Characterization of hMSC-induced inhibition of IFN-γ secretion. (A) hMSCs inhibit the response of SEB-stimulated T cells in a dose-dependent fashion. PBMCs were stimulated with SEB (1 ng/mL) in the absence or presence of increasing numbers of hMSCs. The levels of IFN-γ in the culture media were determined by ELISA (left panel). Proliferation was determined by [³H]thymidine incorporation (right panel). The data represent the mean values of triplicate samples and standard deviations. (B) The inhibitory activity of hMSCs do not correlate with SEB concentration. PBMCs were stimulated with various concentrations of SEB in the absence or presence of hMSCs (5 × 10⁴ cells). IFN-γ secretion was determined in the conditioned medium. (C) Inhibition of IFN-γ secretion by hMSCs requires cell contact. hMSCs and PBMCs were cocultured together or on opposite sides of a transwell in the presence of SEB (1 ng/mL) for 72 hours. IFN-γ secretion was measured in the conditioned media. (D) Proliferation and IFN-γ inhibition by hMSCs are partially restored by adding LPS or anti-CD40 monoclonal antibodies. PBMCs were cocultured with hMSCs (5 × 10⁴ cells) and SEB (1 ng/mL). Either LPS (1μg/mL; left and right panels) or anti-CD40 mAb (1 μg/mL; middle panel) were added and either IFN-γ secretion or incorporation of [³H]thymidine was determined after 72 hours. The data in panels B-D were calculated as the percent inhibition of IFN-γ secretion in the presence of hMSCs compared with PBMCs stimulated in the absence of hMSCs. The results shown are representative of 3 separate experiments.