Stat3-deficient NK cells show enhanced in vitro cytotoxicity against B16F10 melanoma cells. (A-B) A total of 5 × 10⁴ B16F10 melanoma cells were injected IV into Stat3^fl/fl, Stat3^Δ/ΔNcr1-iCreTg, Ncr1-iCreTg, and wild-type mice. After 24 days, the number of tumor nodules in the lung was assessed by 3 independent researchers in a blinded manner. (B) Statistical analysis summarizes 4 independent experiments (n ≥ 11 in total). (C) DNAM-1 expression on Stat3^fl/fl (n = 11) and Stat3^Δ/ΔNcr1-iCreTg (n = 10) NK cells in the blood of B16F10-inoculated mice was analyzed twice a week using flow cytometry (gated on CD3⁻NKp46⁺NK cells). Graph represents mean ± SEM. (D) In vitro cytotoxicity assays of IL-2–cultured NK cells with RMA, RMA-Rae1, YAC-1, v-abl⁺, and B16F10 target cell lines. The E:T cell ratios ranged from 1:1 to 10:1. After 4 to 6 hours of incubation at 37°C, lysis of target cells was analyzed by flow cytometry. Graphs represent mean ± SEM (biological replicates: 2, technical replicates: 3 each). (E) RMA, RMA-Rae1, YAC-1, v-abl⁺ cell line #1 and cell line #2, and B16F10 target cell lines were analyzed for the expression of the DNAM-1 ligand CD155 by flow cytometry. (F) IL-2–cultured Stat3^fl/fl and Stat3^Δ/ΔNcr1-iCreTg NK cells were treated with an anti-DNAM-1 antibody for 1 hour. Control and DNAM-1–blocked NK cells of both genotypes were incubated with B16F10 target cells. The E:T cell ratios ranged from 1:1 to 7.5:1; after 4 hours’ incubation at 37°C, the lysis of the target cells was analyzed by flow cytometry. Symbols represent means and error bars indicate SEM of triplicates. Data are representative for 2 independent experiments.