Additionally, to determine RG-7388 the role of IFN-γ and IL-10 in the inhibitory effect of rSj16-induced Tregs on CD4+CD25− T-cell proliferation, we added anti-IL-10 and anti-IFN-γ neutralizing antibodies in the culture as described above. These results showed that either IL-10 or IFN-γ neutralizing antibodies reduced the inhibitory effect of rSj16-induced Tregs
on CD4+CD25− T-cell proliferation, but only IFN-γ significantly (Figure 3e). Furthermore, to determine the source of IFN-γ, we detected the percentage of IFN-γ+Foxp3+ T cells and IFN-γ+Foxp3− T in CD4+ T cells. The results showed that the percentage of IFN-γ+Foxp3+ T cells increased only in rSj16-treated group. In contrast, the percentage of IFN-γ+Foxp3− T cells in CD4+ T cells did not change significantly between groups (Figure 3f,g). These results suggested that the increased IFN-γ production is from rSj16-induced regulatory T cells. We next investigated the role of APCs in rSj16-induced
CD4+CD25+ regulatory T cells. We first purified CD4+ T cells from naïve mice and cultured with rSj16, OVA, LPS or medium alone, respectively. After 4-day incubation, the cells were buy Pifithrin-�� harvested for FCM analysis. The results showed that there were no significant changes in CD4+CD25+Foxp3+ T cells in each group (Figure 4a). Then, BM-derived DCs (BMDCs) from BALB/c mice were cultured with rSj16, OVA, LPS or medium alone, respectively, and incubated with CD4+T cells from naïve mice for 4 days. The cells were harvested for FCM analysis. The results showed that BMDC pulsed with rSj16, but not OVA, LPS or medium, stimulated a marked increase in CD4+CD25+Foxp3+ T cells (Figure 4b).
Collectively, these findings indicated that rSj16-treated BMDCs favour differentiation of T cells into Clomifene CD4+CD25+Foxp3+ T cells. It has been reported that immature DCs are prone to induce Tregs (27); therefore, we investigated the phenotype of antigen-pulsed BMDC by analysing their surface markers. Compared to LPS-pulsed BMDCs, rSj16-pulsed BMDCs displayed an immature or nonactivated phenotype as their down-regulated MHC II and costimulatory molecule expression (i.e. CD40, CD80 and CD86) on their surface (Figure 5a). Parallel to the increase in CD4+CD25+Foxp3+ T cells, the proliferation of CD4+T cells cocultured with rSj16-pulsed BMDC did not increase significantly compared to CD4+ T-cell proliferation induced by BMDC cocultured with either OVA or LPS (Figure 5b). It suggested that the immature DCs from rSj16-pulsed BMDCs presented weaker ability of antigen presentation. T-bet, a transcription factor that binds to and transactivates the Ifng locus, is required for IFN-γ production by CD4+T cells (28).