IFN-γ production by NK cells on interaction with MSCs. IFN-γ production by polyclonal activated NK cells after interaction with MSCs. Panels A and B represent 2 independent experiments performed in allogeneic and autologous settings, respectively. NK cells were incubated alone or in the presence of MSCs at an E/T ratio of 8:1. MSCs were untreated or treated with 100 U/mL IFN-γ (identified as MSC + IFN-γ) for 48 hours before their use in the experiment. After 5 hours of culture in the presence of 100 U/mL IL-2 and monensin-containing GolgiStop, cells were collected, and double staining was performed. To detect the frequency of IFN-γ-positive cells, NK cells were stained with anti-CD56-PC5 and anti-IFN-γ-PE mAbs and were analyzed by flow cytometry. Events (5 × 10⁴) were acquired and analyzed by gating on low FSC, low SSC cells.