IFN-γ administration rescues HO-1 expression and partially the suppressive potency of MSCs. (A) Relative gene expression (mRNA) of HO-1 and Nrf2 was measured in IFN-γ-treated MSCs with or without priming MLR (n = 7). (B) After an initial pMLR for 5 days with or without IFN-γ (100 U/mL), MSCs were subjected to a secondary MLR for 5 days. Suppressive activity was measured by thymidine incorporation (in responder PBLs) and compared with nonprimed (native) cells (MSC_untr) or MSCs solely treated with 100 U/mL IFN-γ (n = 6). (C) Percentage up- or down-regulation of HO-1 and Nrf2 relative gene expression was assessed in MSCs upon priming with MLRs or IFN-γ and correlation (HO-1/Nrf2) analysis performed (n = 8). (D) Relative gene expression (mRNA) of catalase (Cat), superoxide-dismutase (SOD), and catalytic subunit of glutamylcysteine ligase (GCLC) was assessed in MSCs 5 days after IFN-γ treatment (n = 7). (Ei) MSCs were stained with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein-diacetate-acetylester, mercury orange (MO), and ALM 5 days after IFN-γ treatment to determine intracellular oxidative stress (DCF), intracellular (PE-channel/MO) and extracellular (allophycocyanin-channel/ALM) thiols, respectively, as assessed by flow cytometry (n = 4). (ii) After 5 days of IFN-γ treatment, MSCs were subjected to treatment with 2mM H₂O₂ for 8 hours and subsequently analyzed regarding cell death by 7-AAD and annexin V FITC staining (n = 6). All experimental settings were conducted with MSCs derived from at least 3 individual donors, and n refers to the number of repeated experiments. Bars represent SD. *P ≤ .05.