Single-cell transcriptome analysis of CAR T-cell products reveals subpopulations, stimulation, and exhaustion signatures

Abstract

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) and CAR therapeutic products of 3 healthy donors in the presence and absence of antigen-specific stimulation. Analysis of 53,191 single-cell transcriptomes showed APRIL-based CAR products to contain several subpopulations of cells, with 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 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.

Monitoring CAR T cell generation with a CD8-targeted lentiviral vector by single-cell transcriptomics

“…These findings suggest that under the given experimental conditions exposure of T cells to LV particles results in stronger gene expression profile alterations than presence of the CAR. While CD8+ CAR high cells exhibited an activated TH1 phenotype and an overall profile well in accordance with that observed in previous studies ( Figure 4 ), 16 , 17 , 20 CD8 CAR neg/low cells expressed genes that potentially restricted cell viability, phenotype, and viral entry. These cells had upregulated genes involved in inhibition of T cell activation ( PIK3IP1 ) and proliferation ( CD37, BTG1 ) as well as promoting pyroptosis ( CASP5 ), a type of programmed cell death resulting in inflammatory cytokine release ( Figures 4 A, 4D, and 4H).…”

“…Notably, previous studies have not described this. 16 , 17 , 18 , 19 , 20 Since the expression profiles of CAR low and CAR neg cells were basically identical, they were merged for the downstream analyses ( Figure S2 A). The situation appears more complicated with the CD8A low population.…”

“…These studies focused mainly on the diversity of CAR T cell phenotypes in pre-infusion products and correlated these to activities in patients 16 , 17 , 18 or investigated the consequences of different CAR signaling domains and antigenic stimulation. 19 , 20 …”

Single-cell multiomics dissection of basal and antigen-specific activation states of CD19-targeted CAR T cells

“…With the advent of revolutionized single-cell mRNA sequencing (scRNA-seq), it is now possible to characterize the full spectrum of immune cell functional states and gene programs in a comprehensive and unbiased manner. Although not fully used, several reports have leveraged scRNA-seq to compare the transcriptional states of 4-1BB and CD28 CAR T cells, 11 to understand the clonal kinetics and transcriptional programs preinfusion and postinfusion, 12 to identify pre-existing CD19-negative subclones, 13 to reveal subpopulations, stimulation, and exhaustion signatures, 14 and to track dynamic development of CAR T cell dysfunction in clinical samples. 15 Yet despite some transcriptomic features have been identified associated with the clinical observations, 16 17 how the constitutional properties of these cellular products mediate therapeutic activities is incompletely understood.…”

Single-Cell Analysis Technologies for Immuno-Oncology Research: From Mechanistic Delineation to Biomarker Discovery

“…They have also found an enrichment of central memory cell phenotype and fatty acid metabolism in CD8 + 4-1BB CAR-T cells. In another large-scale single-cell transcriptomic analysis of third-generation CAR-T cells that target both B cell maturation antigen (BCMA) and transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI), Wang et al have found that CAR products from three different donors exhibit a similar cellular composition, the manufacturing process of CAR-T cells induces effector-like transcriptional signatures, and the CAR-specific antigen exposure activates similar pathways as those triggered by TCR [37] .…”