CAR T cell cytotoxic responses are rapidly generated and sensitive to the unligated TCR

Chimeric antigen receptor (CAR) T cells expressing tumor-targeting engineered receptors can robustly eliminate cancer cells through secretion of cytotoxic factors. Durable remission in leukemia and lymphoma treatment has not been matched in solid tumors. Efforts to maximize tumor destruction and minimize toxicities have driven efforts to tune CAR signaling. However, the molecular mechanisms for CAR triggering and thresholds for activation are incompletely understood. Here, we measured the collection of CAR binding interactions that culminate in polarized delivery of lytic granules to the junction with the target. CAR T cells binarized cytotoxic activities in response to a few binding events and population outcomes were dominated by a subset of cells. Activation at the single molecule level matches the sensitivity of the native T cell receptor (TCR) and points to potent downstream signal propagation. Disruption of the unligated TCR with a transmembrane-targeting inhibitory peptide strongly dampened CAR T cell activation, indicating a critical crosstalk between the two receptors. Harnessing CAR T cell efficacy and reduction of toxicity will require new approaches to modify integration of the binding events, collected stochastically, that are rapidly digitized. These sensitive CAR T cell responses provide new insights into driving cytotoxic signaling through surface interaction engineering.