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.