Expansion and Stability Assay of Induced Regulatory T Cells

UCB derived FOXP3⁺ iTregs cells were generated as previous described^{82 (link)}. Ex vivo expansion of FOXP3⁺ iTregs was performed with CD25⁺ cells MACS purified from day 4 TGFβ-induced UCB iTreg. On Day 4 of TGF-β conditioning, CD25 + iTregs were MACS purified, rested for 48 h, and on Day 6, 5 × 10⁵ purified cells were split into expansion cultures with either media containing 100 U/ml IL-2 in suspension or identical media/IL-2 over MSC feeder cells. Cultures were fed every other day with fresh media/IL-2 and MSC during short-term culture (21–28 days). No re-stimulation was performed during the 21–28 day expansion following the initial 4 day stimulation consistent with a previously described UCB Treg expansion protocol^{83 (link)}. For isolation of UCB-derived tTregs, EasySep Human CD4 + CD127lowCD25 + Regulatory T Cell Isolation Kit (Stem Cell Technologies) was used. tTregs were maintained under identical conditions as iTregs. Absolute number of FOXP3⁺ iTreg cells were calculated from the percentage of CD4⁺ cells based on the total viable cell count obtained by trypan blue dye exclusion at each indicated time point. For iTreg stability assays, harvested iTregs were stimulated with CD3 and CD28 antibodies (1 μg ml⁻¹) with IFNγ (10 μg ml⁻¹), IL-6 (10 μg ml⁻¹), and TNF (10 μg ml⁻¹) at concentration of 5 × 10⁵ cells ml⁻¹ for 72 h and analyzed.