Two-week expanded Treg cells were thawed and expanded further for 2 days in DMSO or TAC containing media with IL-2 (1000 U/mL) and with 2 × 104 αCD3/αCD28 beads (1:5 bead:Treg) or without bead stimulation as a negative control. Supernatants were then centrifuged and stored at −80°C until analyzed. IL-10, IL-17A, and IFN-γ levels were determined using enzyme-linked immunosorbent assay according to manufacturers' protocol (BD for IL-10 or eBiosciences for IL-17A and IFN-γ).