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

Boroughs, Angela C.; Larson, Rebecca C.; Marjanovic, Nemanja D.; Gosik, Kirk; Castaño, Ana P.; Porter, Caroline; Lorrey, Selena; Ashenberg, Orr; Jerby, Livnat; Hofree, Matan; Smith-Rosario, Gabriela; Morris, Robert; Gould, Joshua; Riley, Lauren S.; Berger, Trisha R.; Riesenfeld, Samantha J.; Rozenblatt-Rosen, Orit; Choi, Bryan D.; Regev, Aviv; Maus, Marcela V.

doi:10.1016/j.ymthe.2020.07.023

Cited by 73 publications

(91 citation statements)

References 64 publications

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“…Our results show that the CAR product is heterogeneous and that cell cycle and predicted memory status are important factors to define this heterogeneity, in line with previous reports. 8,17 Interestingly, we find that this heterogeneity is present in all donors although at different levels. We also corroborated that all major subpopulations present after T-cell culture were susceptible to CAR transduction as reported before, 18 and moreover found that approximately 7% of CAR-transduced cells already presented an activation signature before antigen-specific stimulation.…”

Section: Discussionmentioning

confidence: 61%

“…This analysis highlighted the presence of eleven clusters ( Figure 2(a) , Table 1 ). We next investigated the nature of the defined clusters using the expression of known T-cell subpopulation marker genes such as CD8A, CD4, CCR7 , and SELL (CD62L) 8 ( Figure 2(b,c) , Supplementary Figures 2A and 2B) and inferred the probability for each cell to stay in a particular cell cycle stage ( Figure 2(d) and Supplementary Figure 2E) using a list of cell cycle genes previously defined. 9 CD8A expression was strongly detected in clusters 9 and 10 whereas CD4 expression was detected in the rest of the clusters.…”

Section: Resultsmentioning

confidence: 99%

“…The expression of CCR7 and SELL has been used in the past to identify different cell types in datasets generated using this technology. 8 The bioinformatic pipeline devised here detects CCR7 expression at the same proportion as identified by flow cytometry thus providing independent validation. The memory status of T-cells before CAR production is thought to represent a key parameter when trying to predict the downstream performance of the therapeutic product.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Discussionmentioning

confidence: 61%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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“…Integrative analysis of scRNA-seq dataset from literature The aligned scRNA-seq data from two 4-1BB CAR T donor samples were selected from Boroughs et al's 11 published work with SRA database accession number PRJNA554339. In their experimental setting, the CAR T cells were generated by transduction with a CD19 targeting 4-1BB-CD3ξ construct in a way similar to our CAR-T cell products.…”

Section: Library Preparation and Sequencingmentioning

confidence: 99%

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

Bai

Lundh

Kim

et al. 2021

J Immunother Cancer

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BackgroundAutologous T cells engineered to express a chimeric antigen receptor (CAR) specific for CD19 molecule have transformed the therapeutic landscape in patients with highly refractory leukemia and lymphoma, and the use of donor-generated allogeneic CAR T is paving the way for further breakthroughs in the treatment of cancer. However, it remains unknown how the intrinsic heterogeneities of these engineered cells mediate therapeutic efficacy and whether allogeneic products match the effectiveness of autologous therapies.MethodsUsing single-cell mRNA sequencing in conjunction with CITE-seq, we performed multiomics characterization of CAR T cells generated from healthy donor and patients with acute lymphoblastic leukemia. CAR T cells used in this study were manufactured at the University of Pennsylvania through lentiviral transduction with a CD19-4-1BB-CD3ζ construct. Besides the baseline condition, we engineered NIH-3T3 cells with human CD19 or mesothelin expression to conduct ex vivo antigen-specific or non-antigen stimulation of CAR T cells through 6-hour coculture at a 1:1 ratio.ResultsWe delineated the global cellular and molecular CAR T landscape and identified that transcriptional CAR tonic signaling was regulated by a mixture of early activation, exhaustion signatures, and cytotoxic activities. On CD19 stimulation, we illuminated the disparities of CAR T cells derived from different origins and found that donor CAR T had more pronounced activation level in correlation with the upregulation of major histocompatibility complex class II genes compared with patient CAR T cells. This finding was independently validated in additional datasets from literature. Furthermore, GM-CSF(CSF2) expression was found to be associated with functional gene productions, but it induced little impact on the CAR T activation.ConclusionsThrough integrated multiomics profiling and unbiased canonical pathway analyses, our results unveil heterogeneities in the transcriptional, phenotypic, functional, and metabolic profiles of donor and patient CAR T cells, providing mechanistic basis for ameliorating clinical outcomes and developing next-generation ‘off- the-shelf’ allogeneic products.

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“…37,[45][46][47][48][49] CD28 and 41BB co-stimulation in the context of CAR T cells has been extensively studied, including detailed phosphoproteomic and single-cell RNA sequencing (RNA-seq) analyses. [50][51][52] They activate different pathways within T cells, with CD28 signaling promoting glycolytic metabolism and an effector memory phenotype, in contrast to 41BB signaling, which induces oxidative metabolism and a central memory phenotype. 53 Depending on the number of co-stimulatory molecules included in the CAR signaling domain, CARs are either designated second (one domain) or third (two domains) generation.…”

Section: Design Of Carsmentioning

confidence: 99%

CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?

et al. 2020

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

Cited by 73 publications

References 64 publications

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

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

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

CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?

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