Uvi3003

All recombinant interleukins (ILs) were purchased from PeproTec (London, UK), except for human IL-2 (Shionogi, Osaka, Japan). All retinoids (all-trans form), LE540, and UVI3003 were purchased from Sigma-Aldrich (St. Louis, MO, USA), Wako (Osaka, Japan), and Tocris Bioscience (Ellisville, MO, USA), respectively. All antibodies used in this study are listed in Supplementary Table 4. Cells were incubated with FcR blocking Reagent (Miltenyi Biotec) and then stained with the appropriate combinations of mAbs and fluorochrome-conjugated streptavidin.
For intracellular cytokine staining, cells were incubated with or without SIINFEKL peptide (1 μM) or PMA + ionomycin in the presence of brefeldin A for 4 h and stained using the Cytofix/Cytoperm Kit (BD Bioscience, Franklin Lakes, NJ, USA). Foxp3-positive cells and apoptotic cells were detected using the BD Foxp3 Staining Kit (BD Bioscience) and Apoptosis Detection Kit (BD Bioscience), respectively. For cell surface-stained samples, 7-aminoactinomycin D (7AAD; eBioscience) was added just before the flow cytometric analysis to exclude dead cells. Flow cytometry was performed using FACSAria. The data were analyzed using FlowJo software (TreeStar, San Carlos, CA, USA).