A single-cell approach to engineer CD8+ T cells targeting cytomegalovirus

Wang, Fei; Xu, Qumiao; Zhuang, Zhenkun; Li, Ziyi; Gao, Qianqian; Huang, Yaling; Luo, Yonglun; Zhang, Xiuqing; Zhu, Linnan; Chao, Cheng‐Chi

doi:10.1038/s41423-020-0466-z

Cited by 5 publications

(8 citation statements)

References 9 publications

(10 reference statements)

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“…The approaches for porcine PBMC isolation were based on the human PBMC isolation protocol in our previous study 102 . In brief, 10 mL of pig blood sample in a citrated vial was gently inverting to mix well.…”

Section: Methodsmentioning

confidence: 99%

Endothelial cell heterogeneity and microglia regulons revealed by a pig cell landscape at single-cell level

et al. 2022

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Pigs are valuable large animal models for biomedical and genetic research, but insights into the tissue- and cell-type-specific transcriptome and heterogeneity remain limited. By leveraging single-cell RNA sequencing, we generate a multiple-organ single-cell transcriptomic map containing over 200,000 pig cells from 20 tissues/organs. We comprehensively characterize the heterogeneity of cells in tissues and identify 234 cell clusters, representing 58 major cell types. In-depth integrative analysis of endothelial cells reveals a high degree of heterogeneity. We identify several functionally distinct endothelial cell phenotypes, including an endothelial to mesenchymal transition subtype in adipose tissues. Intercellular communication analysis predicts tissue- and cell type-specific crosstalk between endothelial cells and other cell types through the VEGF, PDGF, TGF-β, and BMP pathways. Regulon analysis of single-cell transcriptome of microglia in pig and 12 other species further identifies MEF2C as an evolutionally conserved regulon in the microglia. Our work describes the landscape of single-cell transcriptomes within diverse pig organs and identifies the heterogeneity of endothelial cells and evolutionally conserved regulon in microglia.

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Section: Methodsmentioning

confidence: 99%

Endothelial cell heterogeneity and microglia regulons revealed by a pig cell landscape at single-cell level

et al. 2022

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“…To sort MART-1 27-35 -specific T cells, CD8 + CTLs were labeled with FITC-conjugated anti-CD8-antibody (BD biosciences, USA) and APC-conjugated MART-1 27-35 -MHC tetramers. Tetramers were generated via UV light-induced cleavage of MHC-bound ligands from Flex-T™ monomer UVX (Biolegend, USA) as described previously ( 56 ). To sort exogenous TCR-positive cells, TCR-Ts were labeled with APC-conjugated anti-mouse TCRβ-antibody (Biolegend, USA).…”

Section: Methodsmentioning

confidence: 99%

“…scRNA and V(D)J sequencing were performed as described previously ( 56 , 57 ). Briefly, cell suspensions of sorted MART-1 27-35 -specific T cells were loaded onto a GemCode Single-Cell instrument (10x Genomics, Pleasanton, CA) to generate a single-cell emulsion.…”

Section: Methodsmentioning

confidence: 99%

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Single-Cell Transcriptomics Reveals Killing Mechanisms of Antitumor Cytotoxic CD4+ TCR-T Cells

Liang

Liu

et al. 2022

Front. Immunol.

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T cell receptor-engineered T cells (TCR-Ts) have emerged as potent cancer immunotherapies. While most research focused on classical cytotoxic CD8+ T cells, the application of CD4+ T cells in adoptive T cell therapy has gained much interest recently. However, the cytotoxic mechanisms of CD4+ TCR-Ts have not been fully revealed. In this study, we obtained an MHC class I-restricted MART-127-35-specific TCR sequence based on the single-cell V(D)J sequencing technology, and constructed MART-127-35-specific CD4+ TCR-Ts and CD8+ TCR-Ts. The antitumor effects of CD4+ TCR-Ts were comparable to those of CD8+ TCR-Ts in vitro and in vivo. To delineate the killing mechanisms of cytotoxic CD4+ TCR-Ts, we performed single-cell RNA sequencing and found that classical granule-dependent and independent cytolytic pathways were commonly used in CD4+ and CD8+ TCR-Ts, while high expression of LTA and various costimulatory receptors were unique features for cytotoxic CD4+ TCR-Ts. Further signaling pathway analysis revealed that transcription factors Runx3 and Blimp1/Tbx21 were crucial for the development and killing function of cytotoxic CD4+ T cells. Taken together, we report the antitumor effects and multifaceted killing mechanisms of CD4+ TCR-Ts, and also indicate that MHC class I-restricted CD4+ TCR-Ts could serve as potential adoptive T cell therapies.

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“…The genes encoding TCR variable regions from single-cell sequencing were fused with murine TCR-α and -β2 constant regions respectively for reducing the effects of TCR mispairing. The TCR-α and -β chains were linked by a T2A self-cleaving peptide for optimal TCR expression as previously described( 35 ). TCR α/β sequences were codon-optimized, synthesized (GenScript), and cloned into the T7-EGFP vector (Addgene).…”

Section: Methodsmentioning

confidence: 99%

Multi-omics analyses of single cell-derived colorectal cancer organoids reveal intratumor heterogeneity and immune response diversity

Yue

et al. 2022

Preprint

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Colorectal cancer (CRC) organoids have similar genomic and functional characteristics to the original specimens. They have become a novel and promising tumor model. However, systematic research of heterogeneity and evolution based on tumor organoids and unbiased evaluation of CD4+ T cell-mediated tumor recognition is presently lacking. Here, we study the variability in genomic characterization and single-cell transcriptome between different tumor organoid clones derived from the same CRC patient. While clone-specific differences in driver gene mutations and cancer stem cell (CSC) subpopulations were observed, inter-clone heterogeneity was even more prevalent. Using single-cell RNA sequencing, we found that compared with bulk organoids, CRC organoid clones are mainly composed of cancer stem cells and transiently-amplifying cells (TACs), but no epithelial-mesenchymal transition (EMT)-like (S100A4+CDH1-) cells. In addition, we study the phenotypic characteristics and TCR properties of tumor-reactive CD4+ T cells by coupling the organoid-TILs co-culture model, single-cell profiling, and recognition testing of candidate TCRs. We found that the proliferating CD4+ T cells increased significantly from 7.1% to 35.4%, and the TCR clonotypes increased from 110 to 1043 after co-culture with organoids. Moreover, 4 of 10 (40%) candidate TCRs with a significant increase in clone abundance exhibited tumor recognition. These results provide evidence that the proliferating CD4+ T cells with clonal expansion after co-culture with tumor organoids are the potential tumor-reactive T cells. Collectively, we reconstructed intra-patient heterogeneity in single cell-derived organoid models and characterized the phenotypic characteristics and functional properties of tumor-reactive CD4+ T cells.

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A single-cell approach to engineer CD8+ T cells targeting cytomegalovirus

Cited by 5 publications

References 9 publications

Endothelial cell heterogeneity and microglia regulons revealed by a pig cell landscape at single-cell level

Endothelial cell heterogeneity and microglia regulons revealed by a pig cell landscape at single-cell level

Single-Cell Transcriptomics Reveals Killing Mechanisms of Antitumor Cytotoxic CD4+ TCR-T Cells

Multi-omics analyses of single cell-derived colorectal cancer organoids reveal intratumor heterogeneity and immune response diversity

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