Deep phenotypic and functional characterization of NK cells during NK-mediated cytotoxicity

Abstract Natural Killer (NK) cell-mediated cytotoxicity is a critical mechanism of the immune system to respond against harmful stimuli. The development of emerging cell therapies, such as CAR-NK cells, requires a deeper characterization of cultured NK cells. Here we show a phenotyping of ex vivo and in vitro NK cells, and demonstrate how high quality and reliable reagents from BioLegend ensure a more in depth cytotoxic analysis. NK cells were isolated from fresh human PBMCs, using our MojoSort™ Human NK Cell Isolation Kit, and incubated at different ratios with Tag-it Violet™ stained K562 target cells in IMDM + BioLegend’s Cell-Vive™ T-NK Xeno-Free Serum Substitute, in the presence of CD107a-PE. Cells were then harvested for surface phenotyping and the supernatants were collected for cytokine analysis using LEGENDplex™ Human CD8/NK Panel. Tag-it Violet™ and Apotracker™ Green staining of K562 cells demonstrate significant decrease in proliferation and significant increase in apoptosis, respectively. Surface staining of CD107a reveals increased degranulation of both CD56bright CD16− and CD5 + CD16+ NK cells that correlates with increased apoptosis of K562 cells. Supernatant cytokine analysis revealed significant increases in Granzyme A and B, Perforin, and Granulysin, among others. Taken together, these results show a more complete picture of the phenotypical and functional state of NK cells by measuring target cell killing as well as characterizing NK surface markers and secreted molecules involved in NK-mediated cytotoxicity. BioLegend reagents are designed to address ever increasing needs for deeper understanding of immunological processes and support the demand to develop sophisticated and consistent cell-based therapies.