Transwell plates (Nunclon, Rochester, NY) were gently placed in the lower chamber and 2 × 104 CD4+ CD25+ CD127− T cells with or without pre-incubation with RBV were plated in transwell plates with 1 × 105 allogeneic irradiated PBMCs (upper chamber). Soluble OKT3 20 μg/ml was added

to both chambers and incubated for 7 days at 37°. At the end of incubation, the upper chamber was removed gently, and then 1 μCi of thymidine was added to the lower chamber and incubated for an additional 16 hr. Cells were harvested and [3H]thymidine incorporation was measured. Student’s t-test and Bonferroni’s multiple-comparison test were performed to analyse the significance of differences between groups in this study using graphpad prism (GraphPad Software, La Jolla, CA). All experiments were repeated five times, and a P value of < 0·05 was considered to represent selleck chemical a statistically significant difference. Before subsequent analysis, we confirmed the expression of FOXP3 in the isolated CD4+ CD25+ CD127− T cells and found that about 95% of them expressed FOXP3. No FOXP3 expression was seen in CD4+ CD25− T cells (Fig. 1a). The proliferation of CD4+ CD25− T cells was markedly inhibited

when they were incubated for 7 days in Selleckchem JQ1 the presence of CD4+ CD25+ CD127− T cells (Fig. 1b), confirming that the isolated CD4+ CD25+ CD127− T cells were phenotypically and functionally Treg cells. Next, we examined whether RBV affected the characteristics and regulatory activity of CD4+ CD25+ CD127− T cells. The cell viability of CD4+ CD25− and CD4+ CD25+ CD127− T cells was decreased when they were treated with RBV without stimulation. The numbers of viable CD4+ CD25+ CD127−

T cells decreased more than that of CD4+ CD25− T cells (Fig. 2a). For this reason, we counted only the viable cells Methane monooxygenase for use in the subsequent experiments. Intracellular FOXP3 expression in CD4+ CD25+ CD127− T cells was decreased when they were treated with RBV without stimulation (Fig. 2b, upper panels). In addition, the cell surface expression of ICOS was also decreased (Fig. 2b, lower panel). In contrast, CD28 expressed constitutively on the cell surface did not change after RBV incubation (data not shown). Although the proliferation of CD4+ CD25− and CD4+ CD25+ CD127− T cells did not change when they were incubated with RBV (Fig. 2c, left), the proliferation of CD4+ CD25− T cells, which was reduced in the presence of CD4+ CD25+ CD127− T cells, was clearly restored when they were incubated with CD4+ CD25+ CD127− T cells pre-incubated with RBV in an RBV dose-dependent manner when they were stimulated with a sub-optimal dose of human OKT3 (Fig. 2c, centre). A similar result was seen when the cells were stimulated with the maximum dose (5·0 μg/ml) of OKT3 (Fig. 2c, right). Intracellular FOXP3, a specific marker of Treg cells, can be induced in naive CD4+ T cells when stimulated with Tregnat cells.