Viral-induced NK cell proliferation and homeostasis

Our objective is to understand the in vivo regulation of NK cell proliferation and homeostasis during viral infections. NK cells are stimulated by cytokines elicited early during infections as well as by direct recognition of infected cells through activation receptors. In mice, the NK cell activation receptor that mediates resistance to  murine cytomegalovirus (MCMV) is Ly49H. Mouse strains (e.g., C57BL/6) that express this receptor on their NK cells are substantially more resistant to MCMV than mice that lack Ly49H expression. Ly49H recognition of its ligand on infected cells and subsequent signaling through its adaptor molecule DAP12 stimulates NK cell production of immunomodulatory cytokines and killing of infected cells. In addition to these effector functions, NK cells rapidly proliferate during viral infections. This viral-induced proliferative response occurs in three distinct  phases including early nonspecific NK cell proliferation, preferential proliferation of NK cells that are able to recognize infected cells, and cessation of proliferation and contraction of the expanded NK cell population. The regulatory mechanisms involved NK proliferation and homeostasis are poorly characterized.

We are using MCMV infection in mice to 1) delineate the relative contributions of cytokines and NK cell activation receptors to NK cell proliferation during viral infections, 2) determine how NK cell activation receptor signaling augments cytokine driven NK cell proliferation, and 3) define the role of the adaptive immune system in the resolution of viral-induced NK cell proliferation. We propose that a clearer understanding of in vivo NK cell proliferation and homeostasis during viral infections will have broad translational implications and may lead to novel therapeutic interventions to modulate NK cell responses to either stimulate more effective responses (e.g., during intractable viral infections or solid organ malignancies) or down-regulate over-exuberant or inappropriate responses (e.g., during NK cell lymphoproliferative disorders or autoimmune diseases).

Virally-encoded MHC class I-like immunoevasins

Large DNA viruses encode many immunomodulatory proteins that specifically target components in the host immune response. The evolution and retention of these immunoevasins occurs under selective pressure from the host immune response. Due to their binding specificities, immunoevasins can be exploited as affinity labels to identify host-encoded molecules of previously unsuspected importance in defense against the relevant class of virus. NK cell inhibitory and activation receptors bind members of the major histocompatibility (MHC) class-I superfamily. Therefore, we are particularly interested in characterizing the targets and function of virally-encoded MHC class I-like immunoevasins, including m157 from murine cytomegalovirus and OMCP (orthopoxvirus MHC class I-like protein) from cowpox.

MCMV-encoded m157 is recognized by Ly49H+ NK cells in C57BL/6 mice during MCMV infection. We have demonstrated that in the absence of an adaptive immune response, Ly49H+ NK cells exert sufficient selective pressure to facilitate the outgrowth of MCMV with mutations in m157. The question remains as to why the virus has retained m157. m157-deficient MCMV is less virulent than wt virus during infection in mice that lack expression of Ly49H (e.g., BALB/c). These observations suggest that mutations in m157 that were positively selected in the presence of Ly49H may be detrimental to other aspects of modulating the host’s immune response. We are working to understand the selective pressures exerted on the virus and innate immune response that have shaped both MCMV and the host immune response.

OMCP is a secreted poxvirus-encoded immunoevasin that targets NKG2D on cytolytic lymphocytes as well as innate B cells (marginal zone and peritoneal B1 B cells) and macrophages. OMCP binds NKG2D with high affinity blocking recognition of host-encoded ligands, inhibiting NKG2D-mediated killing of target cells. The receptor on murine innate B cells targeted by OMCP has recently been identified as Fc receptor-like 5 (FCLR5). The specificity of OMCP for FCLR5 strongly implicates innate B cells in contributing to anti-viral immunity. We are currently working to identify the receptor targeted on macrophages and to dissect the in vivo role of OMCP during cowpox infection.

Human NK cell functional responses

Although NK cells play a critical role in host defense against viral pathogens, inappropriate or ineffectual NK cell responses to viral infections may contribute to the pathogenesis of pediatric musculoskeletal disorders. We are interested in identifying potential NK cell functional defects in patients with autoimmune disorders and have developed a broad panel of tests to investigate the phenotypes and functional responses of NK cells in pediatric patients. These tests measure the ability of NK cells to proliferate, to degranulate and kill a broad panel of target cells, and to produce immunomodulatory cytokines and chemokines following stimulation through activation receptors or by cytokines.

The relatively rare patients identified with selective NK cell deficiencies have nearly uniformly had difficulty with recurrent, severe herpesvirus infections. Therefore, we have initially utilized our panel of NK cell assays to study pediatric patients identified in our Immunology/Rheumatology and Infectious Diseases Clinics who have experienced severe, recurrent herpes simplex virus (HSV) and cytomegalovirus (CMV) infections or have had unusually severe varicella-zoster virus (VZV) infections and/or recurrent herpes zoster. Currently available clinical assays have failed to identify NK cell defects or other significant immunological abnormalities to explain these patient’s recurrent or severe herpesvirus infections. The identification of specific immune deficiencies in these patients will facilitate improved clinical care and provide mechanistic insights into the function of the innate immune response in mediating resistance to viral infections. However, we anticipate that the utility of robust, reliable tests to assay NK cell phenotypes and functional responses will extend beyond this selected cohort of patients with difficulty with herpesviruses. For example, we envision these tests being useful in screening a wider group of patients with either susceptibility to a broader range of infections or immune dysregulation (i.e., autoimmune or autoinflammatory disorders). We are particularly interested in utilizing this panel of tests in evaluating pediatric patients with new-onset systemic JRA or juvenile dermatomyositis (JDMS). Detecting NK cell deficits in this broader group of patients has the potential to provide significant, novel insights into the role of the innate immune response, and in particular NK cells, in human health and disease and to shed light on the pathophysiology of pediatric musculoskeletal disorders.


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