MSCs conditioned in an MLR depict an increased Treg-promoting capability despite HO-1 down-regulation. (A) Schematic model depicting the experimental setup and time frame for conditioning (priming) of native MSCs in an initial MLR (for 5 days) followed by subjection to a second MLR (for 5 days), including various treatments (INF-γ/SnPP/NS398/1-methyl-tryptophan (1-MT)/NG-monomethyl-L-arginine (L-NMMA)/αTGF-β). (i) Purity of MSCs (> 90%) was assessed after primary MLR for 5 days and subsequent removal of PBLs by flow cytometry as shown in the representative dot plots, MSCs being the CD73⁺/CD3⁻/CD45⁻ cells. (B-C) After initial priming of MSCs in an alloreactive milieu (pMLR) with or without addition of IFN-γ or solely IFN-γ pretreatment of native MSCs for 5 days, harvested MSCs were subjected to a second MLR. After 5 days of coculturing, (B) fold increase of Treg subpopulations and (C) percentile alteration of IL-10 secretion were assessed by flow cytometry and ELISA, respectively (n = 6). (D) Relative gene expression of IL-10 in 4 different MSCs after 48 hours of culturing with or without MLR was determined by real-time PCR. (Ei) The impact of HO-1 inhibition on the IL-10 production was determined by ELISA in the supernatants of MLRs performed in the presence of with SnPP-pretreated native (SnPP prior 1. MLR) or preconditioned (SnPP prior 2. MLR) MSCs (n = 6). (ii) Effects of the HO-1 inhibition on the suppressive potency of by MLR preconditioned (pMLR) MSCs was evaluated by the thymidine incorporation (in responder PBLs) during secondary MLR (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. **P ≤ .01. ***P ≤ .001.