LAT1/LAT2 are required for IFN-γ production and cytotoxicity but are dispensable for phosphorylation of SLP-76, AKT, and ERK and for NK-cell proliferation. Ly49D⁺ splenic NK cells from WT and LAT1/LAT2 DKO mice were enriched and expanded in IL-2 for 6 days. (A) Expanded NK cells were stimulated with plate-bound isotype Ct Ab, anti-Ly49D Ab, or soluble PMA/ionomycin for 4 hours in the presence of Monensin followed by detection of IFN-γ and LAMP-1 expression by flow cytometry. Ly49D expression of cells at time of stimulation is shown. Plots are gated on CD4^–CD8^–NK1.1⁺ lymphocytes. (B) Data compiled from 3 independent experiments were normalized to the control NK-cell response and expressed as mean ± standard error of the mean (SEM). (C) CFSE-labeled T-cell–depleted WT or LAT1/LAT2 DKO splenocytes were stimulated with plate-bound control Ab or anti-Ly49D Ab in the presence of IL-2 (40 to 100 U/ml) for 3 days followed by flow cytometric analysis. Plots are gated on CD4^–CD8^–NK1.1⁺Ly49D⁺ NK cells. (D) The percent of maximal proliferation induced by Ly49D stimulation was calculated from 4 independent experiments and expressed as mean ± SEM. (E) Ly49D-enriched IL-2–expanded NK cells were left unstimulated or stimulated with Ct Ab or anti-Ly49D Ab for 1 or 10 minutes and analyzed for PLC-γ2 (loading control), pSLP-76, pAKT, and pERK. One representative of 3 to 6 independent experiments is shown. * indicates statistical significance of P < .05 by paired t test; n.s., not significant.