Dissecting the role of CAR signaling architectures on T cell activation and persistence using pooled screens and single-cell sequencing.

Chimeric antigen receptor (CAR) T cells offer a promising cancer treatment, yet challenges such as limited T cell persistence hinder efficacy. Given its critical role in modulating T cell responses, it is crucial to understand how the CAR signaling architecture influences T cell function. Here, we designed a combinatorial CAR signaling domain library and performed repeated antigen stimulation assays, pooled screens, and single-cell sequencing to systematically investigate the impact of modifying CAR signaling domains on T cell activation and persistence. Our data reveal the predominant influence of membrane-proximal domains in driving T cell phenotype. Notably, CD40 costimulation was crucial for fostering robust and lasting T cell responses. Furthermore, we correlated in vitro generated CAR T cell phenotypes with clinical outcomes in patients treated with CAR T therapy, establishing the foundation for a clinically informed screening approach. This work deepens our understanding of CAR T cell biology and may guide future CAR engineering efforts.