Naïve T cell differentiation was performed as described previously (Fan et al., 2020). Naïve CD4+ T cells were isolated from murine spleens and lymph nodes using naïve CD4+ T cell purification kit (Miltenyi Biotech, Cat. 130-104-453) according to the manufacturer's instruction. Isolated T cells were activated with anti-CD3 and anti-CD28 (BD Pharmingen, 2 μg/ml) under Th0 (hIL-2, Peprotech, 50 U/ml), Th1 (anti-IL-4, Bioexcel, 10 μg/ml; IL-12, Peprotech, 10 ng/ml; hIL-2, 50 U/ml), Th2 (anti-IFN-γ, Bioexcel, 10 μg/ml; IL-4, Peprotech, 10 ng/ml; hIL-2, 50 U/ml) and Th17 (anti-IFN-γ, 10 μg/ml; anti-IL-4, 10 μg/ml; IL-6, Peprotech, 20 ng/ml; TGF-β, Peprotech, 2 ng/ml) polarizing conditions for 4 days. Then, cells were re-stimulated with anti-CD3–CD28 for 4 h. Supernatant was collected to assess cytokine production by ELISA (R&D Systems) and differentiated cells were used for mRNA expression analysis.

For T cell activation, WT or DHHC21dep T cells were purified using CD4+ T cell isolation kit (Miltenyi Biotech, Cat. 130-104-453) and activated with plate-bound anti-CD3 and/or anti-CD28 (eBioscience). IL-2 production was determined 24 h after T-cell activation by ELISA according to manufacturer's instructions (R&D Systems). CD69 production was analyzed by flow cytometry after 2 days of anti-CD3 and/or anti-CD28 stimulation.