NK cells are activated during a wide variety of viral infections by virus-induced type I IFNs. The importance of the NK cell defense against virus infections is highlighted by the susceptibility of mice depleted of NK cells to many experimental infections and by the invasive or disseminated viral disease that is associated with naturally occurring NK cell deficiencies in humans [16
]. However, many viruses, including WNV, have developed mechanisms to evade the NK cell response. These mechanisms include expression of MHC class I homologs encoded by viruses, selective modulation of MHC class I protein expression, inhibition of activating receptor function and production of cytokine-binding proteins or cytokine-receptor antagonists encoded by viruses [17
]. These mechanisms also include direct viral effects on NK cells, such as infection of NK cells and viral envelope protein blockage of non-class I NK cell inhibitory receptors [2
]. Mice genetically deficient in NK cells or depleted of NK cells by anti-NK cell antibody demonstrated no increased morbidity or mortality compared to controls when infected with WNV [2
]. However, it would seem that this apparent lack of NK protection is only because WNV normally suppresses or bypasses the NK response. Therefore, it is reasonable to expect that blocking the viral evasion process and reinforcing NK cell function might lead to improved control and accelerated clearance of the viral infection.
Co-culture of PBMC with K562D2 not only expanded NK cells over a hundred fold, but also activated NK cells. All of the activating receptors tested including NKG2D, NKp30, NKp44, and NKp46 were upregulated after expansion. Most noticeably, NK cells expressing the natural cytotoxicity receptor, NKp44, were up regulated from only 0.31% before to 64.59% after expanding, which corresponds to a greater than 200-fold increase. Consistent with the upregulated natural killing receptors, the D2NK cells are more potent in killing K562 target cells (Figure ) than either the NK92 or NKL cell lines. The inhibitory markers in D2NK cells showed very small changes, although CD158b appeared to be modestly downregulated. Further studies would be required to determine whether the small CD158a downregulation is statistically significant. The upregulation of the activation receptors combined with the possible downregulation of one of the inhibitory receptors suggest an overall bias towards an activation profile in D2NK cells. The upregulation of the IFN-γ production of D2NK cells (Figure ) with no detectable IL-10 (data not shown) both before and after expansion further confirmed the activation status of the D2NK cells. We have tried to measure TGF-β production by D2NK, but have so far been unable to detect TGF-β above the high background concentration of TGF-β in the culture medium. We have not yet attempted to expand NK cells in serum-free medium.
The NK cell lines, NK92 and NKL, have efficacy disadvantages compared to D2NK. Both cell lines have less natural killing activity than D2NK (Figure ). NK92 has no ADCC activity because it lacks FcR and NKL has only weak ADCC in our assays (not shown). D2NK shows strong ADCC (Figure ). ADCC activity has been shown to play an important role in anti-cancer therapy and in the light of data presented here may warrant assessment for function in combating WNV infection. The WNV infected Vero cells are more susceptible to D2NK killings than normal Vero cells by both ADCC and natural killing. It should be interesting to further study the underneath mechanisms.
The inhibition of WNV replication by NK cells delivered prior or post infection observed in this study, suggests the possibility of controlling WNV infection by adoptive transfer of NK cells expanded in vitro. It remains to be determined whether the direct cytotoxicity and ADCC or the endogenous production of IFN-γ afforded by these expanded NK cells provides advantages over alternative treatments for WNV such as direct administration of IFN-γ.