Figure 2. Immunosuppressive mechanism of human BM-MSCs and HeSCs.

Human MSCs or HeSCs (N=3 donors) were cultured with Staphylococcus Enterotoxin B (SEB) activated PBMCs. Four days post culture, T-cell proliferation was measured by Ki67 intracellular staining. (A) Representative Flow cytometery plot and (B) cumulative dose dependent effect of BM-MSCs or HeSCs effect on T-cell proliferation (%CD3⁺Ki67⁺) is shown. (C) Human BM-MSCs or HeSCs were stimulated with indicated concentrations of IFNγ for 48 h. Expression levels of IDO mRNA were quantitated by quantitative SYBR Green real-time PCR. GAPDH mRNA levels were used as internal controls. (D) Western blot analysis of human BM-MSCs and HeSCs for IDO expression at protein levels. Actin was used as an internal control. Human BM-MSCs or HeSCs were cocultured with SEB activated PBMCs in the presence and absence of IDO blocker 1-MT. Four days after, T-cell proliferation was measured by flow cytometry. (E) Representative flow cytometry plot and dose dependent effect of 1-MT on T cell proliferation in a differential ratio of (F) BM-MSCs or (G) HeSCs with PBMCs is shown with mean and standard deviation. (H) MSCs or HeSCs were stimulated with IFNγ and the expression levels of iNOS were determined through quantitative PCR. Human BM-MSCs or HeSCs were cocultured with SEB activated PBMCs in the presence and absence of 1mM iNOS blocker L-NMMA. Four days after, T-cell proliferation was measured by flow cytometry. (I) Representative flow cytometry plot and (J) cumulative from independent cocultures is shown with mean and standard deviation. Similar results were obtained in at least two experiments with another donor.