Naive CD4+ T cells (CD4+CD25CD62LhiCD44lo) were purified by a FACS Aria II flow cytometer or sorted by the Mouse CD4 Naïve T cell Enrichment Kit (no. 8804-6824-74, Invitrogen). Naive CD4+T cells were cultured with irradiated (30 Gy) anaphase-promoting complex sorted from spleen at a ratio of 1:3 and were activated with anti-CD3 (2 μg/ml) and anti-CD28 (3 μg/ml) in a 48-well plate (5 × 105 T cells per well). T cells were cultured in RPMI 1640 medium supplemented with 10% FBS, sodium pyruvate, penicillin-streptomycin, and 2-mercaptoethanol.

For nonpathogenic TH17 cell differentiation, culture medium was supplemented with IL-6 (20 ng/ml), TGF-β1 (5 ng/ml), anti–IL-4 (10 ng/ml), anti–IL-12 (10 ng/ml), and anti–IFN-γ (10 ng/ml). For pathogenic TH17 cells differentiation, culture medium was supplemented with IL-1β (20 ng/ml), IL-6 (20 ng/ml), and IL-23 (20 ng/ml), anti–IL-4 (10 ng/ml), anti–IL-12 (10 ng/ml), and anti–IFN-γ (10 ng/ml). Other T cell differentiation were performed: TH1, IL-12 (20 ng/ml) and anti–IL-4 (10 mg/ml); TH2, IL-4 (50 ng/ml), anti–IFN-γ (10 ng/ml), and anti–IL-12 (10 mg/ml); iTreg cells, TGF-β1 (5 ng/ml), anti–IL-4 (10 ng/ml), anti–IL-12 (10 ng/ml), and anti–IFN-γ (10 ng/ml). Neutralizing anti–IFN-γ (XMG1.2), anti–IL-4 (11B11), and anti–IL-12 (C17.8) were from BioLegend.

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