Previously, it was demonstrated that, in the presence Adriamycin purchase of signals via TCR and CD28, c-Rel-deficient CD4+ T cells were able to mount normal TH2 responses. However, naive c-Rel−/− CD4+ cells

were unable to develop into TH1 cells and to produce IFN-γ suggesting a selective requirement of c-Rel for TH1 response 12. In view of the evidence that (i) c-Rel controls IL-2 production 15, (ii) IL-2 induces formation of Treg 22, 23, (iii) IL-2 blocks differentiation of TH17 cells 24, and (iv) differentiation of Treg and TH17 cells seems to be interrelated 25, we were interested in exploring the role of c-Rel for TH17 and Treg differentiation. In this study, we report that c-Rel directly regulates conversion of naive CD4+ T cell into inducible Treg cells (iTreg) by regulating intrinsic production of IL-2. On the other hand, c-Rel appears dispensable for TH17 differentiation. To examine the function of c-Rel in differentiation of iTreg, we isolated naive CD4+CD62L+ T cells from spleens and LN of c-Rel-deficient or WT littermate mice and stimulated them for 3 days under iTreg differentiation conditions in the presence

and absence of exogenous IL-2. Without addition of exogenous human IL-2, we observed a striking decrease in the percentage of c-Rel−/− Foxp3+ T cells as compared with WT cells (Fig. 1A and B). Neutralization of endogenous IL-2 by adding anti-mouse IL-2 antibody led to strong reduction in the frequency of WT Foxp3+ cells with almost complete absence MI-503 molecular weight of Foxp3+ in both WT and c-Rel−/− cells (Fig. 1A). Conversely, addition of human IL-2 to mutant and WT cells boosted the generation of CD4+Foxp3+ iTreg irrespective of c-Rel expression Ribonuclease T1 up to 90% after 3 days of culture (Fig. 1A and 1C). These data show that WT naive T cells can differentiate into iTreg even in the absence

of exogenous IL-2, while c-Rel−/− cells are unable to do so. Interestingly, the conversion into Foxp3+ T cells correlated with IL-2 production by the respective cells as determined by ELISA (Fig. 1D): after 24 h of T-cell receptor stimulation under iTreg culture conditions, there was a substantial impairment in IL-2 production of c-Rel-deficient cells as compared with WT TH cells. Further, while the addition of exogenous human IL-2 significantly increased the endogenous production of IL-2 in WT and c-Rel−/− cells, the strong difference in the respective levels still remained. Together, these data demonstrate that c-Rel regulates the expansion of iTreg by mediating the production of IL-2. We next analyzed natural Treg cells (nTreg) in the thymus of c-Rel−/− mice using antibodies against CD4+ and Foxp3.