Opposing functions of MIST in NK- and NKT-cell activation. (A) IL-2-expanded spleen NK cells from wild-type (○) and MIST-deficient mice (•) were assayed for natural cytotoxic activities against YAC-1 and for antibody-mediated cytotoxicity against EL-4 cells either nontreated or pretreated with an anti-Thy1.2 antibody. Data are shown as the mean values and standard deviation (bars) of 1 representative from 3 independent experiments. (B) IL-2-expanded wild-type (+/+) and MIST-deficient (-/-) NK cells were stimulated with anti-NK1.1, anti-Ly49D antibodies, or PMA plus ionomycin (P+I) and assayed for intracellular IFN-γ. The percentages of IFN-γ-positive cells are indicated in the top right quadrants. (C) IL-2-expanded wild-type (□) and MIST-deficient (▪) NK cells were cultured for 24 hours with the indicated antibodies, target cells, or with IL-12, and the amounts of IFN-γ were measured. Data are the mean and standard deviation of at least 4 independent experiments. (D) IL-2-expanded NKT cells from MIST-deficient mice (▪) and wild-type littermates (□) were stimulated with the indicated antibodies, and the amounts of IFN-γ in the supernatants were measured. (E) Freshly isolated spleen cells (5 × 10⁵/200 μL) from MIST-deficient mice (▪) and wild-type littermates (□) were cultured with 50 ng/mL α-GalCer for 72 hours. Culture supernatants were harvested to measure IFN-γ and IL-4 levels as described in “Materials and methods.” (F) Serum IFN-γ and IL-4 levels in MIST-deficient mice (▪) and wild-type littermates (□) at different times after the administration of α-GalCer. Data are the mean and standard deviation of at least 3 independent experiments. The statistical analysis was performed using the Student t test. (^*P < .05; ^**P < .01; ^***P < .001).