Genetic dissection of cytolytic exocytosis from natural killer cells

PROJECT SUMMARY Natural killer (NK) cells are effector lymphocytes in the innate immune system. They destroy transformed and pathogen-infected cells mainly through secreting the contents of membrane-enclosed cytolytic granules, secretory lysosomes containing the pore-forming protein perforin and the serine proteases granzymes. When a NK cell recognizes its target cell, the contact area between the NK cell and the target cell forms a highly organized structure known as the immunological synapse. Cytolytic granules then migrate toward the immunological synapse, where they fuse with the plasma membrane to release their cytolytic molecules, a process known as cytolytic granule exocytosis. Once released from the NK cell, cytolytic molecules enter the target cell and trigger cell death. Imbalances in cytolytic molecule exocytosis cause major forms of human disorder such as immunodeficiency and chronic pathogen infection. The molecular basis of cytolytic granule exocytosis in NK cells is still poorly understood. The proposed research aims to bridge this knowledge gap. In our preliminary studies, we established an assay to quantify cytolytic granule exocytosis in a physiologically relevant NK-like cell line. Furthermore, we developed new platforms to genetically dissect complex exocytic pathways in mammalian cells using unbiased CRISPR screens. In this research, we will take strategic advantage of these assays to dissect cytolytic molecule exocytosis in NK cells using a genome-wide CRISPR screen. Next, we will validate the candidate genes identified in the screen using a pooled secondary screen. Finally, we will validate selected candidate genes using individual gene knockout. If successfully accomplished, this proposed research will offer the first genome-scale view of the cytolytic exocytic pathway and will significantly broaden our knowledge of NK cell functions. Insights acquired from this work will facilitate the development of novel therapeutic strategies for immune diseases caused by defective cytolytic granule exocytosis.