Time-Resolved Study of Triplebody-Mediated Lysis by Natural Killer Cells on Microstructured Target Cell Arrays

Natural Killer (NK) cells are reduced in numbers and cytolytic activity in bone marrow and peripheral blood of Acute Myeloid Leukemia (AML) patients at diagnosis, but generally recover during chemotherapy. Certain antibody-derived therapeutic agents, such as single chain triplebodies, depend on the availability of sufficient numbers of active NK-cells. To monitor recovery of these cells during chemotherapy, sensitive new methods are needed, capable of quantitating key properties, while using only small numbers of primary cells. A new method (single cell cytometry) is presented herein, which uses arrays of isolated target cells to study the ability of triplebody 33-16-123 to recruit Natural Killer (NK) cells. The single cell cytometry (SCC) approach measures the fraction of cells killed by NK cells in an automated high-throughput fashion using time-lapse fluorescence microscopy. We evaluate the fraction of specifically lysed cells, representing the incremental lysis in the presence of the triplebody, mediated by NK cells both as a function of triplebody dose and the effector-to-target cell (ET) ratio. The specifically lysed fractions are in agreement with standard assays. We observed a systematic dependence of killing rates with time, indicating an enhancement and saturation of NK activity at high doses. The results demonstrate the potential of cell arrays for time-resolved studies of immune effector cells interacting with cancer cells and therefore elucidate the role of antibody-derived agents on effector cell activation.