Table 2 Overview of representative studies of single-cell sequencing in research of CAR-T Cell Studies
Disease | Sample | Method | Data and platform | Key points | Reference |
|---|---|---|---|---|---|
Pan-cancer | 18 tissues and organs from healthy human | scRNA-seq | Public data from GEO | Develop a comprehensive single-cell atlas for target antigens of CAR therapy in normal tissues and organs, which helps to capture antigen-expressing rare cell types missed in the assessment of bulk tissues | [216] |
Pan-cancer | Adult cell from HCL and AHCA | scRNA-seq | Two independent cohorts based on Microwell-seq and 10 × Genomics | Report a potential on-target, off-tumor toxicity landscape for CAR targets across a wide range of tissues Develop a user-friendly data portal, CAR target gene toxicity at single-cell level (CARTSC) | [217] |
AML | Single cells from 15 individuals with AML and tissue from 9 healthy individuals | scRNA-seq | Publicly available scRNA-seq data | Identify CSF1R and CD86 as targets for CAR-T cell therapy in AML Extensive in vitro and in vivo validation revealed broad expression on AML blasts, strong and durable treatment responses of newly developed CAR-T cells in vitro and in vivo and minimal toxicities toward relevant healthy cells and tissue | [221] |
B-NHL | Differently prepared CAR-T cells, infusion products, PBMCs from patients | scRNA-seq | 10 × Genomics Illumina | The electroporation method resulted in a high percentage of memory T cells in infusion products, and PD-1 interference enhanced antitumor immune functions, further validating the advantages of non-viral, PD-1-integrated CAR-T cell | [218] |
– | CAR-T cells | scATAC-seq | 10 × Genomics Illumina NextSeq 550 | Develop a method called EpiVIA for the joint profiling of the chromatin accessibility and lentiviral integration site analysis at the population and single-cell levels especially for CAR-T | [222] |
LBCL | CAR-T cells | scRNA-seq | 10 × Genomics Illumina HiSeq | Transcriptional signatures are related to costimulatory domains and signaling domains included in CARs uniquely shape the transcriptional programs of T cells | [219] |
R/R B-ALL | Infusion products | scRNA-seq, CITE-seq | scFTD-seq Illumina HiSeq4000 | Unveil heterogeneities of donor and patient CAR-T cells and provide mechanistic basis for ameliorating clinical outcomes and developing next-generation "off-shelf" allogeneic products | [35] |
– | CAR-T cells | scRNA-seq, scCAR-seq | 10 × Genomics Illumina NovaSeq | Generate a library of 180 unique CAR variants genomically integrated into primary human T cells by CRISPR-Cas9 Identify several variants with tumor killing properties and T cell phenotypes markedly different from standard CARs | [220] |
– | CAR-T cells | Single-cell, 16-plex cytokine profiling | Single-cell barcode chip | Reveal a diverse landscape of immune effector response of CD19 CAR-T cells to antigen-specific challenge Significant subsets of stimulated CAR-T cells exhibit high polyfunctionality with a dominant antitumor effector cytokine profile | [223] |
B cell malignancies | Pre-manufacture T cells from patients | scATAC-seq CITE-seq | 10 × Genomics Illumina Nova-Seq 6000 | Chronic interferon signaling regulated by IRF7 was associated with poor CAR-T cell persistence across T cell subsets, and the TCF7 regulon not only associates with the favorable naive T cell state, but is maintained in effector T cells among patients with long-term CAR-T cell persistence | [214] |
B cell lymphoma | BM cells from one tibia and one femur per mouse | scRNA-seq | 10 × Genomics Illumina HiSeq × 10 | Antitumor activity mediated by CAR-T cells largely relies on cellular cross-talk within the TME. Mechanistically, IFN-γ produced by CAR-T cells and sensed by the host was essential to boost the cytotoxic potential of CAR-T cells and of host NK and T cells. CAR4 and CAR8 T cells exhibited complementary functions, being more efficient at immune activation and tumor killing, respectively | [194] |
B-ALL | Pre-infusion products and post-infusion CD19-CAR-T cells from blood and bone marrow samples | scRNA-seq scTCR-seq | 10 × Genomics Illumina NovaSeq | Pre- and post-infusion CAR-T cells have distinct gene-expression profiles. Pseudotime identifies two distinct trajectories for post-infusion CAR-T cell differentiation: the first trajectory involves effector differentiation characterized by the expression of conventional cytotoxic genes; the other trajectory indicates the rapid development of these same exhaustion and cell death signatures soon after infusion | [187] |
B cell lymphoma | Pre-treatment and post-treatment PBMCs, infusion products | scRNA-seq scTCR-seq | 10 × Genomics Illumina NovaSeq S4 | Cellular dynamics of response differs between the two products: tisa-cel responses were associated with striking expansion of rare CD8+ central-memory-like populations from the IPs, whereas axi-cel treatment revealed less shifting of T cell lineages between IPs and post-treatment. CAR-Treg cells can suppress conventional CAR-T activity and thus facilitate relapse | [224] |
CLL | PBMCs before manufacturing, pre-infusion products and post-infusion CAR-T cells | scRNA-seq scTCR-seq CyTOF CITE-seq | 10 × Genomics Illumina NextSeq 550 Fluidigm Helios mass cytometer | Long-persisting CD4+ CAR-T cells exhibited cytotoxic characteristics along with ongoing functional activation and proliferation | [191] |
B cell malignancies | CD8+ CAR-T cells from infusion products and PB | scRNA-seq scTCR-seq | 10 × Genomics Illumina Hiseq 2500 | Clonal kinetics and transcriptional programs regulate the fate of CAR-T cell after infusion | [186] |
B-ALL | BM | scRNA-seq | 10 × Genomics Illumina NextSeq 500 | A Darwinian-like selection of preexisting CD19neg B-ALL cells is a mechanism for CD19neg B-ALL relapse after CAR-T cell therapy | [189] |
LBCL | Pre-treatment and post-treatment PB samples | scRNA-seq scTCR-seq CITE-seq | 10 × Genomics Illumina NovaSeq 6000 or HiSeq 4000 | Helios-expressing population of circulating CD4+ CAR-T cells on day 7 is associated with clinical progression and reduced neurotoxicity, and this population is non-clonal and manifests hallmark features of T regulatory cells. CAR-Treg cells and tumor burden surrogate can identify patients with clinical progression | [40] |
B-ALL | Infusion products | scRNA-seq CITE-seq | DropSeq Illumina HiSeq 4000 | A deficiency of T helper 2 function was associated with CD19-positive relapse compared with durable responders The frequency of early memory T cells, rather than activation or co-inhibitory signatures, could distinguish the relapse These findings were corroborated by independent functional profiling of 49 patients, and an integrative model was developed to predict the response | [225] |
NHL | CAR-T cells pre- and postinfusion | scRNA-seq combined with TotalSeq™-B antibodies | 10 × Genomics | The evolution of CAR-T cells was toward a non-proliferative, highly differentiated, and exhausted state. An enriched exhaustion profile in CAR-T cells of patients with poor response was marked by TIGIT expression | [158] |
MM | FACS sorted CAR-T cells from PB samples post treatment | scATAC-seq | 10 × Genomics Illumina NovaSeq 6000 | BATF and IRF4 are pivotal regulators in CAR-T cell exhaustion and reducing the expression of BATF or IRF4 had benefits to improve antitumor potency of CAR-T cells | [33] |
LBCL | Infusion products (axi-cel) | scRNA-seq scTCR-seq | 10 × Genomics Illumina HiSeq4000 | The heterogeneity in the cellular and molecular features of CAR-T cell infusion products contributes to variation in efficacy and toxicity after axi-cel therapy in LBCL, and that day 7 molecular response might serve as an early predictor of CAR-T cell efficacy A rare cell population with monocyte-like transcriptional features was associated with high-grade ICANS | [41] |
B cell malignancies | Human brain, lung and PBMC, mouse whole dissociated brain | scRNA-seq | Public data from GEO 10 × Genomics Illumina HiSeq 2500 | CD19, primarily considered as a B cell-specific surface antigen, is expressed in human brain mural cells that are critical for blood–brain-barrier integrity, suggesting that this cell population may contribute to the neurotoxicity of CD19-directed immunotherapy including CAR-T | [125] |
Richter-transformed DLBCL | Pre-treatment and post-treatment PB samples | scRNA-seq scTCR-seq | 10 × Genomics Illumina NovaSeq | Highlight the complex nature of CAR-T-related hematological toxicity and introduce oligoclonal CAR-T cell expansion as a potential contributing pathophysiologic mechanism | [226] |
B cell lymphoma | Infusion products | scRNA-seq | Public data from GSE150992 | Neurotoxicity is associated with decreasing cycling activity, amount of CAR + cells, and expression of cell cycle genes and exhaustion related genes | [227] |