Role of soluble factors secreted by MSCs in NK-cell activation. (A) Secretion of PGE-2 (ng/mL) and HLA-G (U/mL) by MSCs during the culture with NK cells was evaluated by ELISA assay. Results are representative of 5 independent experiments and are shown as the means ± SD of triplicate samples. Statistically significant differences between MSCs cultured or not with NK cells are indicated by asterisks (***P < .001; **P < .01). (B) Freshly isolated NK cells were stimulated for 4 days with IL-2 (250 U/mL) in the presence of MSCs. Inhibitors of IDO activity (1-MT, 1mM), PGE-2 secretion (NS-398, 5μM) and HLA-G neutralizing antibody (87G, 10μg/mL) were added at the start of the culture. Granzyme B expression was determined by Western blot using anti-granzyme B mAb. (C) Secretion of IFN-γ in IL-2–activated NK cells in presence of MSCs and soluble factors inhibitors was evaluated by ELISA assay. Results are representative of 5 independent experiments and are shown as the means ± SD of triplicate samples. Statistically significant differences between NK cells cultured with MSCs in presence or not with soluble inhibitors are indicated by asterisks (***P < .001; *P < .05).