Inhibition of IL-12–induced miR-132, miR-212, or miR-200a blocks the reduction of STAT4 induced by IL-12 and increases the IFN-γ production induced by further IL-12 stimulation. (A) Human NK cells (2 × 105) were transfected with control inhibitor (anti-miR ctrl), anti–miR-132, anti–miR-212, anti–miR-200a, or an equimolar mixture of anti–miR-132, miR-212, and miR-200a inhibitor (anti-miR mix) as indicated at an aggregate final concentration of 50nM. These transfected cells were treated with or without IL-12 (10 ng/mL) for 48 hours in the presence of IL-2, and the expression of STAT4 was measured by flow cytometry. Data are representative of 3 independent experiments with NK cells from different donors. The gray dotted line represents the isotype control staining of NK cells. (B) Summary of data from panel A, mean of IL-12–induced STAT4 reduction in cells transfected with anti-miR as indicated (STAT4 MFI without IL-12 – STAT4 MFI with IL-12) was calculated to determine the relative inhibitory effects of different miRNA inhibitors on IL-12–induced STAT4 reduction. The results are STAT4 MFI decreased mean ± SEM of representative experiment with NK cells from 3 different donors. *P < .05, **P < .01 versus anti-miR ctrl. (C) Cells treated with IL-12 and the indicated mir inhibitors as in panel A (but not harvested) were washed with complete growth medium and secondarily stimulated with 10 ng/mL of IL-12 for 12 hours in the presence of IL-2. Cellular expression of IFN-γ was measured by flow cytometry. Data are representative of 3 independent experiments with NK cells from different donors. The gray dotted line represents the isotype control staining of NK cells. (D) Summary of data from panel C, mean of the inhibitory effect of IL-12 pretreatment on secondary IL-12–induced IFN-γ production in indicated anti-miR–transfected cells (IFN-γ MFI without IL-12 priming – IFN-γ MFI with IL-12 priming) was calculated to determine the relative inhibitory effects of different miRNA inhibitors on the inhibitory effect of IL-12 pretreatment on secondary IL-12–induced IFN-γ production. The results are decreased IFN-γ MFI mean ± SEM of representative experiment with NK cells from 3 different donors. *P < .05 versus anti-miR ctrl.
