Latent infection with MuHV-4 arms NK cells with GzmB protein. Mice were inoculated with media (mock), MuHV-4, or latency-defective MuHV-4 (O73.stop) 31 days before flow cytometric analysis of GzmB expression in NK1.1⁺CD3⁻ NK cells from the spleen and peritoneal cavity. (A) Representative density plots demonstrating GzmB expression within NK1.1⁺CD3⁻ NK cells. (B) Pooled data (10 mice per condition) showing the mean ± SD percentage of NK cells that express GzmB protein. These data are representative of 4 independent experiments, *P < .009 (MuHV-4 vs O73.stop or mock).

NK cells from mice latently infected with MuHV-4 have increased capacity to produce IFN-γ protein. Peritoneal cells from latently infected or control (naive or mock-infected) mice were stained immediately after harvest (No Cx) or after culture ex vivo with phosphate-buffered saline (PBS), IL-12 plus IL-15, or plate-bound antibodies against NK1.1 (Anti-NK1.1). (A) Representative flow cytometric density plots showing IFN-γ expression in NK cells. (B) Pooled data from 10 mice per group showing the mean ± SD percentage of NK cells that express IFN-γ. These data are representative of 3 independent experiments with 10 mice per group per experiment. *P <.001 (MuHV-4 vs control).

NK cells in latently infected mice protect against a lethal RMA-S lymphoma challenge. (A) Mice were inoculated with medium (mock), MuHV-4, or latency-defective MuHV-4 (O73.stop) 29 days before injection with 10³ RMA-S cells intraperitoneally and then followed for survival. These data are pooled from 3 independent experiments with a total of 20 mice per group. *P < .001 (MuHV-4 vs either control). (B) Mice were inoculated with MuHV-4 35 days before injection with anti-NK1.1 or control monoclonal antibody (mAb). One day later, all animals were challenged with RMA-S as in panel A. Antibody injections were continued every 6 to 8 days for the duration of the experiment. These data are pooled from 2 independent experiments with a total of 10 mice per group. *P < .001 (anti-NK1.1 vs control).

Latent infection with MuHV-4 enhances NK-cell degranulation and cytotoxicity. (A) Latent infection with MuHV-4 increases NK-cell degranulation. Peritoneal cells from mock, O73.stop, or MuHV-4 latently infected mice were cultured ex vivo with YAC-1 target cells at the indicated target:effector ratios for 2 hours, followed by flow cytometric analysis of CD107a expression on the surface of NK1.1⁺CD3⁻ NK cells. Control mice were incubated without targets. Bars represent the mean ± SD of 5 to 10 mice per group. *P < .032 (MuHV-4 compared with mock or O73.stop). These data are representative of 2 independent experiments with 15 to 20 total mice per condition. (B) Latent infection with MuHV-4 induces cytotoxic activity by NK cells. Peritoneal cells from control (naive) mice and mice infected 28 days previously with MuHV-4 were enriched for NK cells and then coincubated with RMA-S target cells for 4 hours at the indicated effector:target ratios before analysis of target cell death by 7-AAD. *P < .003 (MuHV-4 vs control). These data are representative of 2 independent experiments with 20 to 30 mice per group per experiment.

NK cells express increased GzmB protein after short-term exposure to the latent MuHV-4 environment in vivo. Latently infected mice (28 days after infection with MuHV-4) were injected intraperitoneally with CFSE-labeled splenocytes from B6.RAG1^−/− donors as a source of naive NK cells. Mock-infected and naive recipient mice were used as controls. At 72 hours peritoneal cells were harvested and analyzed for CFSE and GzmB expression in NK1.1⁺CD3⁻ NK cells. (A) Representative flow cytometric density plots illustrating the expression of GzmB protein in transferred (CFSE⁺) versus endogenous (CFSE⁻) NK cells. (B) Pooled data from 2 independent experiments showing the mean ± SD percentage of NK cells that express GzmB. *P = .027 and **P < .001 (MuHV-4 vs control, 11-14 mice per group). (C) Correlation of GzmB protein expression between transferred versus endogenous NK cells in each mouse. Each dot represents one mouse. Correlation coefficient (r²) = 0.9 in latently infected hosts.