A versatile flow-based assay for immunocyte-mediated cytotoxicity

Cell-mediated cytotoxicity is a critical function of the immune system in mounting defense against pathogens and cancers. Current methods that allow direct evaluation of cell-mediated cytotoxicity suffer from a wide-range of drawbacks. Here, we present a novel strategy to measure cytotoxicity that is direct, sensitive, rapid, and highly adaptable. Moreover, it allows accurate measurement of viability of both target and effector cells. Target cells are fluorescently labeled with a non-toxic, cell-permeable dye that covalently binds to cell proteins, including nuclear proteins. The labeled target cells are incubated with effector cells to begin killing. Following the killing reaction, the cell mixture is incubated with another dye that specifically stains proteins of dead cells, including nuclear proteins. In the final step, cell nuclei are released by Triton X-100, and analyzed by flow cytometry. This results in four nuclear staining patterns that separate target and effector nuclei as well as nuclei of live and dead cells. Analyzing nuclei, instead of cells, greatly reduces flow cytometry errors caused by the presence of target-effector cell aggregates. Target killing time can often be reduced to 2 h and the assay can be done in a high throughput format. We have successfully validated this assay in a variety of cytotoxicity scenarios including those mediated by NK-92 cells, Chimeric Antigen Receptor (CAR)-T cells, and Tumor Infiltrating Lymphocytes (TIL). Therefore, this technique is broadly applicable, highly sensitive and easily administered, making it a powerful tool to assess immunotherapy-based, cell-mediated cytotoxicity.