NK-cell dysfunction is induced by proliferation in the presence of tumor. (A) Killing of chromium-labeled A20 cells by allogeneic C57BL/6 sorted NK that had been cultured at a 1:1 ratio with or without irradiated A20 for 5 days, in presence of IL-2 750 U/mL; effector:target ratios for the chromium assay are indicated on the x-axis. (B) Killing of chromium-labeled A20 cells by C57BL/6 NK cells that had been cultured at the indicated ratios with irradiated A20 tumor cells for 5 days, effector:target ratio for the chromium release assay was 2.5:1; P = .0009, ANOVA with Dunnett multiple comparison test. (C) Exposure to tumor leads to loss of IFNγ production and degranulation. After a 5 day exposure to irradiated A20 cells, C57BL/6 NK cells were stimulated with plate-bound anti-NK1.1 antibody and stained for IFNγ or CD107a. (D) Quantification of the events in panel C; P < .001 for IFNγ production and P < .01 for CD107a degranulation; 2-tailed unpaired Student t test. (E) Tumor coculture leads to increased NK-cell proliferation. (F) Proliferated, CFSE-low NK cells undergo more marked dysfunction than unproliferated cells. C57BL/6 NK cells were labeled with CFSE and cultured with irradiated A20 tumor cells for 4 days, then sorted on the basis of CFSE dilution; sorted cells were then cultured with chromium-labeled A20 cells at a 1:1 ratio for 18 hours; P = .03 (2-tailed unpaired Student t test). Results were done in triplicate and are representative of 3 (A,C,D) or 2 (B,E,F) experiments.