Investigation of cytotoxic mechanisms in human NK cells

Natural killer cells (NK) are part of the innate immune system. They form an immunological synapse (IS) with infected or tumorigenic target cells and kill them via death receptors (Fas ligand CD95 and Fas receptor CD178) or by releasing perforin and granzyme from lytic granules. Fas-FasL binding induces apoptosis. Perforin release can either trigger direct lysis of the target cells (necrosis) or lead to the penetration of granzymes into the target cell, which induces apoptosis. Both NK-dependent target cell deaths have been described in the literature, but population-wide analyses have only been performed using endpoint assays. The dynamic regulation of NK-dependent apoptosis and necrosis of target cells at the single-cell level has not yet been investigated. To study dynamic single-cell processes, target cells that can be killed by human NK cells were stably transfected with a cell death sensor. This assay allows the classification of single-cell apoptosis and necrosis by different fluorescence patterns of the FRET-based sensor.