Transcription factors Eomesodermin and T-bet are down-regulated on exposure to tumor and on proliferation. (A) Levels of Eomesodermin and T-bet fall with cell proliferation. Gates were set using isotype controls as shown in the top row for each analysis. Naive NK cells were analyzed fresh and hence were not CFSE-labeled. NK cells reisolated at D+ 15 after adoptive transfer into nontumor-bearing hosts were isolated using the expression of congenic markers, as shown in the left panel. (B) Eomes expression and IFNγ production decrease over time. NK cells were reisolated 1 or 10 days after transfer without or with 1 × 10⁵Hoxa9-Meis1 leukemia cells. Gates are set using isotype controls, as shown in the top row. Naive spleens are depicted for comparison in the left column. (C) Sorted C57BL/6 NK cells were transduced with Eomes, T-bet, or control vector and injected with A20 cells at an E/T ratio of 5:1 into irradiated Balb/c recipients. Overexpression of Eomes, and not T-bet, leads to a significantly prolonged survival compared with control treated NK cells. (D) Tumor burden is reduced in mice receiving Eomes-transduced NK cells. Sorted C57BL/6 NK cells were transduced with Eomes, T-bet, or control vector and injected with luc⁺ A20 cells at a 5:1 ratio into irradiated Balb/c recipients. BLI on D+8 after injection shows a significant reduction in tumor burden among mice receiving Eomes-transduced NK cells compared with no NK cells. T-bet or control vector treated NK cells did not show a significant reduction in tumor burden. Results are representative of 3 (A) or 2 (B-D) individual experiments, or are a composite of 3 independent experiments with a total of 13 to 14 mice per group (C).