Naive T-cell isolation and in vitro differentiation

Naive T cells were isolated by sorting CD4⁺CD25⁻CD62L^hiCD44^low cells from spleens and lymph nodes (seen gating strategy in Supplementary Fig. ^10a), and stimulated with the plate-bound anti-CD3 (clone 17A2; BioXCell, 5 μg ml⁻¹) and anti-CD28 (clone 37.51; BioXCell, 5 μg ml⁻¹) under different cytokine cocktails. The T cells were cultured with anti-IFN-γ (clone XMG1.2; BioXCell, 10 μg ml⁻¹) and anti-IL-4 (clone 11B11; BioXCell, 10 μg ml⁻¹) for Th0 differentiation, mIL-12 (Peprotech; catalog 210-12, 10 ng ml⁻¹) and anti-IL-4 (10 μg ml⁻¹) for Th1 differentiation, mIL-4 (Peprotech; catalog 214-14, 10 ng ml⁻¹) and anti-IFN-γ (10 μg ml⁻¹) for Th2 cell differentiation, hTGF-β1 (R&D Systems; catalog 240-B-010, 2 ng ml⁻¹) with IL-2 (Peprotech; catalog 212-12, 10 U ml⁻¹) for iTreg differentiation. The Th17 differentiation was performed using hTGF-β1 (0.5 ng ml⁻¹) and IL-6 (Peprotech; catalog 216-16, 20 ng ml⁻¹), with or without IL-1β (Peprotech; catalog 211-11B, 10 ng ml⁻¹) and IL-23 (R&D Systems; catalog 1887-ML, 50 ng ml⁻¹) as indicated.