Project Summary Natural killer (NK) cells constitute an important arm of the host immune system that detects and eliminates virus-infected cells. Newborns and immunocompromised patients lack NK cells and are extremely susceptible to viral infection, and in particular, human cytomegalovirus (HCMV) can cause severe health complications or be life-threatening in these individuals. Mouse cytomegalovirus (MCMV) is an accurate and robust model for HCMV infection, and represents a good system to study antiviral NK cell responses. Using MCMV infection in mice, we have discovered that NK cells possess novel adaptive immune features such as clonal expansion and long-lived memory. From the funding in the initial R01 period, our lab has uncovered many of the cellular and molecular mechanisms underlying NK cell memory. Our long-term goals are to understand the general biology of NK cells and the molecular basis by which these powerful effector cells can mediate protection against pathogen invasion. To this end, we have recently identified a novel role for the transcription factor IRF8 in the NK cell response against MCMV infection. Based on this exciting preliminary data, our current R01 renewal proposes to use a newly-generated transgenic mouse where Irf8 is specifically ablated in NK cells to study the impact of this transcription factor in antiviral responses. In Aim 1, we will perform epigenetic profiling of activated NK cells to determine how IRF8-mediated NK cell expansion and effector function are controlled following MCMV infection. In Aim 2, we will identify the binding partners and gene targets of IRF8 in antiviral NK cells using proteomic and transcriptomic analyses, respectively. In Aim 3, we will measure mitochondrial health and metabolism in antiviral NK cells to investigate the mechanisms behind IRF8-mediated survival of effector and memory NK cells during the contraction phase following MCMV clearance. Together, the studies in this R01 renewal will not only advance our understanding of the transcriptional mechanisms whereby NK cells contribute to host defense during viral infection, but also establish novel clinical paradigms for how the NK cell compartment may be harnessed for immunization and therapeutic strategies against infectious disease.