Mechanical force determines chimeric antigen receptor microclustering and signaling

Abstract

The chimeric antigen receptor (CAR)-T cells are activated to trigger the lytic machinery after antigen engagement, and this has been successfully applied clinically as therapy. The mechanism by which antigen binding leads to the initiation of CAR signaling remains poorly understood. Here, we used a set of short double-stranded (ds) DNA tethers with mechanical forces ranging from ∼12 to ∼51 piconewtons (pN) to manipulate the mechanical force of antigen tether and decouple the microclustering and signaling events. Our results revealed that antigen binding-induced CAR microclustering and signaling are mechanical force-dependent. Additionally, the mechanical force delivered to the antigen tether by the CAR for microclustering is generated by autonomous cell contractility. Mechanistically, the mechanical force-induced strong adhesion and CAR diffusion confinement led to CAR microclustering. Moreover, cytotoxicity may have a lower mechanical force threshold than cytokine generation. Collectively, these results support a model of mechanical force-induced CAR microclustering for signaling.