CD4+ splenic T cells were purified from mice. Cells (5 × 105) were cultured in 1 ml of RPMI 1640 in 48-well plates coated with anti-CD3 (2 μg/ml) and anti-CD28 (3 μg/ml) antibodies. For TH17 differentiation, splenic CD4+ cells were cultured in the medium containing IL-23 recombinant proteins (50 ng/ml; R&D Systems), IL-6 recombinant proteins (20 ng/ml; R&D Systems), TGF-β recombinant proteins (5 ng/ml; R&D Systems), and anti–IL-4 (5 μg/ml; BioLegend) and anti–IFN-γ (5 μg/ml; BioLegend) antibodies. For TH1 differentiation, CD4+ splenic cells were cultured in the medium containing IL-12 recombinant proteins (5 ng/ml; R&D Systems) and anti–IL-4 antibodies (1 μg/ml; BioLegend).

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