T Cells and DCs Coculture

Naïve CD4+ T cells were isolated from splenic cells of normal mice and PBMCs of healthy volunteers by naïve CD4+ T cells negative selection (Miltenyi Biotech, Bergisch Gladbach, Germany). Then the naïve CD4+ T cells were labeled with CFSE and then were cocultured with BMDCs or MoDC. A soluble anti-mouse CD3 or anti-human CD3 (0.5 μg/ml) antibody was added to the T cells/DCs cultural medium. The cells and supernatants were harvested after 5 days and followed by proliferation assay by flow cytometry. The Th1 and Treg cells differentiation were analyzed by intracellular flow cytometry analysis. The 1-MT (25 μM) purchased from Sigma-Aldrich (Shanghai, China) was added to some culture medium for inhibiting the IDO activity. CD103− DCs were isolated from DCs generated under low concentration of IL-4 by negative magnetic beads by the following steps. Biotin-conjugated anti-CD103 antibody was used, then anti-biotin-beads (Miltenyi Biotech, Bergisch Gladbach, Germany) were performed in accordance with manufactory instructions.