Dendritic cell generation from human peripheral blood precursors
PBMCs were isolated from healthy HLA-A2+ donors by Ficoll–Hypaque density gradient centrifugation (TBC company, Tianjing, China) and then seeded into culture flasks in RPMI-1640 medium supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), and 10 % FBS. After monocytes adhered (incubation for 2 h), the nonadherent cells were collected and frozen in freeze medium (60 % RPMI-1640 and 30 % FBS, 10 % DMSO) for later use in CTL assays. The adherent cells were cultured for 5 days in RPMI-1640 containing 1,000 U/ml of granulocyte–macrophage colony-stimulating factor (R&D Systems, Inc., Minneapolis, MN) and interleukin-4 (IL-4; R&D Systems, Inc.) and were the cultured for an additional 2 days in the presence of 1,000 U/ml of tumor necrosis factor α (R&D Systems, Inc.) to induce final maturation. After 7 days of culture, the mature DCs were harvested and analyzed for DC typical phenotypes by FACS analysis.