Primary total T cells were isolated from peripheral venous blood by negative selection and kept in RPMI 1640 medium supplemented with 10% FBS. Naive (CD4+ CD45RA+) and memory (CD4+ CD45RO+ ) T cells were purified using T cell isolation kits from Miltenyi Biotec according to the manufacturer’s instructions.
Naive and memory CD4+ T cells (1 3 106 /well) were differentiated into Th17 cells in serum-free X-VIVO 10 medium (BioWhittaker) by the addition of IL-6 (25 ng/ml), TGF-b1 (5 ng/ml), IL-1b (12.5 ng/ml), IL-21 (25 ng/ml), and IL-23 (25 ng/ml) for up to 7 days.