In vitro induction of NK cytoxicity and IFNγ mRNA after CD69 stimulation. Splenocytes from 8-week-old wt mice were incubated in vitro with mAb CD69.2.2 or isotype control mAb (IgG1) and after 18 hours cytotoxic activity was assayed with RMA-S tumor cells as target cells. Results of standard ⁵¹Cr cytotoxic assay are shown for total spleen cells (A) and purified NK cells (B). Results shown are representative of 3 independent experiments. (C) mAb CD69.2.2 increases IFNγ mRNA in splenocytes in vitro. mAb CD69.2.2 or isotype control mAb were added at 20 μg/mL and levels of IFNγ, MIF, and TGF-β mRNA were determined by RPA. Results are expressed in arbitrary densitometric units normalized for the expression of GAPDH in each sample. Four animals were used per experimental group, and results shown are representative of 4 separate experiments. (D) mAb CD69.2.2 induces IFNγ production in mouse NK cells. Cells were incubated in serum-free medium for 18 hours and IFNγ was determined by intracellular staining. Dot plots represent the distribution of side scatter (SSC) versus IFNγ–expressing cells among gated DX5⁺ NK cells. Numbers in left quadrants represent the percentage of INF-γ positive cells. Results are representative of 2 independent experiments and 4 animals were used per experimental group. Error bars represent standard deviation (^*P < .05).