A simple and rapid method for measuring human natural killer cell function in whole blood by anti-NKp30-conjugated nanoparticles

Abstract

Abstract Natural killer (NK) cells play a central role as innate immune effector cells to virus infected cells and tumor cells. The standard assay to evaluate NK activity is 51Cr release assay using radio-labeled K562 cells, however, new methods using fluorescence-labeled targets or flow cytometry to examine degranulation markers have been developed recently. In addition to cytotoxicity, NK cells enable to produce various cytokines to regulate the adaptive immune responses in infection, inflammation and cancer. Here we generated nanoparticles conjugated with various antibody for NK receptors and examined to stimulate human whole blood with these nanoparticles instead of K562 target cells. We found that various cytokines such as IFN-γ, IL-6, IL-1β and CCL2 were produced by NK cells in whole blood by anti-NKp30-conjugated nanoparticles in dose-dependent manner more than by anti-NKp46 or anti-NKG2D nanoparticles. Production of IFN-γ was detected within 4 hours and reached maximum level at 24 hours after stimulation. IFN-γ production was relatively high at incubation temperature of 37°C, but that was significantly decreased over 39°C. Importantly, we could evaluate the reproducible cytokine production in same donors at another blood collection date because of the stable stimulus of nanoparticles and less variation data by human manipulation for lymphocyte separation. Additionally, cytokine secretion did not correlate with NK cell-mediated lysis of K562 target cells and NK cell numbers in whole blood. These data indicated that this method using NKp30 nanoparticles might be useful for measurement of individual differences of NK function especially correlated in adaptive immune response and inflammation.