Tumor-infiltrating NK cells down-regulate activating receptors and show impaired effector functions. (A) Purified CD3⁻DX5⁺ or CD3⁻NK1.1⁺ NK cells from CD45.1⁺ congenic donors were transferred into CD45.2⁺ recipients. All analyses are gated on live singlet lymphocytes. Within the spleen and tumor of tumor-bearing mice (rows 2 and 3), transferred CD45.1⁺ NK cells down-regulated NK1.1, NKG2D, and DX5 compared with ex vivo expanded CD45.2⁺ NK cells (top row). (B) Statistical comparison of events in panel A, showing marked loss of activating receptors on intratumoral NK cells compared with cultured and splenic NK cells; ANOVA with the Dunnett multiple comparison test. (C) IFNγ production is diminished within reisolated NK cells. Gating is based on nonstimulated controls (not shown). (D) Statistical comparison of events in panel C, showing loss of cytokine production on intratumoral NK cells compared with cultured or splenic NK cells; ANOVA with Dunnett multiple comparison test. (E) CD45.1⁺ cells were sorted from spleen or tumor and assessed for cytotoxicity against chromium-labeled A20 cells. Analyses are gated on reisolated CD45.1⁺ or control CD45.2⁺ live NK cells. Results are representative of at least 3 (A-D) or 2 (E) independent experiments with at least 3 mice per group.