tolerance by naive T cells in vivo, strongly support the hypothesis that donor-specific peripheral T cells are being converted to a regulatory phenotype by exposure to graft-derived Ag under the cover of coreceptor blockade. This is further supported by the observation that anti-CD4 blockade in vitro also leads to the induction of foxP3 and that this, like the tolerance induction, is dependent on TGF-. This is consistent with recent data by Chen et al. (35) who have shown that CD4+CD25– cells can be converted to CD4+CD25+ regulatory cells due to a TGF--dependent induction of foxP3.
The fact that foxP3 is highly enriched in the tolerated grafts and is only transiently detected in the spleen suggests that such regulatory T cells are accumulating and perhaps acting locally within the tolerated tissue. This result is particularly intriguing with respect to how foxP3+ regulators are found within the secondary challenge skin grafts given to already tolerant recipients. It remains to be determined whether these foxP3+ regulators are recruited from the original tolerizing graft, or some lymphoid reservoir of tolerant foxP3+ T cells outside of the spleen or draining lymph nodes, or whether they derive from a foxP3– T cell, perhaps via the influence of modulated APCs (50, 51) within the tolerant mouse. Others have also shown that regulatory T cell activity can be generated in the periphery of Ag-specific TCR-transgenic mice (46), but these were CD25– and of unknown foxP3 status.
Although the expression of foxP3, CD25, and GITR suggests that the maintenance of tolerance in this model may involve mechanisms related to the natural CD4+CD25+foxP3+ regulatory cell population, we cannot exclude a role for Tr1-like or CD25– cells, as we do not know the proportion of T cells in the tolerated grafts that expressed foxP3, and only about half of the CD3+CD4+ T cells coexpressed CD25 and GITR. In addition, we did detect the presence of ROG mRNA, a marker we have found previously associated particularly with Tr1-like cells (28), but this was not differential between tolerant and rejecting grafts. It may be that tolerance is maintained by a combination of different regulatory T cells, including CD25+, foxP3+, CD25–, or ROG+ populations, even in a monospecific TCR-transgenic mouse.
The ability to block CD4 mAb-induced tolerance by neutralizing TGF-, but not by blocking the IL-10R, is of considerable interest, as we have previously been unable to break established tolerance or linked suppression by neutralizing either TGF- (52) or IL-10 (23). This would suggest that TGF- plays a specific role in inducing foxP3-mediated differentiation to regulatory cells (35), but that neither TGF- nor IL-10 may be a critical component of their mechanism of action. There has been some debate over the role of TGF- in CD4+CD25+ regulatory T cells, with evidence from in vitro suppression assays both for (53) and against any such requirement (54). However, TGF- has been found essential to induce tolerance to allografts in rats by donor-specific blood transfusions (55), and in the induction of regulatory T cells by anti-CD3 treatment of either IL-2-deficient (56) or diabetic nonobese diabetic (57) mice.
In summary, peripheral tolerance can be induced in TCR-transgenic mice where every T cell is CD4+ and directed to the donor transplantation Ag. A nondepleting CD4 mAb was able to induce foxP3 during Ag stimulation in vitro and tolerance to skin grafts in vivo, and in both cases this was dependent on TGF-. CD4+ T cells from tolerant mice still made proliferative and cytokine-producing responses to donor Ag in vitro, and systemically to fresh donor-type skin grafts, even though these were not rejected. Tolerance was associated with the presence of regulatory T cells that were able to resist the rejection normally caused by the infusion of donor-specific naive T cells. In the TCR-transgenic mice, putative regulatory T cells were concentrated in tolerated skin and expressed CD4, CD25, GITR, and foxP3, and would appear to have arisen de novo in the periphery.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stephen P. Cobbold, Reader in Cellular Immunology, Therapeutic Immunology Group, Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, U.K. E-mail address: stephen.cobbold{at}path.ox.ac.uk
3 Current address: Laboratorio de Medicina Internal, Hospital Universitario Puerta de Hierro, c/San Martín de Porres, 4. 28035 Madrid, Spain.
4 Current address: Centre National de Génotypage, 2 rue Gaston, Cremieux, 91057 Evry Cedex, France.
5 Abbreviations used in this paper: GITR, glucocorticoid-induced TNFR superfamily member 18; 7-AAD, 7-aminoactinomycin D; bmDC, bone marrow-derived dendritic cell; HPRT, hypoxanthine phosphoribosyltransferase; RAG-1–/–, recombination-activating gene-1–/–; ROG, repressor of GATA.
Received for publication December 29, 2003. Accepted for publication March 3, 2004.
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 J. Yokokawa, V. Cereda, C. Remondo, J. L. Gulley, P. M. Arlen, J. Schlom, and K. Y. Tsang Enhanced Functionality of CD4+CD25highFoxP3+ Regulatory T Cells in the Peripheral Blood of Patients with Prostate Cancer Clin. Cancer Res., February 15, 2008; 14(4): 1032 - 1040. [Abstract] [Full Text] [PDF]
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J.-G. Chai, D. Coe, D. Chen, E. Simpson, J. Dyson, and D. Scott In Vitro Expansion Improves In Vivo Regulation by CD4+CD25+ Regulatory T Cells J. Immunol., January 15, 2008; 180(2): 858 - 869. [Abstract] [Full Text] [PDF]
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C. Siewert, U. Lauer, S. Cording, T. Bopp, E. Schmitt, A. Hamann, and J. Huehn Experience-Driven Development: Effector/Memory-Like {alpha}E+Foxp3+ Regulatory T Cells Originate from Both Naive T Cells and Naturally Occurring Naive-Like Regulatory T Cells J. Immunol., January 1, 2008; 180(1): 146 - 155. [Abstract] [Full Text] [PDF]
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N. J. Robertson, F. A. Brook, R. L. Gardner, S. P. Cobbold, H. Waldmann, and P. J. Fairchild Embryonic stem cell-derived tissues are immunogenic but their inherent immune privilege promotes the induction of tolerance PNAS, December 26, 2007; 104(52): 20920 - 20925. [Abstract] [Full Text] [PDF]
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J. Wei, O. Duramad, O. A. Perng, S. L. Reiner, Y.-J. Liu, and F. X.-F. Qin Antagonistic nature of T helper 1/2 developmental programs in opposing peripheral induction of Foxp3+ regulatory T cells PNAS, November 13, 2007; 104(46): 18169 - 18174. [Abstract] [Full Text] [PDF]
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O. Bestard, J. M. Cruzado, M. Mestre, A. Caldes, J. Bas, M. Carrera, J. Torras, I. Rama, F. Moreso, D. Seron, et al. Achieving Donor-Specific Hyporesponsiveness Is Associated with FOXP3+ Regulatory T Cell Recruitment in Human Renal Allograft Infiltrates J. Immunol., October 1, 2007; 179(7): 4901 - 4909. [Abstract] [Full Text] [PDF]
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S. Brouard, E. Mansfield, C. Braud, L. Li, M. Giral, S.-c. Hsieh, D. Baeten, M. Zhang, J. Ashton-Chess, C. Braudeau, et al. Identification of a peripheral blood transcriptional biomarker panel associated with operational renal allograft tolerance PNAS, September 25, 2007; 104(39): 15448 - 15453. [Abstract] [Full Text] [PDF]
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S. R. Daley, J. Ma, E. Adams, S. P. Cobbold, and H. Waldmann A Key Role for TGF-beta Signaling to T Cells in the Long-Term Acceptance of Allografts J. Immunol., September 15, 2007; 179(6): 3648 - 3654. [Abstract] [Full Text] [PDF]
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J. Wong, D. Mathis, and C. Benoist TCR-based lineage tracing: no evidence for conversion of conventional into regulatory T cells in response to a natural self-antigen in pancreatic islets J. Exp. Med., September 3, 2007; 204(9): 2039 - 2045. [Abstract] [Full Text] [PDF]
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J. L. Coombes, K. R.R. Siddiqui, C. V. Arancibia-Carcamo, J. Hall, C.-M. Sun, Y. Belkaid, and F. Powrie A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-{beta} and retinoic acid dependent mechanism J. Exp. Med., August 6, 2007; 204(8): 1757 - 1764. [Abstract] [Full Text] [PDF]
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M. J. Benson, K. Pino-Lagos, M. Rosemblatt, and R. J. Noelle All-trans retinoic acid mediates enhanced T reg cell growth, differentiation, and gut homing in the face of high levels of co-stimulation J. Exp. Med., August 6, 2007; 204(8): 1765 - 1774. [Abstract] [Full Text] [PDF]
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S. F. Yates, A. M. Paterson, K. F. Nolan, S. P. Cobbold, N. J. Saunders, H. Waldmann, and P. J. Fairchild Induction of Regulatory T Cells and Dominant Tolerance by Dendritic Cells Incapable of Full Activation J. Immunol., July 15, 2007; 179(2): 967 - 976. [Abstract] [Full Text] [PDF]
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M. Habib, M. N. Rivas, M. Chamekh, S. Wieckowski, W. Sun, A. Bianco, N. Trouche, O. Chaloin, H. Dumortier, M. Goldman, et al. Cutting Edge: Small Molecule CD40 Ligand Mimetics Promote Control of Parasitemia and Enhance T Cells Producing IFN-{gamma} during Experimental Trypanosoma cruzi Infection J. Immunol., June 1, 2007; 178(11): 6700 - 6704. [Abstract] [Full Text] [PDF]
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Z. Wang and J. D. Davies CD8 Blockade Promotes Antigen Responsiveness to Nontolerizing Antigen in Tolerant Mice by Inhibiting Apoptosis of CD4+ T Cells J. Immunol., May 15, 2007; 178(10): 6148 - 6157. [Abstract] [Full Text] [PDF]
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 Y. Peng, H. Shao, Y. Ke, P. Zhang, G. Han, H. J. Kaplan, and D. Sun Minimally Activated CD8 Autoreactive T Cells Specific for IRBP Express a High Level of Foxp3 and Are Functionally Suppressive Invest. Ophthalmol. Vis. Sci., May 1, 2007; 48(5): 2178 - 2184. [Abstract] [Full Text] [PDF]
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S. You, B. Leforban, C. Garcia, J.-F. Bach, J. A. Bluestone, and L. Chatenoud Adaptive TGF-beta-dependent regulatory T cells control autoimmune diabetes and are a privileged target of anti-CD3 antibody treatment PNAS, April 10, 2007; 104(15): 6335 - 6340. [Abstract] [Full Text] [PDF]
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K. N. Couper, D. G. Blount, J. B. de Souza, I. Suffia, Y. Belkaid, and E. M. Riley Incomplete Depletion and Rapid Regeneration of Foxp3+ Regulatory T Cells Following Anti-CD25 Treatment in Malaria-Infected Mice J. Immunol., April 1, 2007; 178(7): 4136 - 4146. [Abstract] [Full Text] [PDF]
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C. J. Callaghan, F. J. Rouhani, M. C. Negus, A. J. Curry, E. M. Bolton, J. A. Bradley, and G. J. Pettigrew Abrogation of Antibody-Mediated Allograft Rejection by Regulatory CD4 T Cells with Indirect Allospecificity J. Immunol., February 15, 2007; 178(4): 2221 - 2228. [Abstract] [Full Text] [PDF]
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K. V. Tarbell, L. Petit, X. Zuo, P. Toy, X. Luo, A. Mqadmi, H. Yang, M. Suthanthiran, S. Mojsov, and R. M. Steinman Dendritic cell-expanded, islet-specific CD4+ CD25+ CD62L+ regulatory T cells restore normoglycemia in diabetic NOD mice J. Exp. Med., January 22, 2007; 204(1): 191 - 201. [Abstract] [Full Text] [PDF]
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H. H. Uhlig, J. Coombes, C. Mottet, A. Izcue, C. Thompson, A. Fanger, A. Tannapfel, J. D. Fontenot, F. Ramsdell, and F. Powrie Characterization of Foxp3+CD4+CD25+ and IL-10-Secreting CD4+CD25+ T Cells during Cure of Colitis J. Immunol., November 1, 2006; 177(9): 5852 - 5860. [Abstract] [Full Text] [PDF]
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 J. A. Kapp, K. Honjo, L. M. Kapp, X. y. Xu, A. Cozier, and R. P. Bucy TCR transgenic CD8+ T cells activated in the presence of TGF{beta} express FoxP3 and mediate linked suppression of primary immune responses and cardiac allograft rejection Int. Immunol., November 1, 2006; 18(11): 1549 - 1562. [Abstract] [Full Text] [PDF]
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M. Mahic, S. Yaqub, C. C. Johansson, K. Tasken, and E. M. Aandahl FOXP3+CD4+CD25+ Adaptive Regulatory T Cells Express Cyclooxygenase-2 and Suppress Effector T Cells by a Prostaglandin E2-Dependent Mechanism J. Immunol., July 1, 2006; 177(1): 246 - 254. [Abstract] [Full Text] [PDF]
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K. Wing, Z. Fehervari, and S. Sakaguchi Emerging possibilities in the development and function of regulatory T cells Int. Immunol., July 1, 2006; 18(7): 991 - 1000. [Abstract] [Full Text] [PDF]
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 M. J. Jasper, K. P. Tremellen, and S. A. Robertson Primary unexplained infertility is associated with reduced expression of the T-regulatory cell transcription factor Foxp3 in endometrial tissue Mol. Hum. Reprod., May 1, 2006; 12(5): 301 - 308. [Abstract] [Full Text] [PDF]
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D. C. Neujahr, C. Chen, X. Huang, J. F. Markmann, S. Cobbold, H. Waldmann, M. H. Sayegh, W. W. Hancock, and L. A. Turka Accelerated Memory Cell Homeostasis during T Cell Depletion and Approaches to Overcome It. J. Immunol., April 15, 2006; 176(8): 4632 - 4639. [Abstract] [Full Text] [PDF]
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H. Geng, G.-M. Zhang, D. Li, H. Zhang, Y. Yuan, H.-G. Zhu, H. Xiao, L.-F. Han, and Z.-H. Feng Soluble Form of T Cell Ig Mucin 3 Is an Inhibitory Molecule in T Cell-Mediated Immune Response J. Immunol., February 1, 2006; 176(3): 1411 - 1420. [Abstract] [Full Text] [PDF]
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 S. A. Nanji, W. W. Hancock, B. Luo, C. D. Schur, R. L. Pawlick, L. F. Zhu, C. C. Anderson, and A.M. J. Shapiro Costimulation Blockade of Both Inducible Costimulator and CD40 Ligand Induces Dominant Tolerance to Islet Allografts and Prevents Spontaneous Autoimmune Diabetes in the NOD Mouse Diabetes, January 1, 2006; 55(1): 27 - 33. [Abstract] [Full Text] [PDF]
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A. S. Krupnick, A. E. Gelman, W. Barchet, S. Richardson, F. H. Kreisel, L. A. Turka, M. Colonna, G. A. Patterson, and D. Kreisel Cutting Edge: Murine Vascular Endothelium Activates and Induces the Generation of Allogeneic CD4+25+Foxp3+ Regulatory T Cells J. Immunol., November 15, 2005; 175(10): 6265 - 6270. [Abstract] [Full Text] [PDF]
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 Z. Fehervari and S. Sakaguchi CD4+ regulatory cells as a potential immunotherapy Phil Trans R Soc B, September 29, 2005; 360(1461): 1647 - 1661. [Abstract] [Full Text] [PDF]
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S. M Metcalfe Axotrophin and leukaemia inhibitory factor (LIF) in transplantation tolerance Phil Trans R Soc B, September 29, 2005; 360(1461): 1687 - 1694. [Abstract] [Full Text] [PDF]
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S. P Cobbold T cell tolerance induced by therapeutic antibodies Phil Trans R Soc B, September 29, 2005; 360(1461): 1695 - 1705. [Abstract] [Full Text] [PDF]
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N. Beyersdorf, S. Gaupp, K. Balbach, J. Schmidt, K. V. Toyka, C.-H. Lin, T. Hanke, T. Hunig, T. Kerkau, and R. Gold Selective targeting of regulatory T cells with CD28 superagonists allows effective therapy of experimental autoimmune encephalomyelitis J. Exp. Med., August 1, 2005; 202(3): 445 - 455. [Abstract] [Full Text] [PDF]
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J. C. Ochando, A. C. Yopp, Y. Yang, A. Garin, Y. Li, P. Boros, J. Llodra, Y. Ding, S. A. Lira, N. R. Krieger, et al. Lymph Node Occupancy Is Required for the Peripheral Development of Alloantigen-Specific Foxp3+ Regulatory T Cells J. Immunol., June 1, 2005; 174(11): 6993 - 7005. [Abstract] [Full Text] [PDF]
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I. Lee, L. Wang, A. D. Wells, M. E. Dorf, E. Ozkaynak, and W. W. Hancock Recruitment of Foxp3+ T regulatory cells mediating allograft tolerance depends on the CCR4 chemokine receptor J. Exp. Med., April 4, 2005; 201(7): 1037 - 1044. [Abstract] [Full Text] [PDF]
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P. Zhou, S. J. Balin, M. Mashayekhi, K. W. Hwang, D. A. Palucki, and M.-L. Alegre Transplantation Tolerance in NF-{kappa}B-Impaired Mice Is Not Due to Regulation but Is Prevented by Transgenic Expression of Bcl-xL J. Immunol., March 15, 2005; 174(6): 3447 - 3453. [Abstract] [Full Text] [PDF]
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L. Fahlen, S. Read, L. Gorelik, S. D. Hurst, R. L. Coffman, R. A. Flavell, and F. Powrie T cells that cannot respond to TGF-{beta} escape control by CD4+CD25+ regulatory T cells J. Exp. Med., March 7, 2005; 201(5): 737 - 746. [Abstract] [Full Text] [PDF]
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D.-X. Chen, H. He, and R. M. Gorczynski Synthetic peptides from the N-terminal regions of CD200 and CD200R1 modulate immunosuppressive and anti-inflammatory effects of CD200-CD200R1 interaction Int. Immunol., March 1, 2005; 17(3): 289 - 296. [Abstract] [Full Text] [PDF]
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 M. K. Levings, S. Gregori, E. Tresoldi, S. Cazzaniga, C. Bonini, and M. G. Roncarolo Differentiation of Tr1 cells by immature dendritic cells requires IL-10 but not CD25+CD4+ Tr cells Blood, February 1, 2005; 105(3): 1162 - 1169. [Abstract] [Full Text] [PDF]
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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. [Abstract] [Full Text] [PDF]
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D. Winsor-Hines, C. Merrill, M. O'Mahony, P. E. Rao, S. P. Cobbold, H. Waldmann, D. J. Ringler, and P. D. Ponath Induction of Immunological Tolerance/Hyporesponsiveness in Baboons with a Nondepleting CD4 Antibody J. Immunol., October 1, 2004; 173(7): 4715 - 4723. [Abstract] [Full Text] [PDF]
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 H. Waldmann and S. Cobbold Exploiting Tolerance Processes in Transplantation Science, July 9, 2004; 305(5681): 209 - 212. [Abstract] [Full Text] [PDF]
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). After >100 days, each group was then given second challenge male CBA/Ca skin grafts (rejecting group, n = 9, 
; mAb control group, n = 11, 
; tolerized group, n = 9, 
). Only in the tolerized group did both first and second grafts survive indefinitely (>200 and >100 days, respectively; p < 0.001 for first or second accepted grafts compared with anti-CD4 mAb-only control second grafts).
