Although it is well-established that NK cells play an important role in immune protection to viral infection, relatively little is known about the kinetics of NK cell responses to most viral pathogens. In the current study we investigated the response of NK cells to respiratory viral infections. We found that upon infection, frequencies of NK cells increased in the airways, however, NK cells did not detectably proliferate there. Instead, proliferation occurred preferentially in the BM. We furthermore used an adoptive transfer model to generate mature long-lived NK cells from respiratory virus-infected donor mice and found that a proportion of long-lived NK cells migrated to the BM and there underwent both homeostatic and respiratory virus infection-induced proliferation. Thus, although the BM harbors high amounts of immature, developing NK cells, our data indicate that it is also the central site of proliferation for long-lived NK cells.
Previous studies on interactions between NK cells and influenza virus infected cells by the group of Mandelboim (20
) showed that NK cells recognized influenza virus HA through the activating receptor NKp46, which led to target cell killing. Mice that lacked NKp46 died more readily of influenza virus infection than wt
mice, despite similar increases in NK cells numbers in the lungs (16
). These data suggested that NKp46 ligation leads to activation but not proliferation. Our finding that NK cells barely proliferated in the lungs upon intranasal influenza virus infection further confirms this proposition. In contrast, stimulation of the activating receptor Ly49H through MCMV m157 led to selective proliferation of Ly49H positive NK cells after infection (6
). These different outcomes of receptor ligation might have resulted from differences in the signal pathways used by Ly49H and NKp46, which signal through the adaptor protein DAP12 (25
) and the FcεRIγ and CD3ζ (26
As our results showed that upon influenza virus infection, NK cells proliferated mostly in the BM and not at the site of infection, we conclude that the increased NK cell numbers in the airways (), at least in part, were the result of migration. A similar situation is seen during Listeria monocytogenesis
infections. There monocytes proliferate in the BM and emigrate CCR2-dependent into the blood (27
). Interestingly, in addition to monocytes, NK cells localized in the BM also express CCR2 (28
). Whether NK cells migrate CCR2-dependent from the BM to the site of infection during respiratory virus infection will be the subject of further investigation.
Recent reports have shown that NK cells can mount recall-responses up to several months following sensitization (9
). This adaptive trait requires the preservation of antigen-specific NK cells for a long period of time. In the present study, we did not directly address the role of specific cytokines involved in the maintenance of mature NK cells in the BM; however, NK cell homeostatic proliferation has been assessed on NK cells isolated from RAG−/−
mice that were adoptively transferred to RAG−/−
mice backcrossed on an IL-7−/−
). In addition to having a role in survival of naïve NK cells (30
), IL-15 played a dominant role in survival of transferred NK cells (29
). Interestingly, when transferred into RAG−/−
mice, NK cell proliferation was reduced three-fold (29
). Thus, both IL-15 and Il-7 might play an important role in the maintenance of long-lived NK cells.
Our finding that mature NK cells undergo homeostatic proliferation in the BM dovetails well with the maintenance of immunological memory that has extensively been studied for T and B cells and, in the absence of antigen, is believed to depend on survival signals, homeostatic proliferation or a combination of the two (31
). The BM is known to play a key role in preservation of immunological memory by being a niche for memory T cells and plasma cells (32
) and by producing the cytokines needed for survival (33
). Memory CD4+
T cells have shown to be in close contact with IL-7 expressing stroma cells, where they are maintained in a low proliferative state and receive IL-7 to survive (33
). Memory CD8+
T cells rely both on IL-7 and IL-15 for homeostatic proliferation (30
). Thus, like other ‘classic’ cells of the adaptive immune system, we here show that a proportion of NK cells behaves in a similar way by migrating back to the BM to undergo homeostatic proliferation
In addition to the cytokines necessary for homeostatic proliferation, a recent studied showed that IL-12 is indispensable for the generation of memory NK cells. Compared to their co-transferred wt
counterparts, adoptively transferred IL-12 receptor deficient NK cells could not be recovered 2 weeks after MCMV infection (40
). It is currently unknown which cells provide IL-12 for NK cell survival and at what locations.
Taken together, based on our data, we propose that following virus entry and inflammation in the lungs, NK cells migrate from remote storage sites to the site of infection, while the remaining NK cells proliferate at these sites probably to effect their replenishment. After becoming activated, NK cells in the airways produce cytokines and participate in viral clearance. A proportion of NK cells becomes long-lived and can migrate back to the BM where they undergo homeostatic proliferation and rapidly proliferate following re-infection.