Lytic granule convergence is essential for NK cells to promote targeted killing while preventing collateral damage

Abstract NK cell activation triggers serial events leading to lysis of diseased cells: 1) granule convergence, preformed lytic granules rapidly cluster to the microtubule-organizing center (MTOC), 2) granule/MTOC polarization to the immunological synapse, and 3) degranulation, exocytosis of lytic contents onto the target cell. Granule convergence requires LFA-1 signaling and dynein motor function. Yet, it remains elusive how lytic granule convergence contributes to NK cell cytotoxicity. Using Drosophila S2 cells, we regulate NK cell signal inputs to precisely control granule convergence and degranulation. In the absence of LFA-1 signaling, the average distance of lytic granules to the MTOC increased >1.3 fold, indicating diffuse granule localization. Using imaging flow cytometery, we determined that without LFA-1, the synaptic accumulation and degranulation of lytic granules decreased by 3.3 and 2.5 fold, respectively suggesting undirected granule release. Using an ultrasound-guided-acoustic-trap-microscopy system to enforce live single NK cell with multiple target cell contacts, we determined killing efficiency with real-time granule tracking. NK cells activated by CD16 alone without co-engaging LFA-1 showed ~40% lower targeted killing and ~30% higher non-specific killing of neighboring unlabeled “bystander” S2 cells. Finally, we used ciliobrevin D, a dynein inhibitor, to physically block granule convergence in NK cells conjugated with physiologically relevant target cells. Dynein-inhibited NK cells caused ~36% increase in bystander killing. Thus, we demonstrated that NK cells converge lytic granules to improve the efficiency of targeted lytic granule secretion and prevent collateral damage to neighboring tissue.