Role of ITAM-bearing subunits in NK cell effector functions. (A-B) polyIC NK cells were purified from control (CTR) and mutant mice and used as effectors against indicated tumor cells. Data shown are representative of at least 4 independent experiments. (C-D) Mice were injected intravenously with 3 × 10⁵ cells of B16, B16-H60, or B16-Rae1β melanoma cells, and total lung metastases were counted 2 weeks after tumor challenge. Dots indicate data from individual mice: ○, control mice (CTR); •, DAP12^KI mice. Horizontal bars indicate mean values. Statistical analysis was performed using unpaired Student t test. (E) NKG2D tetramer staining (filled histograms) is represented in comparison with staining with an irrelevant KIR2DL3 tetramer (empty histograms).⁶³  Cytotoxicity experiments were performed as described in panels A and B. Blocking experiments were performed by adding anti-NKG2D (MI-6, rat IgG2a) or isotype control mAb at a final concentration of 50 μg/mL, during the 4-hour cytotoxicity assay. (F) Percentages of IFN-γ⁺ cells in gated CD3^–NK1.1⁺ cells; □ indicate polyIC NK cells from C57BL/6 mice; ▪, polyIC NK cells from DAP12^KI mice. IFN-γ⁺ NK cells were not detectable in cells stimulated with B16 melanoma cells. (G) Percentages of IFN-γ⁺ cells in mixed bone marrow chimera reconstituted with RAG^KO and ZGR precursors were analyzed on NK cell subsets phenotypically defined, as described in legend to Figure S1; □ indicate percentages of IFN-γ⁺ cells gated in CD3^–NK1.1^bright2B4⁺ (NK cells from RAG^KO); ▪, percentages of IFN-γ⁺ cells gated in CD3^–NK1.1^dim2B4^– (NK cells from ZGR mice). Stimulation with target cells was performed at an effector-target (E/T) ratio of 1:1. Ly49D triggering was performed on high-affinity 96-well plates (Immulon, Labsytems, VWR, France) precoated with isotype control antibodies or with anti-Ly49D (4E5) at the final concentration of 50 μg/mL. Values obtained with control antibodies were subtracted from values obtained with Ly49D stimulation. Data are representative of at least 2 experiments. Error bars represent SEM.