HO-1 is involved in the MSC-mediated induction of Tr1 and Th3 cells. (A) After an MLR for 5 days in the presence or absence of MSCs, the (v) fold increase of (i) CD3⁺CD4⁺ T cells expressing (ii) CD25⁺FOXP3⁺ (nTreg-like), (iii) CD25⁺IL-10⁺ (Tr1), and (iv) TGF-β⁺FOXP3⁺ (Th3) was assessed by flow cytometry (n = 20). (B) HO-1 in MSCs was blocked before primary MLR using 50μΜ SnPP. Subsequently, (i) frequencies of nTreg-like, Tr1, and Th3 cell subsets were determined by flow cytometry (n = 6) and (ii) gene expression of FOXP3, IL-10, and TGF-β in the retrieved lymphocytes by quantitative real-time PCR (n = 8). The Treg frequency and the gene expression levels detected in the presence of untreated MSCs (MSC_untr) represent the 100% baseline. (C) MSCs treated with 50μΜ SnPP were subjected to a 5-day coculture with purified CD4⁺ T cells from single healthy donors (n = 6), activated by CD2, CD3, and CD28 bead-coupled antibodies; and subsequently, frequencies of nTreg-like, Tr1, and Th3 cell subsets were determined by flow cytometry. The Treg frequency detected in the presence of untreated MSCs (MSC_untr) represents the 100% baseline. (Di) MSCs expressing high (MSC097 and MSC101) and low (MSC104 and MSC074) HO-1 levels as assessed by flow cytometry were (ii) subjected to MLR for 5 days and Treg promotion was evaluated subsequently. Results are representative for 2 independent MLRs performed with each MSC. 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 ≤ .01. ***P ≤ .001.