Fig. 6.

S1P promotes IDO1-dependent immune regulatory properties in fHASCs. fHASCs were preconditioned with S1P or TGF-β1, or their combination, using vehicle-stimulated fHASCs as a control. A) Scheme of treatment and analysis. B) Representative results of CD4⁺FOXP3⁺ cell frequency (top right quadrants) among total CD4⁺ T cells, cultured for 5 days with fHASCs treated as indicated. Representative results from one experiment of three. C) CD4⁺ FOXP3⁺ cell frequency in cultures established as in b; n = 5. Means ± S.D. (three independent experiments). ***P < 0.001; Dunnett test (treatments vs. vehicle control). D) IDO1 protein expression in fHASCs treated with different concentrations of S1P or TGF-β1, or their combination, as determined by immunoblotting. Representative results from one of three independent experiments. E) Densitometric analysis of IDO1 immunoblots in three independent experiments. Data are represented as means ± S.D. (n = 5); **P < 0.01; ANOVA followed by Bonferroni multiple comparison test. β-tubulin was used as protein loading control. F) IDO1 enzymatic activity by kynurenine quantification in fHASCs treated with different concentrations of S1P or TGF-β1, or their combination, as determined by HPLC. ***P < 0.001; Dunnett test (treatments vs. vehicle control). G) Representative results of CD4⁺ FOXP3⁺ cell frequency (top right quadrants) among PBMC cells, cultured for 5 days with fHASCs (preconditioned with S1P and TGF-β1) that had been transfected with an IDO1 siRNA (IDO1 siRNA) or a control siRNA (c siRNA). H) CD4⁺ FOXP3⁺ cell frequency in cultures established as in g; n = 5. Means ± S.D. (three independent experiments). Data are means ± S.D. of three independent experiments performed in triplicate. ***P < 0.001; Student's t-test (IDO1 siRNA vs. vehicle c siRNA).