5b). These data suggest that demethylation of this CpG island of the Foxp3 promoter region correlates with Foxp3 expression. The methylation status of this region was evaluated in Foxp3− T cells that were activated for 72 hr in the presence of TGF-β alone, simvastatin alone, and the combination of TGF-β/simvastatin (Fig. 5c). After 72 hr, 48% and 42% of the CpGs of dimethylsulphoxide-treated or simvastatin-treated cells were methylated, respectively. However, this region in TGF-β-treated cells was less methylated (26%) than in dimethylsulphoxide-treated

or simvastatin-treated cells and the lowest level of methylation (16%) was observed in the cells treated with simvastatin/TGF-β. Seventy-two hours after activation, the extent of demethylation correlated well with the level of Foxp3 expression PD-0332991 cell line detected by FACS analysis (bottom boxes in Fig. 5c). These results suggest that the synergistic action of simvastatin on TGF-β-mediated induction of Foxp3 may be mediated by co-operative control of methylation of the Foxp3 promoter. To directly examine the effects of simvastatin on TGF-β-mediated signal transduction, we measured phosphorylation of Smad3. Significant phosphorylation of

Smad3 was observed 24 hr after activation of cells cultured in the presence of LBH589 TGF-β, but not simvastatin alone, and the levels of Smad3 phosphorylation were not modulated when the cultures were stimulated with both TGF-β and simvastatin (Fig. 6a). In addition, the total amount of Smad4 was comparable in all treatment groups. The lack of an effect of Interleukin-2 receptor simvastatin on Smad3 phosphorylation is consistent with its late time of action and raised the possibility that simvastatin might block steps in the negative-feedback regulation of TGF-β signalling. Smad6 and Smad7 are the major inhibitory Smad proteins in the negative feedback regulation

of the TGF-β signalling pathway. In contrast to Smad3 phosphorylation, we could not detect Smad7 by Western blot analysis 24 hr after T-cell activation and only low levels of Smad6 were observed. The levels of Smad6/7 increased after 48 hr and were maximal at 72 hr after activation (Fig. 6b). Importantly, when combined with TGF-β, simvastatin markedly inhibited the induction of Smad6 at 48 and 72 hr, and completely blocked Smad7 induction at both 48 and 72 hr. Simvastatin alone also decreased levels of Smad6 and completely blocked Smad7 expression at 72 hr. As TGF-β has been reported to play an important role in Foxp3+ Treg homeostasis,16 we also examined the expression of Smad6/7 in nTregs that were activated under conditions similar to those used in our iTreg induction cultures. Foxp3− and Foxp3+ CD4+ T cells were FACS-sorted from Foxp3gfp mice and activated with anti-CD3/CD28 and IL-2 in the absence or presence of TGF-β for 72 hr (Fig. 6c).