Boroughs and Larson et al. transduced human T cells with CARs comprising a CD19 or EGFR scFV and a CD3ζ, 4-1BB-CD3ζ (BBζ) or CD28-CD3ζ (28ζ) costimulatory domain. Bulk and single-cell RNAseq showed that resting and activated CAR T cells made similar, but weaker responses when induced via their CAR versus by anti-CD3. Cell activation amplified the tonic signaling profiles shared among CD3ζ CAR T cells that were distinct from those shared among BBζ CAR T cells. A Th1-polarizing and a TCM CCR7+ gene profile, and MHC-II, IL-21, and IL-21R, but not PD-1 expression were increased in BBζ compared to 28ζ CAR T cells, which had increased Th2-polarizing genes.
Contributed by Paula Hochman
ABSTRACT: 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.