Functional and molecular characterization of single serial killer CAR+ T cells demonstrates adaptive modification of behavior and fate based on tumor cell density

T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) for the investigational treatment of B cell malignancies comprise a heterogeneous population, and their ability to persist and participate in serial killing of tumor cells is a predictor of therapeutic success. We developed Timelapse Imaging Microscopy In Nanowell Grids (TIMING) to dynamically analyze thousands of interactions between T cells and tumor cells. Our results demonstrate that while CAR T cells launch fullycompetent anti-tumor responses as defined by polarized motility, serial-killing and IFN-g secretion, the cytolytic functionality of CAR T cells must be quantified in the context of their ability to resist apoptosis. The ability of killer CAR T cells to adaptively modify both cell-intrinsic (polarization and motility) and extrinsic (conjugation and kinetics of tumor-cell death) behavior was modulated by number of tumor cell encounters. By comparing both the serial killer and single killer CAR T cells it appears that the propensity and kinetics of effector apoptosis was markedly different, effectors underwent apoptosis at higher frequencies (50%, single target killers, vs 28%, serial killers) and faster kinetics (time to apoptosis: 256 ± 12 minutes, single target; vs. 396 ± 25 minutes, multiple targets; p-value < 0.0001) (Figure 1). These data indicate that activation for lysis through multiple targets as opposed to prolonged contact with a single target reduces the propensity for effector apoptosis. Although the mechanistic basis for the responsiveness of these T cells to