Role of IDO and PGE2 in the MSC-mediated inhibition of NK-cell proliferation and cytotoxic activity. Freshly isolated NK cells were cultured with IL-2 either alone or in the presence of MSCs. In some wells, coculture of NK cells and MSCs was set in the presence of inhibitors, namely, 1-M-Trp (1 mM, inhibitor of IDO enzymatic activity) and NS-398 (5 μM, inhibitor of PGE2 synthesis), used singularly or in combination. To evaluate NK-cell proliferation (A), after 6 days, cells were pulsed with ³H-thymidine for 18 hours, and then ³H-thymidine incorporation was measured after harvesting. Results are expressed as percentage of proliferation of NK cells cultured with MSCs, in the presence or in the absence of inhibitors of IDO or PGE2, with respect to NK cells cultured alone (100%). Bars represent mean plus or minus SD of 9 independent experiments. NK-mediated cytotoxic activity (B) was analyzed after 6-day culture using the ⁵¹Cr-specific release method. Data represent the percentage of lysis mediated by NK cells cultured with MSCs in the presence or in the absence of IDO and/or PGE2 inhibitors, with respect to NK cells cultured alone (100%). Bars represent means plus or minus SD obtained from 5 independent experiments. The E/T ratio used was 5:1 for all targets analyzed. **P < .01; ***P < .001.