Coculture with OT-II T cells

Spleen cells were isolated from class II MHC-restricted TCR transgenic OT-II mice, and CD4 T cells were purified using anti-CD4 microbeads and MACS cell separation reagents (Miltenyi Biotec, Seoul, Korea). OT-II T cells were covalently fluorescein-labeled using the CellTrace Violet Cell Proliferation Kit (ThermoFisher, Waltham, MA, USA) and cultured with BMDMs in the presence of 1 μg/ml OVA_323–339 peptides for 4 days. For intracellular cytokine staining, the cells were restimulated with 50 ng/ml PMA and 1 μg/ml ionomycin for 6 h in the presence of the Golgi-stop (BD Bioscience) followed by surface FACS staining. After fixed with 4% paraformaldehyde and permeabilized with 1% saponin, the cells were stained with antigen presenting cell (APC)-conjugated anti-IFN-γ and analyzed by flow cytometry.