A number of Treg-associated

molecules, including the inhibitory molecules PD-1 and CTLA-4, as well as CD38 and CD25 were shown to be increased following exposure to 1α25VitD3, although the expression of the Treg-associated marker, GITR, and also CD62L, were inhibited by 1α25VitD3 (Fig. 3). We have previously shown that IL-10 expression is reduced when IL-10 signaling is neutralized in culture [12, 13]. Cells were stimulated in the absence or presence of 10−8 –10−6 M 1α25VitD3 together with either an anti-IL-10R antibody or the appropriate isotype control reagent. In a representative donor shown in Fig. 4A, a high frequency of Foxp3+ cells was observed following culture with 10−6 M 1α25VitD3 and the presence of anti-IL-10R antibody in culture did not alter this. In contrast, considerably less Foxp3+ Trichostatin A order cells were detected in cell cultures containing 10−7 M or 10−8 M 1α25VitD3, and the addition of anti-IL-10R to these cultures resulted in a marked increase in the frequency of Foxp3+ cells (Fig. 4A; mean data from four healthy donors depicted in Fig. 4C). These data were also replicated at the mRNA level using real time RT-PCR where addition of anti-IL-10R antibody resulted in a significant increase in Foxp3 transcripts, with a reciprocal decrease in IL-10 transcripts (Fig. 4B). To confirm these

findings of the effects of IL-10 on 1α25VitD3-enhanced Foxp3 expression, a complimentary approach was used. CD4+ T-cell stimulation cultures were established with high 10−6 M 1α25VitD3 in the Vincristine mouse presence or absence of recombinant IL-10. As predicted, the Thalidomide presence of IL-10 significantly inhibited the frequency of Foxp3+ T cells compared with 10−6

M 1α25VitD3 alone (Fig. 4D). TGF-β is required for the peripheral induction of Foxp3, both alone and in conjunction with retinoic acid (RA) [16-20]. Note in this study, no significant increase in Foxp3 expression was observed when exogenous TGF-β alone was added to cultures containing 10−6 M or 10−7 M 1α25VitD3 (data not shown). However, neutralization of endogenous TGF-β (by the addition of an antibody specific for TGF-β to the culture) decreased 1α25VitD3-enhanced Foxp3 expression (Supporting Information Fig. 1), suggesting a possible role for TGF-β. Human CD4+CD25high cells, which are largely Foxp3+, are known to lose expression of Foxp3 over time upon culture in vitro. To determine if 1α25VitD3 acted to maintain the expression of Foxp3 in this population, CD4+CD25high (>99% CD25+; 86% Foxp3+; Fig. 5A) T cells were isolated by cell sorting and cultured for 7 days with or without 1α25VitD3. The frequency of Foxp3+ cells diminished from 86 to 11.7% upon culture with anti-CD3 and low dose IL-2 alone, shown in a representative plot in Figure 5B.