Isolation and Characterization of CD4+ T Cells

CD4⁺ T cells were magnetically isolated from lymph nodes and/or spleen cells by a previously described method (Hashimoto et al.2017 (link)). Magnetic sorting was performed using Dynabeads Mouse CD4 (Invitrogen, Thermo Fisher Scientific, MA, USA) followed by treatment with DETACHaBEAD Mouse CD4 (Invitrogen). CD4⁺ T cells (1 × 10⁶ well⁻¹) were co-cultured in a 24-well plate with mitomycin C-treated BMDCs (0.5 × 10⁶ well⁻¹) in a total volume of 2 mL in the presence of C. albicans cell fractions for 6 days. The differentiated T cells were washed and re-stimulated with 50 ng mL⁻¹ phobol myristate acetate (PMA; Sigma-Aldrich) and 500 ng mL⁻¹ ionomycin (Sigma-Aldrich) in the presence of 10 μg mL⁻¹ brefeldin A (Sigma-Aldrich) at 37°C for 4 h. The cells were stained with anti-CD4 antibody (Ab) (APC, clone RM4-5, BD Biosciences) and permeabilized with 0.1% saponin (Nacalai Tesque). Intracellular cytokines were stained with anti-IL-17A Ab (PE, clone TC11-18H10, BD Biosciences) and anti-IFNγ Ab (fluorescein isothiocyanate, clone XMG1.2, BD Biosciences). All data were obtained using FACSCalibur (BD Biosciences).