Cell isolation and culture
Peripheral blood mononuclear cells (PBMCs) from heparinized blood were isolated with Ficoll-Paque. Monocyte-derived DCs were generated from the adherent fraction of PBMCs and cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum (FBS) for 7 days in the presence of 50 ng/ml human recombinant granulocyte-macrophage colony stimulating factor (Peprotech, USA), 20 ng/mL h IL‑4 (Peprotech) and 25 ng/mL human TNF-α (PeproTech) for more 2 days to stimulate DC maturation according to an established method [12]. The maturation markers cluster of differentiation CD80, CD86, CD83, and human leukocyte antigen-DR isotype (HLA-DR) of DCs were analyzed with flow cytometry as described previously [13]. CD4+ T cells were separated from PBMCs by magnetic cell separation using a commercial CD4+ T cell isolation kit (Miltenyi Biotec, Germany) according to the manufacturer’s instructions. The purity of the cells was confirmed by flow cytometry. Purified CD4+ T cells (6×105/mL) and DCs (2×105/mL) were co-cultured 3:1 in 24-well plates with 0.4 μm transparent PET membrane inserts (BD Falcon, USA) as described by the manufacturer for 3 days in the presence of 0.1 ug/mL human anti-CD3 antibody (eBioscience, USA) and 20 ng/ml human IL-15 (Peprotech, USA). Stimulated cells were irradiated at 25Gy before co-incubation.