Effect of Human Mesenchymal Stem Cells on Xenogeneic T and B Cells Isolated from Lupus-Prone MRL.Fas^lpr Mice

Figure 4

IFN-hMSCs inhibit MRL.Fas^lpr B cells in an indoleamine 2,3-dioxygenase-dependent manner. (a) IFN-γ-activated hMSCs (IFN-hMSCs; cells/well) were added to the upper (U) or lower (L) wells of transwell plates and MRL.Fas^lpr mouse B cells ( cells/well) to the L wells. After incubation with LPS for 72 h, the level of IgM accumulated in culture medium was measured by ELISA. (b) Total RNA was isolated from IFN-hMSCs, and the expression levels of COX-2, TGF-β, and IDO were analyzed by RT-PCR. (c) IDO protein level in IFN-hMSCs analyzed by western blotting. (d) Levels of PGE₂ and TGF-β accumulated in IFN-hMSC culture medium over 24 h measured by ELISA (d). (e, f) hMSCs were treated with IFN-γ (10 ng/ml) for 7 days and the levels of IDO, STAT1, and p-STAT1 were analyzed by western blotting (e). In (f), the cells were treated with the STAT1 inhibitor fludarabine (10 nM) for 7 days. (g–i) IFN-hMSCs were transfected with IDO siRNA for 48 h (g) or treated with the IDO inhibitor 1-methyltryptophan (1MT, 100 μM) for 7 days (h) or fludarabine (10 nM) for 7 days (i). The level of IgM accumulated in culture medium was measured by ELISA. (). NS: not significant. (b, c, e, and f) Band areas were analyzed by ImageJ software (NIH, Bethesda, MD, USA), and data were presented as ratios versus UN (b, c), day 0 (e), and IFN/fludarabine-untreated group (f) ().

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