Directing Function at the Natural Killer Cell Secretory Immunological Synapse

Project Summary Cytotoxic lymphocytes, including Natural Killer (NK) cells, are at the forefront of a therapeutic revolution in which the power of the cytotoxic cell is harnessed to kill tumor or virus-infected cells in patients with otherwise hopeless medical diagnoses. For nearly two decades, our laboratory has studied NK cell biology, learning that NK cells use a tightly controlled series of cell biological steps to precisely target their potent cytotoxic contents onto a diseased cell. This process is initiated and directed through the lytic immunological synapse, a specialized interface formed between the NK cell and its triggering target. In previous programs, we have shown that positioning of the NK cell’s destructive organelle, the lytic granule, is key to precise control of NK cell killing. When NK cells meet a target cell, lytic granules rapidly converge to the microtubule organizing center (MTOC); as activation proceeds, the MTOC and its granules move to the lytic synapse where they traverse through a dynamic actin meshwork to be secreted onto the target cell (degranulation). Lytic granule convergence is dynein- dependent and key to limiting killing to a single target cell. When convergence is disrupted, NK cells degranulate in all directions, killing “innocent” bystander cells. While convergence makes sense for surveillance cells who seek to destroy a single infected or malignant cell without causing damage to surrounding healthy tissue, it is limiting in the context of immunotherapy for an established tumor. We propose that blocking convergence without interfering with degranulation will convert NK cells into efficient non-directional destroyers, better suited for solid tumor cancer therapy. We predict that inhibitors and mutations that durably promote non-directional degranulation will promote killing efficiency and afford broader killing (escaping cells, stroma, and tumor- promoting immune cells) in complex pathogenic environments. In the proposed work, we test known and novel inhibitors of convergence, using high through-put small molecule inhibitor and genetic screens as well as a physical “synapsome mapping” approach to identify novel candidates. We ask whether we can achieve non- directional degranulation in a sustainable way (Aim 1A) without disrupting other critical NK cell functions (Aim 1B). In Aim 2, we confirm relevance of non-directional degranulation in a new, tumor-relevant, high-resolution, 3-dimensional model system we’ve called TheCOS (Thermal Collapse of Stroma). This hydrogel-based system allows us to generate instantaneous cell interactions in a manipulable complex environment suitable for high resolution live-cell imaging and functional assessment. We expect our insights to inform an immunotherapy strategy wherein a cytotoxic cell could be maximally efficient, destroying cells it normally wouldn’t, and potentially even avoiding exhaustion within the tumor environment. This would be of tremendous therapeutic value as broadly targeting the pathogenic environment with present cell-based immunotherapies has been a challenge. Equally important, the detailed “map” of synaptic function and novel tools we develop herein will provide critical resources for comprehending and harnessing the function of these powerful cytotoxic cells.