Single-cell genomics links targeted functional manipulations to efficacy-associated chromatin signatures in CAR T cells.

Chimeric antigen receptor (CAR) T cells produce extraordinary remission rates in some hematologic tumors, results inconsistently replicated across malignancies and/or target antigens. Anti-tumor efficacy can be enhanced by modifying CAR architecture or T cell differentiation state. Pooled screening methods to identify effects of modifications are often restricted to in vitro readouts of abundance or transcriptome, limiting ability to interrogate biology and project long-term T cell fates. We use genetically-encoded barcodes to track the effects of diverse functional manipulations on pooled murine and human CAR T cell chromatin states using scATAC-seq. We report stable and transient transcription factor activities programmed by cytokine concentration during in vitro expansion and altered in vivo effector differentiation programs driven by modifications to CAR antigen binding domain construction. These data establish genetic barcoding to tie targeted functional manipulations to single CAR T cell chromatin profiles in vitro and in vivo, providing insights toward augmented therapeutic efficacy.