Enhanced NKG2D expression on primary human NK cells by IL-10 and IL-21 is STAT3 dependent. (A) To assess the activation of STAT1, STAT3, and STAT5, NK cells were treated with 20 ng/mL of IL10 or IL21, with or without 0.1 µM of JSI-124 for 24 hours. Total and tyrosine-phosphorylated STAT were evaluated by western blot of cell lysates using STAT- or phospho–STAT-specific antibodies as indicated. (B) To assess cytokine-mediated induction of NKG2D expression, primary NK cells were treated with IL-10 and IL-21 at the indicated concentrations. After treatment of 24, 48, and 72 hours, NKG2D expression was evaluated by flow cytometry. (C) To assess the contribution of STAT3 signaling to cytokine-mediated induction of NKG2D, primary NK cells were treated with 20 ng/mL IL-10 or IL-21 with or without 0.1 µM of JSI-124 for 24 hours. NKG2D surface expression on NK cells was then determined by flow cytometry. (A,C) Representative of at least 3 independent experiments. (B) Pooled data from 4 donors.