Flow cytometry and sorting

Surface markers were stained in HBSS with 0.1% BSA for 30 min at 4°C (antibodies are listed above). For cytokine staining, cells were stimulated with 50 ng/ml PMA (Sigma-Aldrich) and 1 mg/ml ionomycin (Merck) in the presence of 1,000× brefeldin A (eBioscience) for 4 h at 37°C. Cells were first stained with antibodies against indicated cell surface markers followed by 10 min of fixation with 2% formaldehyde solution in PBS at room temperature and 5 min of permeabilization in Perm/Wash Buffer (BD Biosciences) at 4°C. For Foxp3 staining, cells were fixed and permeabilized with the Foxp3 Staining Buffer Set according to the manufacturer’s instructions (eBioscience). To track cell division, T reg cells were labeled in vitro with CFSE (Sigma-Aldrich) or CellTrace Violet (Life Technologies) according to the manufacturer’s protocol. Cell fluorescence was acquired on a four-laser BD LSR Fortessa II or a two-laser BD FACS Calibur and was analyzed with FlowJo software (TreeStar). Cell sorting was carried out by a BD FACSAria II after surface staining. Sorted cell purity was over 95%.