Regulation of natural killer cell cytotoxic pathways during serial killing activity

Abstract

Natural Killer (NK) cells act as the front line of the body´s defense in the immune system and they are key players in efficiently recognizing and eliminating virus-infected and tumor cells. Individual NK cells can eliminate multiple target cells in a sequential manner during a process named serial killing. There are two major pathways NK cells use to induce apoptosis in their target cells: By releasing the content of cytotoxic granules containing the serine protease granzyme B (GrzB) and the pore-forming protein perforin or by the engagement of death receptors, which initiates caspase cascades via Caspase-8 (Casp8). The contribution of both cell death processes to NK cell cytotoxicity and serial killing remains poorly understood. Therefore, investigating the interplay between these pathways in more detail is important for a better understanding of NK cell cytotoxicity. To visualize the GrzB and death receptor-mediated target cell death in a time-dependent manner, we used fluorescent localization reporters that enabled us to simultaneously measure the activities of GrzB and of Casp8 in target cells upon contact with NK cells by life cell imaging. In this study we observed that NK cells kill their initial targets via the fast GrzB-induced pathway and switch to a slow death receptor-mediated killing for the final target. During the target cell contact NK cells lost GrzB and perforin, whereas the expression of CD95L, a main death cell ligand, was increased on the NK cell surface. The reduction of the lytic granules can be efficiently restored by the stimulation with different cytokines such as IL- 15, IL-2 or IL-21. Perforin deficient NK cells or ILC3 cells were unable to perform GrzB-mediated killing and no serial killing could be mediated without this pathway. In contrast, the absence of death receptor CD95 on the target cell and/or the absence of death receptor ligands on the NK cell had no direct influence on the GrzB-mediated serial killing. This demonstrates that the use of GrzB vs. death receptor-mediated target cell killing is differentially regulated during the serial killing activity of NK cells. Taken together, we observed a rapid target cell death which was induced by GrzB and originated from early established NK : target contacts. In contrast, cell death mediated by Casp-8 was a result of later target cell engagements and took much longer from NK : target cell contact to target cell death.