IFN-γ production by NK cells from the spleens of WT and BCAP^−/− mice as a function of SRIR expression. (A) Fresh splenocytes from C57BL/6 background mice were incubated alone or with plate-bound anti-NK1.1, 500 U/mL IL-2, and 10 ng/mL IL-12; 10 μg/mL brefeldin A was added 1 hour later, and the cells were cultured 4 more hours. Cells were surface stained, then fixed, permeabilized, and stained for intracellular IFN-γ. The thicker line represents BCAP^−/− mice, and the dashed line represents unstimulated control. Percentages of IFN-γ⁺ cells in WT and BCAP^−/− NK cells are listed on gates. Percentages on arrows are the percentage of NK cells on each mouse that are either negative for both Ly49C and Ly49I (left arrow) or express at least one of these SRIRs (right arrow). (B) Mean IFN-γ responses in NK-cell subsets from 3 C57BL/6 background mice analyzed, as described in panel A. The mean percentages plus or minus SD of IFN-γ⁺ wild-type (■) and BCAP^−/− NK cells (□) are shown. P values were calculated by applying a 2-tailed Student t test (*, **). (C) Mean IFN-γ responses of 7 pairs of MHC-I–deficient mice as described in panel A. The mean percentages plus or minus SD of IFN-γ⁺ wild-type (■) and BCAP^−/− NK cells (□) within indicated subsets (NKG2A of Ly49I expression) are shown (*).