IFNγ prelicensed senescent MSCs display enhanced immunosuppressive activity through IDO. αCD3αCD28 Dynabeads-stimulated PBMCs were cocultured in the presence and absence ± IFNγ prelicensed (20 ng/mL for 48 hours) fit and senescent MSCs. Four days post culture, T-cell proliferation was measured by Ki67 intracellular staining. (A) Representative FACS plot and (B) dose-dependent T-cell inhibitory effect of ± IFNγ prelicensed fit and senescent MSC pairs from 5 independent donors is shown. (C) Fit and senescent MSCs were stimulated with IFNγ for 48 hours. Expression level of IDO mRNA relative to GAPDH was evaluated by quantitative SYBR green real-time PCR. δ-δ CT method was applied to calculate the fold induction of IDO over the unstimulated control. (D) IDO expression at protein level is shown by western blot analysis, and actin was used as an internal control. Similar results were obtained in a repeat experiment with an additional independent MSC donor. (E) ± IFNγ prelicensed fit and senescent MSCs were cocultured with activated PBMCs in the presence and absence of IDO blocker, 1MT. Four days after, T-cell proliferation was measured by flow cytometry. Similar results were obtained in a repeat experiment with another MSC donor pair.