Natural Killer Cell Licensing Delineates NK “Helper/Repair” and NK “Effector/Suppressor” Subsets During Viral Infections

Natural killer (NK) cells are innate lymphocytes with anti-viral and anti-tumor capabilities that can be divided into subsets based on differential receptor expression patterns. NK cells that express inhibitory receptors that can bind to the MHC class I molecules present in the host are considered to be “licensed,” fully functional NK cells with normal production of cytokines and cytotoxicity in response to targets. In contrast, “unlicensed” NK cells are unable to strongly bind to host MHC class I molecules and are in turn hyporesponsive to targets in terms of cytotoxicity and cytokine production. Recent data suggest that NK cells also regulate antigen-specific adaptive immune responses during the course of viral infection, playing a significant role in viral clearance and immunopathology. The specific populations of NK cells that may mediate these differential effects during the course of viral infection have not been identified.

Here, we demonstrate differential effector and immunoregulatory functions of licensed versus unlicensed NK cells during influenza and murine cytomegalovirus (MCMV) infections in mouse models. We hypothesize that licensed NK cells serve a dual role as both effector and suppressor populations depending on the stage of viral infection. Similarly, unlicensed NK cells serve a dual role as helper and repair populations during the early and late stages of viral infection, respectively. We performed licensed and unlicensed NK cell subset depletions and then infected mice with influenza or MCMV and ascertained effects on: viral titers, antigen-specific T cell responses, and tissue pathology. Our data show that after influenza or MCMV infection, there is a significant reduction in antigen-specific CD4+ and CD8+ T cell populations in the presence of licensed NK cells as determined by tetramer-positive cells. Targeting of these T cells by the NK “effector/suppressor” licensed population appears later in the time course of infection and to be through NKG2D recognition and perforin-mediated lysis based on upregulation of NKG2D ligands Rae-1 and MULT1 on the T cells and the loss of T cell regulation with NKG2D blockade or perforin knockout mice. Depletion of the unlicensed NK “helper/repair” subset reduced the number of DCs in the lymph nodes and reduced total antigen-specific T cells. The unlicensed NK cells were found to home to the lymph node and produce increased levels of GM-CSF early during infection resulting in DC expansion. Additionally, the unlicensed NK cells are the primary producers of IL-22 based on intracellular staining in the damaged tissues in the late stages of viral infection, aiding in tissue regeneration. Adoptive transfer of unlicensed NK cells with IL-22 silencing through siRNA transfection into immunodeficient mice showed increased tissue damage and pathology as compared to transfer of non-IL-22 silenced NK cells.

Collectively, these data suggest differential roles of licensed versus unlicensed NK cells that are both tissue and time-specific. At early stages of infection, licensed NK cells serve as direct anti-viral cells at the sites of infection while unlicensed cells promote DC expansion in the lymph nodes promoting antigen-specific T cell responses. Conversely, at the late stages of infection, licensed NK cells serve an immunoregulatory role by lysing antigen-specific T cells at the site of infection and in the lymph nodes, while unlicensed NK cells travel to the sites of injury to aid in tissue repair through production of IL-22. Importantly, a similar functional polarization of resting human NK cells was also observed after PMA/ionomycin stimulation, with a small population of unlicensed NK cells producing IL-22 and a bias towards GM-CSF secretion over IFNγ production by the unlicensed NK cell subset. We conclude that licensed NK cells have an effector/suppressor function while the unlicensed NK cells function as the helper/repair population suggesting distinct roles of NK cell subsets throughout the course of infection. By understanding the functions and characteristics of these NK cell populations, specific subsets can either by adoptively transferred or therapeutically targeted clinically to aid in different stages of immunological response including elimination of the virus, inhibiting the adaptive immune response, or aiding in tissue repair and regeneration.