Leukemic blast cells in acute myeloid leukemia (AML) often evade elimination by cytotoxic immune cells, including natural killer (NK) cells, contributing to high relapse rates. This immune resistance is partly due to the impaired ability of NK cells to effectively polarize lytic granules at the immunological synapse, preventing the formation of a functional cytolytic synapse. Calcium signaling is critical to lymphocyte cytotoxic function, enabling the effective elimination of cancer cells, with precise calcium dynamics essential for the proper depolarization of lytic granules. Our research seeks to elucidate the role of calcium signaling in NK cell cytotoxicity, aiming to inform strategies for enhancing NK cell activation and overcoming leukemic cell resistance in immunotherapeutic applications.

We follow two approaches to achieve these goals. First, we optimized a protocol to efficiently isolate and culture NK cells and primary AML blasts, ensuring high purity and functionality. This approach enables the extension of calcium signaling studies to primary NK-leukemic blast systems, providing more physiologically relevant insights. Second, we established a panel of AML cell lines with differential sensitivity to NK-mediated killing, incorporating a FRET-based caspase reporter system. This setup facilitates real-time monitoring of AML target cell death during co-culture with primary NK cells from healthy donors.

By combining time-resolved, microscopy-based single-cell NK cytotoxicity assays with ratiometric calcium measurements, we can correlate calcium signaling dynamics in NK cells with differences in the susceptibility of target cells to the cytotoxic attack by NK cells. Preliminary findings reveal distinct calcium signaling patterns in NK cells, particularly in peak intensity and duration, that correspond with the sensitivity of target cells being killed by NK cells.