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CD4(+) CD25(+) Foxp3(+) regulatory T (Treg) cells mediate immunological self-tolerance and suppress immune responses. Retinoic acid (RA), a natural metabolite of vitamin A, has been reported to enhance the differentiation of Treg cells in the presence of TGF-β. In this study, we show that the co-culture of naive T cells from C57BL/6 mice with allogeneic antigen-presenting cells (APCs) from BALB/c mice in the presence of TGF-β, RA, and IL-2 resulted in a striking enrichment of Foxp3(+) T cells. These RA in vitro-induced regulatory T (RA-iTreg) cells did not secrete Th1-, Th2-, or Th17-related cytokines, showed a nonbiased homing potential, and expressed several cell surface molecules related to Treg-cell suppressive potential. Accordingly, these RA-iTreg cells suppressed T-cell proliferation and inhibited cytokine production by T cells in in vitro assays. Moreover, following adoptive transfer, RA-iTreg cells maintained Foxp3 expression and their suppressive capacity. Finally, RA-iTreg cells showed alloantigen-specific immunosuppressive capacity in a skin allograft model in immunodeficient mice. Altogether, these data indicate that functional and stable allogeneic-specific Treg cells may be generated using TGF-β, RA, and IL-2. Thus, RA-iTreg cells may have a potential use in the development of more effective cellular therapies in clinical transplantation.

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long-term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti-CD4 antibody (aCD4). Here, we investigated whether adding TGF-β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg-cell generation and function. Murine CD4(+) T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF-β+RA or aCD4+Rapa. Addition of TGF-β+RA or Rapa resulted in an increase of CD25(+)Foxp3(+)-expressing T cells. Expression of CD40L and production of IFN-γ and IL-17 was abolished in aCD4+TGF-β+RA aTreg cells. Additionally, aCD4+TGF-β+RA aTreg cells showed the highest level of Helios and Neuropilin-1 co-expression. Although CD25(+)Foxp3(+) cells from all culture conditions displayed complete demethylation of the Treg-specific demethylated region, aCD4+TGF-β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF-β+RA aTreg cells suppressed effector T-cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF-β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.

International audience; The long-term stability of renal grafts depends on the absence of chronic rejection. As T cells play a key role in rejection processes, analyzing the T-cell repertoire may be useful for understanding graft function outcomes. We have therefore investigated the power of a new statistical tool, used to analyze the peripheral blood TCR repertoire, for determining immunological differences in a group of 229 stable renal transplant patients undergoing immunosuppression. Despite selecting the patients according to stringent criteria, the patients displayed heterogeneous T-cell repertoire usage, ranging from unbiased to highly selected TCR repertoires; a skewed TCR repertoire correlating with an increase in the CD8(+)/CD4(+) T-cell ratio. T-cell repertoire patterns were compared in patients with clinically opposing outcomes i.e. stable drug-free operationally tolerant recipients and patients with the "suspicious" form of humoral chronic rejection and were found significantly different, from polyclonal to highly selected TCR repertoires, respectively. Moreover, a selected TCR repertoire was found to positively correlate with the Banff score grade. Collectively, these data suggest that TCR repertoire categorization might be included in the calculation of a composite score for the follow-up of patients after kidney transplantation.

Interferon (IFN)-gamma was originally characterized as a pro-inflammatory cytokine with T helper type 1-inducing activity, but subsequent work has demonstrated that mice deficient in IFN-gamma or IFN-gamma receptor show exacerbated inflammatory responses and accelerated allograft rejection, suggesting that IFN-gamma also has important immunoregulatory functions. Here, we demonstrate that ex vivo IFN-gamma conditioning of CD4 T cells driven by allogeneic immature dendritic cells (DC) results in the emergence of a Foxp3(+) regulatory T-cell (Treg)- dominant population that can prevent allograft rejection. The development of this population involves conversion of non-Treg precursors, preferential induction of activation-induced cell death within the non-Treg population and suppression of Th2 and Th17 responses. The suppressive activity of IFN-gamma is dependent on the transcription factor signal transducer and activator of transcription 1 and is mediated by induced nitric oxide. These data indicate not only how IFN-gamma could be used to shape beneficial immune responses ex vivo for possible cell therapy but also provide some mechanistic insights that may be relevant to exacerbated inflammatory responses noted in several autoimmune and transplant models with IFN-gamma deficiency.

CD4(+) CD25(+) Foxp3(+) regulatory T (Treg) cells mediate immunological self-tolerance and suppress immune responses. Retinoic acid (RA), a natural metabolite of vitamin A, has been reported to enhance the differentiation of Treg cells in the presence of TGF-β. In this study, we show that the co-culture of naive T cells from C57BL/6 mice with allogeneic antigen-presenting cells (APCs) from BALB/c mice in the presence of TGF-β, RA, and IL-2 resulted in a striking enrichment of Foxp3(+) T cells. These RA in vitro-induced regulatory T (RA-iTreg) cells did not secrete Th1-, Th2-, or Th17-related cytokines, showed a nonbiased homing potential, and expressed several cell surface molecules related to Treg-cell suppressive potential. Accordingly, these RA-iTreg cells suppressed T-cell proliferation and inhibited cytokine production by T cells in in vitro assays. Moreover, following adoptive transfer, RA-iTreg cells maintained Foxp3 expression and their suppressive capacity. Finally, RA-iTreg cells showed alloantigen-specific immunosuppressive capacity in a skin allograft model in immunodeficient mice. Altogether, these data indicate that functional and stable allogeneic-specific Treg cells may be generated using TGF-β, RA, and IL-2. Thus, RA-iTreg cells may have a potential use in the development of more effective cellular therapies in clinical transplantation.

The beneficial effect on graft survival achieved by pretransplant blood transfusions is well established. However, the type of major histocompatibility complex (MHC) mismatch between transfusion donor and recipient seems to play a role in determining the outcome. The hypothesis that this sharing of MHC antigens is correlated with the level of sensitization or tolerization was studied in mice by pretreatment with semi-allogeneic (F1) or with fully allogeneic whole blood transfusions. Limiting dilution analysis (LDA) in vitro for donor-specific T helper (Thp) and cytotoxic T lymphocyte precursors (CTLp) performed on splenocytes isolated from transfused recipients 2 or 4 weeks after transfusion showed that the duration and magnitude of the response was reduced after a semi-allogeneic compared to a fully allogeneic transfusion. After a semi-allogeneic transfusion, both Thp and CTLp frequencies had returned to naive levels 4 weeks after transfusion, whereas after infusion of fully allogeneic blood, they remained elevated after 4 weeks. When a fully allogeneic heart was transplanted 2 or 4 weeks after transfusion, a small but significant improvement in graft prolongation (2 weeks, not significant, 4 weeks: p < 0.01) was observed following pretreatment with a semi-allogeneic transfusion (2 weeks: median survival time (MST) 30 days, 4 weeks: MST 29 days) compared to that obtained after fully allogeneic transfusion (2 weeks: MST 23 days, 4 weeks: MST 12 days). The semi-allogeneic transfusions were correlated with a statistically significant prolonged (7 days) persistence of donor-derived MHC class II+ cells in the recipient and with reduced levels of anti-donor MHC class I-specific antibody formation compared to these responses after transfusion with fully allogeneic cells. These results demonstrate that pretreatment with a semi-allogeneic blood transfusion is more tolerizing and less sensitizing than pretreatment with a fully allogeneic blood transfusion. These findings may be explained by the sharing of MHC antigens between recipient and transfusion donor.

Regulatory T cells (Treg) have been shown to play a role in the prevention of autoimmune diseases and transplant rejection. Based on an established protocol known to generate alloantigen reactive Treg in vivo, we have developed a strategy for the in vitro selection of Treg. Stimulation of unfractionated CD4(+) T cells from naive CBA.Ca (H2(k)) mice with C57BL/10 (H2(b)) splenocytes in the presence of an anti-CD4 antibody, YTS 177, resulted in the selection of Treg able to inhibit proliferation of naive T cells. In vivo, the cells were able to prevent rejection of 80% C57BL/10 skin grafts when co-transferred to CBA.Rag(-/-) mice together with naive CD45RB(high)CD4(+) cells. Purification of CD62L(+)CD25(+)CD4(+) cells from the cultures enriched for cells with regulatory activity; as now 100% survival of C57BL/10 skin grafts was achieved. Furthermore, differentiation of Treg could be also achieved when using purified CD25(-)CD4(+) naive T cells as a starting population. Interestingly, further in vitro expansion resulted in a partial loss of CD4(+) cells expressing both CD62L and CD25 and abrogation of their regulatory activity in vivo. This study shows that alloantigen stimulation in the presence of anti-CD4 in vitro provides a simple and effective strategy to generate alloreactive Treg.

Direct injection of foreign antigen into the adult thymus is a potent route of antigen delivery for the induction of tolerance in vivo. In this report, we demonstrate that tolerance to C57BL/10 (H2b/BL10) alloantigens can be induced in CBA/Ca (H2k/CBA) mice by intrathymic (IT) administration of BL10 spleen leukocytes coincident with transient peripheral immunomodulation of CD4+ T cells using a depleting anti-CD4 monoclonal antibody. T cell receptor (TCR) transgenic mice (BM3.6; H2k) expressing a CD8-independent TCR specific for H2Kb were used as recipients to facilitate investigation of the mechanisms responsible for tolerance induction by allowing visualization of events in the thymus following IT injection. IT administration of 5 x 10(7) BL10 spleen leukocytes and concomitant transient peripheral T cell depletion in BM3.6 mice resulted in a substantial H2Kb-specific deletion of transgenic-TCR+ (tg-TCR) thymocytes which was dependent on the level of tg-TCR expression. IT deletion and the failure to export CD8+ T cells to the peripheral lymphoid organs correlated with the induction of tolerance to H2Kb; TCR transgenic mice that had received IT injection of BL10 splenocytes and peripheral T cell depletion accepted a H2Kb+ cardiac allograft indefinitely. Analysis of tolerant BM3.6 mice revealed that there were low numbers of CD8+ T cells in the periphery giving rise to a substantially reduced reactivity in vitro despite the fact that no donor cells or IT deletion were observed in the thymi of the majority of tolerant mice. These results demonstrate for the first time that IT injection of foreign alloantigen into an adult thymus results in the deletion of thymocytes expressing a TCR specific for the injected alloantigen and suggest that this is an important mechanism of tolerance induction following IT injection of alloantigen in vivo. Furthermore, analysis of tolerant TCR-transgenic mice suggests that IT deletion is not required for the maintenance of tolerance, and that peripheral mechanisms enforce continued hyporesponsiveness to H2Kb following transplantation.

Adult mice pretreated with donor-specific transfusion and depleting anti-CD4 antibody 28 days before transplant accept fully allogeneic heart grafts and become specifically tolerant without further treatment. The induction of tolerance in this model is not simply a function of CD4+ T cell ablation, but appears to depend on residual CD4+ T cells which escape depletion and engage donor alloantigen during a transient period of antibody blockade. To test the hypothesis that these CD4+ T cells might be responsible for regulating immune responses toward the graft, mice were reconstituted with naive recipient leukocytes at various times after pretreatment. Reconstitution either shortly after pretreatment or shortly after transplant had little effect on graft survival. However, when pretreated mice were given an additional dose of depleting anti-CD4 antibody at the time of transplant to target putative regulatory cells, naive leukocytes were able to cause acute graft rejection. These data suggest that in clinical transplantation specific T cell regulation might develop following pretreatment with antigen and non-depleting anti-CD4 antibodies. Such an approach could provide donor-specific unresponsiveness prior to transplant without the risks associated with sustained CD4+ T cell depletion.