Cytokine Detection Protocol for T Cells

For cytokine detection, cells were stimulated with phorbol myristate acetate (50 ng/mL; Sigma-Aldrich), ionomycin (1 μM; Sigma-Aldrich) for 4 hours with protein transport inhibitor (GolgiPlug 1 μl/ml, GolgiStop 2/3 μl/ml; BD Bioscience) in complete medium (CM) before staining. Cells were first stained extracellularly with specific antibodies against human CD3, CD4, CD8, CD45RA, CD45RO (BD Bioscience), then were fixed and permeabilized with Fixation/Permeabilization solution (eBioscience), and finally were stained intracellularly with anti-IL-2, anti-IL-17, anti-tumor necrosis factor-α (TNF-α), anti-interferon-γ (IFN-γ), anti-Granzyme B, anti-Ki67 and anti-EZH2 (BD Biosciences). Samples were acquired on flow cytometry (LSR II; Becton Dickinson) and data were analyzed with DIVA software (BD Biosciences) [35 (link)]. The gating strategy was shown in Supplementary Figure S2. KLRG1⁺ and KLRG1⁻ T cells were purified and sorted with FACSAria cell sorter (Becton Dickinson).