Natural Killer (NK) cells play an important role in the first-line defense against tumor and virus-infected cells. Studies suggest NK cells have a dichotomous role in transplantation with evidence supporting the role of NK cells in both graft rejection and tolerance. The activity of NK cells is tightly regulated by a repertoire of cell-surface expressed inhibitory and activating receptors. NKp46 is a major NK cell activating receptor that is involved in the elimination of HCV and other viral infected cells and has been shown to regulate interactions of NK cells with other immune cells including T cells and dendritic cells (DC). Recent studies have shown that DC-mediated activation of NK cells is dependent upon signaling through the NKp46 activation receptor. Rapid advances in microscopy techniques have significantly contributed to our ability to examine and analyze the NK immune synapse, the interface between NK cells and other cells. NK cells can form different types of synapses; cytotoxic, inhibitory, and regulatory. However the distinct nature by which NKp46 participates in NK immunological synapse formation and function remains unknown. In this study we show that NKp46 is arranged in microcluster structures at the immune synapse between NK cells and target cells. Labeling of F-actin with Rh-Phalloidin showed that higher expression of NKp46 is correlated with increased accumulation of actin mesh at the immune synapse and that knock-down of NKp46 in donor-derived NK cells abridged recruitment of F-actin to the synapse. NK92 cells transfected with NKp46-IRES-GFP in combination with LysoTracker to image acidic spherical organelles demonstrated that higher expression of NKp46 contributes to rapid polarization of lytic granules toward target cells. We developed a live imaging cytotoxicity assay with NKp46-IRES-GFP expressing NK cells to show that NKp46 expression is correlated with a more rapid lysis of target cells. These results suggest that NKp46 on NK cells activates and regulates cells of the immune system resulting in the promotion or prevention of an alloimmune response. NK cell functions are generally refractory to immunosuppressive drugs thus understating these fundamental aspects of NKp46 function will contribute to our ability to manipulate NK cell activity post-transplant.

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