A Distinct Transcriptional Program in Human CAR T Cells Bearing the 4-1BB Signaling Domain Revealed by scRNA-Seq

T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have produced impressive outcomes for the treatment of B cell malignancies, but different products vary in kinetics, persistence, and toxicity profiles based on the co-stimulatory domains included in the CAR. In this study, we performed transcriptional profiling of bulk CAR T cell populations and single cells to characterize the transcriptional states of human T cells transduced with CD3ζ, 4-1BB-CD3ζ (BBζ), or CD28-CD3ζ (28ζ) co-stimulatory domains at rest and after activation by triggering their CAR or their endogenous T cell receptor (TCR). We identified a transcriptional signature common across CARs with the CD3ζ signaling domain, as well as a distinct program associated with the 4-1BB co-stimulatory domain at rest and after activation. CAR T cells bearing BBζ had increased expression of human leukocyte antigen (HLA) class II genes, ENPP2, and interleukin (IL)-21 axis genes, and decreased PD1 compared to 28ζ CAR T cells. Similar to previous studies, we also found BBζ CAR CD8 T cells to be enriched in a central memory cell phenotype and fatty acid metabolism genes. Our data uncovered transcriptional signatures related to costimulatory domains and demonstrated that signaling domains included in CARs uniquely shape the transcriptional programs of T cells. Maus, Regev, and colleagues performed a deep transcriptional analysis of CAR T cells bearing different signaling domains, at rest and after activation. Findings include CAR-specific signatures and a distinct program activated in CARs with 4-1BB domains that was characterized by expression of central memory markers, MHC class II, and IL-21, among others