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, integrin-influenced 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-like (CrkL) protein controls NK cell activation and inhibition. The proposed work will bring cutting-edge single molecule imaging technology to the field of NK cell research. Experiments will determine where and when Crk is phosphorylated at the IS, as well as how it interacts with key receptors, signaling molecules, and the actin cytoskeleton; 2) Determine the role of CrkL in NK cells from patients with partial DiGeorge syndrome (pDGS). By studying one of the most common (1 in 3,000 births) immunodeficiency diseases, pDGS (mainly caused by CrkL haploinsufficiency), we will determine how loss of CrkL function affects NK cell-mediated cytotoxicity; 3) Determine whether Crk or CrkL is required for NK function in vivo. Leveraging novel NK cell-specific Crk knockout mice that we have already generated, we will determine Crk's in vivo roles in NK cell-mediated immune responses to viral infection and cancer, including a newly identified role in memory NK cell generation. The proposed work involves key signaling players and regulatory mechanisms and generates a novel model system in which to determine Crk's role as a master regulatory molecule. It is broadly relevant with direct clinical implications for the treatment of primary immunodeficiency diseases, viral infections, and cancer. !

Public Health Relevance

Natural killer (NK) cells play a critical role in human host defense against viruses and cancer. Here, cutting- edge imaging techniques, a common human immunodeficiency (partial DiGeorge syndrome), and NK cell- specific mouse ?knockouts? will be used to define how Crk (an adaptor protein) and its phosphorylation act as a molecular switch to turn NK cell function on or off. These techniques and the knowledge they yield will set the stage for identifying specific phosphorylated Crk (pCrk) inhibitors that can harness the power of the immune system (including NK cell and other killer cells) to fight malignancies and viral infections. !

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Innate Immunity and Inflammation Study Section (III)
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Lapham, Cheryl K
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Rutgers University
Schools of Medicine
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Xiong, Wei; Chen, Yuhui; Kang, Xi et al. (2018) Immunological Synapse Predicts Effectiveness of Chimeric Antigen Receptor Cells. Mol Ther 26:963-975
Liu, Dongfang; Tian, Shuo; Zhang, Kai et al. (2018) Erratum to: Chimeric antigen receptor (CAR)-modified natural killer cell-based immunotherapy and immunological synapse formation in cancer and HIV. Protein Cell 9:902
Nabekura, Tsukasa; Chen, Zhiying; Schroeder, Casey et al. (2018) Crk Adaptor Proteins Regulate NK Cell Expansion and Differentiation during Mouse Cytomegalovirus Infection. J Immunol 200:3420-3428