Induction of COX-2 in MSCs during conditioning in MLRs. (A) MSCs were treated with IFN-γ or cocultured with MLRs (pMLR) in regular or trans-wells. Relative gene expression of COX-2 was evaluated after 2 and 5 days (n = 9). (B) After initial priming of MSCs in an alloreactive milieu (pMLR) with or without administration of IFN-γ (100 IU/mL) or solely IFN-γ pretreatment for 5 days, MSCs were applied to a secondary MLR. After 5 days, levels of secreted PGE₂ were assessed using ELISA (n = 6). (C-E) After initial alloreactive priming (pMLR), COX-2 in the MSCs was inhibited by NS398 (10μM) before performing secondary MLR. After 5 days, (C) IL-10 secretion, (D) suppressive activity, and (E) nTreg-like/Tr1 induction were evaluated by ELISA, thymidine incorporation (responder PBLs), and flow cytometry, respectively (n = 6). (F) To rule out any potential effect of NS398 on the HO-1 expression in MSCs, cells were analyzed with regard to HO-1 levels 5 days on treatment by flow cytometry (n = 6). (G) The effect of blocking COX-2 (NS398, 10μM), IDO (1-methyl-tryptophan [1-MT], 1mM), inducible nitric oxide synthase (NG-monomethyl-L-arginine [L-NMMA], 1mM), or neutralizing TGF-β (anti–αTGF-β antibodies, 10 μg/mL) on the suppressive activity of native versus prelicensed MSCs (n = 12) was assessed by thymidine incorporation in a 5-day (secondary) MLR. 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.