Cell purification and flow cytometry

Single-cell suspensions were prepared from spleen and peripheral lymph nodes for staining or cell purification. CD4⁺ T cells and CD11c⁺ DCs were purified with Dynabeads Untouched Mouse CD4 Cells Kits (Invitrogen, 11415D) or CD11c MicroBeads (Miltenyi Biotec, 130-108-338), respectively. Indicated T-cell populations were sorted from purified CD4⁺ T cells with FACSAria III (BD Biosciences), and the sorted populations were >98% pure unless otherwise specified. Flow cytometry of cell surface molecules was performed as previously described⁵⁵, and the antibody (Supplementary Data 5) was diluted as the instructions of the manufacturers suggested. Intracellular staining of Foxp3, cytokines (spleen cells were stimulated with phorbol myristate acetate (50 ng ml⁻¹) and ionomycin (500 ng ml⁻¹) for 4 h before analysis of cytokine expression in indicated populations) and other proteins were performed with Foxp3 staining kits (eBioscience, 00-5523). Intracellular staining of phosphorylated proteins was performed on cells fixed with methanol and permeabilized with Triton X-100. The antibodies were obtained from eBioscience, Biolegend, BD Biosciences, Cell Signalling Technology, R&D Systems and Invitrogen, and listed in Supplementary Data 5. Flow cytometry data were acquired on LSR II, LSRFortessa (BD Biosciences) or FACSCanton II (BD Biosciences) and analysed with Flowjo software (Tree Star).