Human MSCs inhibit allogeneic T-cell responses and express IDO. (A) Human MSCs inhibit allogeneic T-cell responses in a dose-dependent manner. Responding T cells (1 × 10⁵) were incubated for 5 days with 5 × 10⁴ allogeneic PBMCs as stimulator cells in the presence (+) or absence (-) of 5 × 10³ or 2 × 10⁴ human MSCs. Each experiment was performed in triplicate. T-cell proliferation is expressed as averaged means of triplicates ± SEM obtained from a series of 11 experiments with 5 × 10³ MSCs from 7 different donors and 7 experiments with 2 × 10⁴ MSCs from 6 different donors. ^*P value of difference as calculated by Student t test. (B) Human MSCs express IDO protein upon stimulation with IFN-γ. MSCs were cultured in the absence (-) or presence (+) of 500 U/mL IFN-γ. After 3 days, cells were harvested and subjected to Western blot analysis using an IDO-specific monoclonal antibody. As a control, expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was analyzed in parallel. (C) Human MSCs express functional IDO activity upon stimulation with IFN-γ. MSCs (2 × 10⁴) were stimulated with increasing amounts of IFN-γ. After 3 days of culture, supernatant was harvested and IDO activity was measured by determining kynurenine concentration as absorbance (OD) at 492 nm. Background OD (approximately 0.15) was subtracted. Data obtained from 4 independent experiments each using MSCs from different individual donors are presented as mean OD ± SEM.