NK-cell dysfunction is induced by homeostatic proliferation. (A) IFNγ production in restimulated NK cells reisolated 1 or 5 days after in vivo transfer with or without disseminated tumor; gates were set using nonstimulated controls (not shown). Injection into Rag2^−/−γ_c^−/− recipients in addition to irradiated recipients was performed to account for the effects of radiation. (B) Killing of chromium-labeled A20 cells by freshly isolated naive NK cells or C57BL/6 CD45.1⁺ NK reisolated 18 hours after transfer into irradiated hosts bearing Hoxa9-Meis1 leukemia, and cultured with A20 tumor cells at a 1:1 ratio for 16 hours. Control cells were sorted CD3⁻NK1.1⁺ NK cells from naive spleens; P = .0035 (2-tailed unpaired Student t test). (C) Killing of chromium-labeled A20 cells by freshly isolated naive NK cells or C57BL/6 CD45.1⁺ NK reisolated 17 days after transfer into irradiated hosts bearing Hoxa9-Meis1 leukemia, and cultured with A20 tumor cells indicated ratios for 16 hours. Control cells were sorted CD3⁻NK1.1⁺ NK cells from naive spleens; P = .001 for difference between naive and reisolated NK cells at a 6:1 ET ratio. (D) CFSE-labeled C57BL6 NK cells were injected into Rag2^−/−γ_c^−/− recipients alone or with 5 × 10⁶ A20 tumor cells and sorted into CFSE high (unproliferated) or CFSE low (proliferated) populations; these cells were then cultured with chromium-labeled A20 cells for 12 hours at an effector:target ratio of 2:1; ANOVA with the Dunnett multiple comparison test. Results are representative of 2 to 3 experiments with 3 to 4 mice pooled per experiment.