Visualizing and manipulating hyperstabilization of T cell microvilli contacts by engineered chimeric antigen receptors

T cells typically recognize their ligands using a defined cell biology – the scanning of their membrane microvilli to palpate their environment – while that same membrane scaffolds T cell receptors (TCRs) that can signal upon ligand binding. Chimeric antigen receptors (CARs) present both a therapeutic promise as well as a tractable means to study the interplay between receptor affinity, microvillar dynamics and T cell function. CARs are often built using single-chain variable fragments (scFvs) with far greater affinity than that of natural TCRs. We used high resolution lattice lightsheet (LLS) and total internal reflection fluorescence (TIRF) imaging to visualize microvillar scanning in the context of variations in CAR design. This demonstrated that conventional CARs hyper-stabilized microvillar contacts relative to TCRs. Reducing the affinity and/or avidity of binding brought synapse microvillar dynamics into natural ranges, normalized synapse resolution and improved downstream effector function. This work highlights the importance of understanding the underlying cell biology when designing receptors for optimal antigen engagement. ### Competing Interest Statement K.T.R. is a cofounder, consultant, SAB member, and stockholder of Arsenal Biosciences. K.T.R. is on the SAB of Ziopharm Oncology and an Advisor to Venrock.