Monocyte-Derived DC and CD4+ T Cell Interaction

CD14 and CD4 mAb-conjugated magnetic microbeads (Miltenyi Biotec, Germany) were used to isolate CD14⁺ monocytes and CD4⁺ T cells from VKH and the healthy subjects (purity >90%). The RPMI 1640 medium with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin was used to culture the cells. To generate the monocyte-derived DCs, CD14⁺ monocytes were stimulated with 100 ng/ml granulocyte macrophage colony stimulating factor (GM-CSF, Acro Biosystems, Newark, DE, USA) and 50 ng/ml interleukin-4 (IL-4, Acro Biosystems, Newark, DE, USA) for 6 days. Half of the medium was then refreshed at the 4^th day. Mature DCs were subsequently generated by stimulation with 100 ng/ml lipopolysaccharide (LPS) for 24 hours. The co-culture experiment was performed as previously described^{32 (link)}. Briefly, the DCs were co-cultured with CD4⁺ T cells (DC: T cell ratio = 1:5) with or without DAC for 5 days. The flow cytometry tests were performed to detect the intracellular IFN-γ and IL-17. The culture supernatants were used for ELISA assay.