Project Summary Natural killer (NK) cells play an important role in the human immune response to infection and malignancy. How these cells effectively distinguish between diseased and healthy tissue is one of the key unsolved problems in immunology today. The proposed work seeks to identify the mechanism(s) by which the small adaptor protein CT10 regulator of kinase (Crk), and its phosphorylation, control NK cell activation and inhibition by using both human NK cells and novel NK cell-specific conditional knockout mice. The long-term goal is to use this knowledge and the novel imaging techniques developed herein to uncover the molecular basis of NK cell activation and inhibition, and to develop new treatments for human primary immunodeficiency diseases and chronic diseases such as cancer and viral infection. NK cells kill target cells through the polarized release of lytic granules through a specialized region of cell-cell contact known as the immunological synapse (IS). Through previous studies of the cytotoxic (Liu, D. et al., Immunity, 2009, Cover Article) and inhibitory (Liu, D. et al., Immunity, 2012) IS, we discovered that Crk plays an essential upstream role at the IS, influencing signaling events required for both activation and inhibition. The molecular mechanisms underlying this dual role, however, remain unclear. We hypothesize that receptor-driven phosphorylation of Crk acts as a molecular switch, driving a conformational change, which in turn determines Crk's ability to interact with critical downstream signaling molecules and ultimately shapes the actin cytoskeleton into a functional IS. Guided by strong preliminary data, we will test these hypotheses via three Specific Aims: 1) Define the precise molecular mechanisms by which Crk controls NK cell activation and inhibition. The proposed work capitalizes on the recent explosion in single molecule imaging and will bring cutting-edge single molecule imaging technology to the field of NK research, with a focus on human primary immunodeficiency diseases; 2) Determine the role of Crk-like protein (CrkL) in NK cells from patients with partial DiGeorge syndrome (pDGS). Using one of most common (1 in 4,000 births) immunodeficiency diseases, pDGS (mainly caused by CrkL haploinsufficiency), we will determine how loss of CrkL function affects NK cell-mediated cytotoxicity in pDGS and its molecular mechanisms; 3) Determine whether Crk is required for NK function in vivo. Leveraging novel NK cell-specific Crk knockout mice that we have already generated, we will determine the roles that Crk molecules play in NK cell-mediated immune responses to viral infection and cancer in vivo. The proposed work involves key signaling players and novel regulatory mechanisms and is broadly relevant with direct clinical implications for the treatment of primary immunodeficiency diseases and cancer because elevated expression and increased phosphorylation of Crk are common to various cancers (e.g., lung cancer, breast cancer, glioblastoma, sarcomas, ovarian cancer, and hematopoietic cancers). The proposed work also generates a novel model system in which to determine Crk's role as a master regulatory molecule. !