IPS-MSCs and H9-MSCs are more resistant to activated NK cytolysis than BM-MSCs. NK cells stimulated with IL-2 for 48 hours were cocultured with MSCs for 4 hours. Activated NK cells were tested in a 4-hour 51Cr-release assay against H9-MSCs (♦), IPS-MSCs (■) or BM-MSCs (●) at different NK/MSC ratios (A). (B) MSCs were cultured for 4 hours with activated NK cells (ratio NK/MSC, 10:1) and then stained with an anti-Apo2.7–PE for 30 minutes at 4°C to evaluate their apoptosis. All experiments were done in triplicate. K562 cells (▴) were used as a positive control for NK cells. The interaction between NK cells and MSCs was visualized by fluorescence microscopy. (C) IPS-MSCs or H9-MSCs were mixed with activated (IL-2) or MSC-cultured NK cells (preIPS-MSCs or preH9-MSCs; ratio 2:1) and cultured for 20 minutes at 37°C on a poly-lysine–coated slide. Granule polarization, as defined by the accumulation of granzyme B (green) staining in the contact area between NK cells and MSC, after 30 minutes of coculture, was analyzed by fluorescence microscopy using a specific mAb. Nuclei were stained with DAPI (blue stain). In the differential interference contrast micrograph (DIC), on the left (63×), a human NK cell is shown conjugated to a MSC. One representative experiment, of at least 4 independent experiments, is shown. (D) NK cells were tested in a 4-hour 51Cr-release assay against IPS-MSCs or H9-MSCs at different E/T ratios. The panels show IL-2–activated NK cells against IPS-MSCs or H9-MSCs. Pretreatment with IPS-MSCs or H9-MSCs strongly decreased the killing potential of NK cells. 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 or with MSCs are indicated by asterisks (***P < .001; **P < .01).
