Single Cell Transcriptomic Characterization of CAR T-Cell Products Reveals Subpopulations, Stimulation and Exhaustion Signatures

Chimeric antigen receptor (CAR) T-cell adoptive therapy is set to transform the treatment of a rapidly expanding range of malignancies. Although the activation process of normal T cells is well characterized, comparatively little is known about the activation of cells via the CAR. Here we have used flow cytometry together with single cell transcriptome profiling to characterize the starting material (peripheral blood mononuclear cells –PBMCs-) and CAR therapeutic products of 3 healthy donors in the presence and absence of antigen specific stimulation. Bioinformatic analysis of the 57,676 single cell transcriptomes showed the CAR products to contain several subpopulations of cells, with the cellular composition reproducible from donor to donor, and all major cellular subsets compatible with CAR expression. Only 50% of CAR-expressing cells displayed transcriptional changes upon CAR-specific antigen exposure. The resulting molecular signature for CAR T-cell activation provides a rich resource for future dissection of the underlying mechanisms. Targeted data interrogation also revealed that a small proportion of antigen-responding CAR-expressing cells displayed an exhaustion signature, with both known markers and genes not previously associated with T-cell exhaustion. Comprehensive single cell transcriptomic analysis thus represents a powerful way to guide the assessment and optimization of clinical-grade CAR-T-cells, and inform future research into the underlying molecular processes. Overall design: To characterize CAR-T cell products