Library-based single-cell analysis of CAR signaling reveals drivers of in vivo persistence

Caleb R. Perez; Andrea Garmilla; Avlant Nilsson; Hratch M. Baghdassarian; Khloe S. Gordon; Louise G. Lima; Blake E. Smith; Marcela V. Maus; Douglas A. Lauffenburger; Michael E. Birnbaum

doi:10.1016/j.cels.2025.101260

Library-based single-cell analysis of CAR signaling reveals drivers of in vivo persistence
Journal article, 2025

The anti-tumor function of engineered T cells expressing chimeric antigen receptors (CARs) is dependent on signals transduced through intracellular signaling domains (ICDs). Different ICDs are known to drive distinct phenotypes, but systematic investigations into how ICD architectures direct T cell function—particularly at the molecular level—are lacking. Here, we use single-cell sequencing to map diverse signaling inputs to transcriptional outputs, focusing on a defined library of clinically relevant ICD architectures. Informed by these observations, we functionally characterize transcriptionally distinct ICD variants across various contexts to build comprehensive maps from ICD composition to phenotypic output. We identify a unique tonic signaling signature associated with a subset of ICD architectures that drives durable in vivo persistence and efficacy in liquid, but not solid, tumors. Our findings work toward decoding CAR signaling design principles, with implications for the rational design of next-generation ICD architectures optimized for in vivo function.

T cell signaling

persistence

tonic signaling

CAR T cells

pooled screens

intracellular signaling domains

chimeric antigen receptors

T cells

single-cell RNA sequencing

immunotherapy