Latent herpesvirus infection arms NK cells

Abstract

Natural killer (NK) cells were identified by their ability to kill target cells without previous sensitization. However, without an antecedent "arming" event, NK cells can recognize, but are not equipped to kill, target cells. How NK cells become armed in vivo in healthy hosts is unclear. Because latent herpesviruses are highly prevalent and alter multiple aspects of host immunity, we hypothesized that latent herpesvirus infection would arm NK cells. Here we show that NK cells from mice latently infected with Murid herpesvirus 4 (MuHV-4) were armed as evidenced by increased granzyme B protein expression, cytotoxicity, and interferon-gamma production. NK-cell arming occurred rapidly in the latently infected host and did not require acute viral infection. Furthermore, NK cells armed by latent infection protected the host against a lethal lymphoma challenge. Thus, the immune environment created by latent herpesvirus infection provides a mechanism whereby host NK-cell function is enhanced in vivo.

Figure 1

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).

Figure 2

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.

Figure 3

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).

Figure 4

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.

Figure 5

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).