tTreg and naïve CD4+ T-cells were purified from apheresis products using magnetic beads, stimulated with a cell line expressing CD86 and CD64 (KT64/86) to which anti-CD3 mAb is bound, and cultured for 14 days. Low Trp+Kyn iTreg (and Teff) were re-stimulated on day 7. (A) Suppressive function was determined using a CFSE-based proliferation assay at 1:16 (expanded cell:PBMC) and stimulated with anti-CD3 mAb beads for 4 days. (B) Calculated yield of tTreg and Low Trp+Kyn iTreg from a 12 billion cell apheresis product. (C) Representative expression of CD25, Foxp3, and Helios. (D) Representative metabolic analysis showing oxygen consumption rate and extracellular acidification rate for tTreg Low Trp+Kyn iTreg and Teff. (E) Kaplan-Meier survival curves for mice receiving expanded tTreg, and Low Trp+Kyn iTregs and an equal number of HLA-mismatched PBMC. Low dose IL-2 was administered to a cohort of mice receiving iTreg. Mice were bled on day 6 and tTreg and iTreg in circulation quantitated by flow. (F) As in E, but IL-2 given to cohorts of mice receiving PBMC only or PBMC + iTreg for 18 days (n=8 per cohort).