Visualization and quantification of NK cell-mediated cytotoxicity over extended time periods by image cytometry

Natural killer (NK) cell-mediated cytotoxicity is traditionally measured using the chromium release assay, which measures the fraction of radioactive ⁵¹Cr released from dying target cells co-cultured with NK cells. However, the time frame of ⁵¹Cr release assays is limited to approximately 4 h due to spontaneous release of ⁵¹Cr. In the tumor microenvironment, interactions between NK cells and tumor cells occur over extended time periods, and NK cell-mediated cytotoxicity is modulated by cytokines produced by tumor cells and other immune cells. Here we demonstrate that the interaction of NK cells and tumor cells can be imaged and quantified over an extended period of time using a novel image cytometry method. Specifically, we imaged killing of human ZsGreen⁺ melanoma cells by primary human NK cells in the presence of an antibody targeting MICA and MICB on the tumor cell surface. The number of live ZsGreen⁺ A375 cells was counted in 96-well plates over a three day time frame, and the results were used to first calculate % specific killing at the 4 h time point to compare to ⁵¹Cr release assay. Analysis of data from the 4 h time point demonstrated that both ⁵¹Cr and image cytometry enable sensitive detection of NK cell-mediated killing of tumor cells. Image cytometry demonstrated that the combination of the MICA/B antibody and IL-2 induced near-complete eradication of A375 melanoma cells by NK cells at later time points. This novel image cytometry based approach will be suitable for the discovery of combination therapies that enhance the cytotoxic function of NK cells against tumor cells.