Human MSC Suppression Correlates With Cytokine Induction of Indoleamine 2,3- Dioxygenase and Bystander M2 Macrophage Differentiation  Moïra François, Raphaëlle Romieu-Mourez, Mengyang Li, Jacques Galipeau  Molecular Therapy  Volume 20, Issue 1, Pages 187-195 (January 2012) DOI: 10.1038/mt.2011.189 Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 The immunosuppressive potential of human mesenchymal stromal cells (MSC) varies between donors. (a) T cell proliferation assays were performed using carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled human peripheral blood mononuclear cell (PBMC) activated with 0.2 µg/ml anti-CD3 and CD28 antibodies and cocultured with or without MSC for 4 days at MSC:PBMC ratios of 1:3, 1:9, and 1:27. T cell proliferation was performed on seven MSC donors (donor 303. 304, 305, 306, 307, 308, and 311) and MSC were preactivated for 18 hours with 10 ng/ml of interferon (IFN)-γ and 3 ng/ml of tumor necrosis factor-α (TNF-α) before their addition to the coculture. Cell proliferation was determined by flow cytometry after gating the leukocyte population on the forward and side scatter plot and measuring the percentage of CFSElow T cells (figure show representative results). (b) T cell proliferation was measured as in a using MSC:PBMC ratios of 1:3, 1:9, and 1:27. MSC were either left (i) untreated or (ii) preactivated with IFN-γ and TNF-α before their addition to the coculture. Results show means ± s.d. of three independent experiments using different allogeneic PBMC donors. (c) Levels of IFN-γ were measured by enzyme-linked immunosorbent assay (ELISA) in the supernatant of a T cell proliferation assay performed as in a with (i) untreated or (ii) cytokine preactivated MSC. Results show duplicates ± SD. Molecular Therapy 2012 20, 187-195DOI: (10.1038/mt.2011.189) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Inhibition of indoleamine-2,3-dioxygenase (IDO) activity of mesenchymal stromal cells (MSC) suppresses their immunosuppressive action on T cells. (a) mRNA expression level of (i) interleukin (IL)-10, (ii) transforming growth factor-β (TGF-β), (iii) Cox2, and (iv)indoleamine 2,3-dioxygenase (IDO) encoding genes were measured by real-time quantitative PCR (qPCR) in nontreated MSC or MSC stimulated for 24 hours with 10 ng/ml interferon (IFN)-γ and 3 ng/ml tumor necrosis factor-α (TNF-α). Total RNA extracts from the adherent cell population of human peripheral blood mononuclear cell (PBMC), constituted mostly of monocytes (Mo), stimulated for 24 hours with IFN-γ and TNF-α was used for comparison and attributed a value of 1. Relative quantification (RQ) was calculated by normalizing to 18S mRNA levels. Results shows mean of duplicates ± SD. (b) T cell proliferation was measured as in (a) in cocultures with TNF-α and IFN-γ preactivated MSC at ratio MSC: PBMC of 1:3 in the presence of 10 µmol/l indomethacin, 1 mmol/l 1-methyl-DL-tryptophan (1-MT), or 20 µg/ml anti-TGF-β neutralizing antibodies. Figure shows representative results of 1 experiment out of 2. (c) T cell proliferation was measured in a proliferation assay performed with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled PBMC activated with anti-CD3/CD28 antibodies in the presence of conditioned media from preactivated MSC with or without 20 µg/ml of anti-IL-6 neutralizing antibody. Conditioned media was obtained by stimulating MSC with the TNF-α and IFN-γ for 4 hours; washing and culturing in fresh medium for 24 hours. Figure shows means ± SD from representative results of 1 experiment out of 2. Molecular Therapy 2012 20, 187-195DOI: (10.1038/mt.2011.189) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Correlation between the amount of indoleamine 2,3-dioxygenase (IDO) produced and the T cell inhibitory potential of mesenchymal stromal cells (MSC) donor. (a) (i) IDO protein expression was analyzed by immunoblot in nontreated MSC and MSC pretreated for 24 hours with 10 ng/ml interferon (IFN)-γ and 3 ng/ml tumor necrosis factor-α (TNF-α). (ii) IDO protein expression after IFN-γ and TNF-α stimulation by each MSC donor was analyzed by western blot. Relative intensity of each IDO band was estimated by densitometry analysis and normalized to its GAPDH loading control band. (iii) The correlation between the amount of IDO produced by a given MSC donor and its percentage of proliferation inhibition was determined by one-tailed Pearson's test. Figure shows results from three different assays performed with all seven MSC donors and therefore figure shows 21 points in total and the fit by linear regression. (b) (i) Level of STAT-1 phosphorylation was analyzed by immunoblot and (ii) quantified by densitometry using total STAT-1 for normalization in human MSC stimulated for 20 minutes with 10 ng/ml of IFN-γ. Molecular Therapy 2012 20, 187-195DOI: (10.1038/mt.2011.189) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Upregulation of CD206 and interleukin (IL)-10 expression in monocytes following coculture with mesenchymal stromal cells (MSC). (a) Nontreated MSC or TNF-α and interferon (IFN)-γ preactivated MSC from donor 304 were cultured in combination with peripheral blood mononuclear cell (PBMC)-purified CD14+ monocytes, PBMC-purified CD3+ T cells or both with or without 1 mmol/l 1-methyl-DL-tryptophan (1-MT). All cultures were activated with anti-CD3/CD28 antibodies. Levels of IL-10 in the supernatant of MSC/monocytes/T cells cocultures were measured by enzyme-linked immunoabsorbent assay (ELISA). Results show means of duplicates ± SD and 1 representative experiment out of 2. (b) (i) Expression of CD206 in the CD14+ monocyte population was measured by flow cytometry in coculture with TNF-α and IFN-γ preactivated MSC from donor 304, 306, 307, 308, and 311 in combination with CD3/CD28-activated PBMC and (ii) level of IL-10 in supernatants measured by ELISA. Molecular Therapy 2012 20, 187-195DOI: (10.1038/mt.2011.189) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Involvement of the monocyte population in the mesenchymal stromal cells (MSC)-mediated T cell inhibition. (a) T cell proliferation was measured in proliferation assays as detailed in Figure 1a and using tumor necrosis factor-α (TNF-α) and interferon (IFN)-γ-preactivated MSC at MSC:PBMC ratios of 1:3 and 1:9 with (i) whole or CD14-depleted peripheral blood mononuclear cell (PBMC) or (ii) CD14-depleted PBMC with or without 1 mmol/l 1-methyl-DL-tryptophan (1-MT). Figure show 1 representative experiment out of 2. (b) T cell proliferation assay using TNF-α and IFN-γ-preactivated MSC at MSC: PBMC ratios of 1:9 and measured as in Figure 1a. Assays were performed with total PBMC to which PBMC donor-derived purified monocytes were added. Dose 1 and dose 2 represent 0.5× and 1.5× the total amount of CD14+ monocyte/PBMC added, respectively. Panels show (i) total and (ii) normalize percentage of T cell proliferation as well as the level of (iii) interleukin (IL)-2 and (iv) IFN-γ detected in the supernatant after 4 days. Figure shows means ± SD from representative results of 1 experiment out of 2. Molecular Therapy 2012 20, 187-195DOI: (10.1038/mt.2011.189) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Mesenchymal stromal cells (MSC)-mediated T cell immunosuppression mechanisms. Our proposed mechanism of MSC-meditated T cell immunosuppression involves both a direct and indirect pathway. Indoleamine 2,3-dioxygenase (IDO) protein expression and enzymatic activity induced in MSC following exposure to interferon (IFN)-γ from the inflammatory milieu catalyze the degradation of tryptophan (TRP) present in the surrounding environment into kynurenine (KYN). The catabolic activity of IDO can directly suppress T cell proliferation or act indirectly through the surrounding monocytes. Tryptophan depletion or metabolites of the kynurenine pathway and possibly other unknown factors secreted by MSC mediate the differentiation of monocytes into interleukin (IL)-10 secreting, CD206+ immunosuppressive M2 macrophages which in turn contribute to T cell suppression in IL-10-independent mechanism in vitro. Molecular Therapy 2012 20, 187-195DOI: (10.1038/mt.2011.189) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions