Physical and molecular mechanisms of engineered T cell activation

Cytotoxic T cells expressing engineered chimeric antigen receptors (CARs) consolidate key components of native T cell triggering machinery into a single transmembrane receptor capable of scanning for specific pathogenic markers. These programmable therapeutics are capable of identifying and destroying target cells but have had limited clinical success and often exhibit off-target toxicities. The mechanistic determinants for threshold setting in CAR T cells are still poorly understood and are expected to inform future improvements of the therapies. Signaling onset, at the cell surface, may be tuned by spatiotemporal, topographic, mechanical, and chemical parameters. Using an in vitro cell:cell killing assay using low CAR T cell:target cell ratios, we show that small numbers of binding interactions by CAR T cells are sufficient to induce target cell death. This single cell sensitivity is enhanced by mechanical features of the microenvironment. To explore the potency of individual cells, we mapped CAR T cell inputs to cellular activation and cytotoxic responses using a single molecule in vitro reconstitution assay. We monitored the collection of binding interactions that culminate in polarization of lytic granules that are trafficked to the intercellular junction and may undergo exocytosis to release cytolytic factors. We found that, surprisingly, some CAR T cells mobilize cytotoxic responses to a small number of antigenic binding events, suggesting a different molecular activation threshold than previously appreciated. These characteristics bear an uncanny resemblance to the native T cell receptor (TCR), leading us to explore how the native receptor may be contributing to CAR T cell function. I will discuss how mechanochemical features of the CAR T cell:target cell junction tune the activation setpoint and our efforts to map the molecular mechanisms underlying the mobilization of the cytotoxic response.