Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT: Quantifying gene expression in individual cells can substantially improve our understanding about complex genetically engineered cell products such as chimeric antigen receptor (CAR) T cells. Here we designed a single-cell RNA sequencing (scRNA-seq) approach to monitor the delivery of a CD19-CAR gene via lentiviral vectors (LVs), i.e. the conventional VSV-LV and the CD8-targeted CD8-LV. LV exposed human donor PBMC were evaluated for a panel of 400 immune-response related genes including LV-specific probes. The resulting data revealed a trimodal expression for the CAR and CD8A demanding for a careful distribution-based identification of CAR T cells and CD8+ lymphocytes in scRNA-seq analysis. The fraction of T cells expressing high CAR levels was in concordance with flow cytometry results. More than 97% of the cells hit by CD8-LV expressed the CD8A gene. Remarkably, the majority of the potential off-target cells were in fact on-target cells resulting in a target cell selectivity of above 99%. Beyond that, the differential gene expression analysis revealed the upregulation of restriction factors in CAR-negative cells thus explaining their protection from CAR gene transfer. In summary, we provide a workflow and subsetting approach for scRNA-Seq enabling reliable distinction between transduced and untransduced cells during CAR T cell generation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE184895 | GEO | 2021/10/01

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

CAR gene delivery by T-cell targeted lentiviral vectors is enhanced by rapamycin induced reduction of antiviral mechanisms

Project description:Lentiviral vectors (LV) have become the dominant tool for stable gene transfer into lymphocytes including chimeric antigen receptor (CAR) gene delivery to T cells, a major breakthrough in cancer therapy. Yet, room for improvement remains, especially for the latest LV generations delivering genes selectively into T cell subtypes, a key requirement for in vivo CAR T cell generation. Towards improving gene transfer rates with these vectors, we conducted whole transcriptome analyses on human T lymphocytes after exposure to CAR-encoding conventional vector VSV-LV, and vectors targeted to CD8+ (CD8-LV) or CD4+ T cells (CD4-LV). Genes related to quiescence and antiviral restriction were found to be upregulated in CAR-negative cells exposed to all types of LVs. Down-modulation of various antiviral restriction factors including the interferon-induced transmembrane proteins (IFITMs) was achieved with rapamycin as verified by mass spectrometry (LC-MS). Strikingly, rapamycin enhanced transduction by up to 7-fold for CD8-LV and CD4-LV without compromising CAR T cell activities, but did not improve VSV-LV. When administered to humanized mice, CD8-LV resulted in higher rates of GFP gene delivery as well as faster in vivo CAR T cell generation and tumor control. The data favors multi-omics approaches for improvements in gene delivery.

2023-12-20 | GSE228824 | GEO

scRNA-seq of mouse thymocyte CD4 CD8 lineage choice and differentiation

Project description:CD4 and CD8 identify helper and cytotoxic T cell lineages, and serve as co-receptors for MHC-restricted TCR recognition. Despite decades of investigation, questions remain about how TCR specificity and co-receptor gene expression are matched during T cell repertoire selection. Here we address the order and logic of CD4/CD8 lineage choice and differentiation by scRNA-seq. Maturation, activation, and lineage specification emerged as principle components, in this order. Co-receptor gene expression uncovered waves of Cd4+Cd8a+, Cd4+Cd8a- and Cd4-Cd8a+ selection intermediates and revealed gene expression programs associated with each pattern, including TCR- and cytokine signaling genes, lineage markers and transcription factors. While in the unperturbed thymus early Cd4+Cd8a+ selection intermediates were followed first by CD4-like Cd4+Cd8a- and later by CD8-like Cd4-Cd8a+ selection intermediates, the order of co-receptor gene expression was not inherently fixed. These data provide a benchmark for models of CD4/CD8 lineage choice, and a rich resource for interrogating T cell differentiation.

2020-10-25 | GSE149207 | GEO

Targeting pathogenic CD8+ tissue-resident T cells with chimeric antigen receptor therapy in murine autoimmune cholangitis

Project description:Primary biliary cholangitis (PBC) is a cholestatic autoimmune liver disease secondary to an autoreactive T cell response against intrahepatic small bile ducts. Multiple murine models have demonstrated the critical role of effector CD8+T cells in this response and our work has used IL-12p40-/-IL-2Rα-/- mice (DKO mice) to study this issue. Herein we first demonstrated that use of either a CD8a knock-out or an anti-CD8a antibody prevents/reduces biliary immunopathology. Bulk and single-cell RNA sequencing analysis identifies CD8+ tissue resident memory T cells (Trm) in the liver of DKO mice, which highly express activation and cytotoxicity associated markers and have the ability to induce apoptosis of bile duct epithelial cells. Liver CD8+Trm cells also upregulate expression of several immune checkpoint molecules and in particular we identified PD-1 as a specific liver CD8+Trm cell marker in this model. Based on these data we constructed a novel chimeric antigen receptor containing a PD-L1 extracellular domain to target PD-1-expressing CD8+Trm cells. Importantly, treatment of DKO mice with PD-1 targeting CAR-T cells selectively depleted liver CD8+Trm cells in vivo and alleviated autoimmune cholangitis.

2023-09-05 | GSE241014 | GEO

Tumor cell in response to CAR T cell treatment

Project description:To examine the response of 4662 tumor cell on MSLN-CAR T cells. Series of genes of 4662 cells treated with the conditioned media (CM) from MSLN-CAR T cells and untransduced (UTD) T cells.

2024-05-07 | GSE266446 | GEO

Expression data from chimeric antigen receptor transduced (CAR) human CD4+ T cells during expansion

Project description:In this data set we include expression data from human CD4+ T cells isolated on day 0, 6, 11 and 24 follow anti-CD3/anti-CD28 magnetic bead stimulation and chimeric antigen receptor transduction. 30 samples were submitted. Samples represented three biological replicates of normal donors transduced with various CARs. CARs used were a cMet 28z specific CAR comprised of the IgG4 hinge, CD28 transmembrane and CD28 and CD3zeta intracellular domains. A CD19 CD28 CAR was specific to CD19, and was comprised of a CD8a hinge, CD28 transmembrane and CD28 and CD3zeta intracellular domain. A third CAR, the CD19 BBz, was used that was specific to CD19 was comprised of a CD8a hinge, CD8a transmembrane and 4-1BB and CD3zeta intracellular domains. Expression data was analyzied on day 0, 6, 11 and 24.

2015-01-14 | E-GEOD-64914 | biostudies-arrayexpress

A genome-scale gain-of-function CRISPR screen in CD8 T cells identifies proline metabolism as a means to enhance CAR-T therapy(Part 1)

Project description:Chimeric antigen receptor (CAR)-T cell-based immunotherapy for cancer and immunological diseases has made great strides, but it still faces multiple hurdles. Finding the right molecular targets to engineer T cells toward a desired function has broad implications for the armamentarium of T cell-centered therapies. Here, we developed a dead-guide RNA (dgRNA)-based CRISPR activation screen in primary CD8+ T cells, and identified gain-of-function (GOF) targets for CAR-T engineering. Targeted knock-in or overexpression of a lead target, PRODH2, enhanced CAR-T-based killing and in vivo efficacy in multiple cancer models. Transcriptomics and metabolomics in CAR-T cells revealed that augmenting PRODH2 expression re-shaped broad and distinct gene expression and metabolic programs. Mitochondrial, metabolic and immunological analyses showed that PRODH2 engineering enhances the metabolic and immune functions of CAR-T cells against cancer. Together these findings provide a system for identification of GOF immune boosters, and demonstrate PRODH2 as a target to enhance CAR-T efficacy.

2022-02-12 | ST002085 | MetabolomicsWorkbench

A genome-scale gain-of-function CRISPR screen in CD8 T cells identifies proline metabolism as a means to enhance CAR-T therapy(Part 2)

2022-02-14 | ST002084 | MetabolomicsWorkbench

scRNA-seq analysis of mouse splenic CD8 T cells 1 month post transfer of CD8 T cells from 3 months old donor into 24 months old recipient

Project description:Aging is associated with systemic chronic inflammation (inflammaging) that leads to impaired physiological functions and vulnerability to several diseases. However, underlying alterations in aged immune system resulting in gradual loss of immune fitness remain unclear. Using a combination of CD8 T cell transfer from young to old and from old to young mice and single-cell RNA sequencing, we characterized the age-associated alterations in CD8 T cells. We transferred 2 millions of purified CD8+ T cells pooled from 3 CD45.1 C57BL/6J 3 months old female mice (donor) by i.v. injection into CD45.2 C57BL/6J 24 months old female mouse (recipient) and sorted CD45.1+CD3e+CD8a+ and CD45.2+CD3e+CD8a+ T cells from the spleen 1 month post transfer to perform scRNA/TCR-seq.

2020-12-06 | GSE155005 | GEO

Gene expression analysis of thymocyte subsets

Project description:Mouse thymocytes can be classified into four major subsets based on expression of CD4 and CD8 co-receptors. CD4-CD8- (double negative, DN) cells become CD4+CD8+ (double positive, DP) cells following productive T cell receptor (TCR) beta chain rearrangement. A small proportion of DP cells are selected through interaction of clonal TCRalpha/beta and MHC self peptide complex expressed on thymic stromal cells. DP cell expressing MHC class I-restricted TCR become CD4-CD8+ cells, which will finally differentiate into cytotoxic T cells, while MHC class II restricted selection generates CD4+CD8- helper lineage T cells. We used microarrays to identify genes important for thymocyte differentiation and lineage determination by profiling gene expression in different thymocyte subsets. Mouse thymocytes were divided into four subsets based on CD4, CD8a, and TCRb expression and purified by flw cytometry. FACS purified DN (CD4-CD8a-TCRb-), DP (CD4+CD8a+), CD4SP (CD4+CD8a-TCRbhi) and CD8SP (CD4-CD8a+TCRbhi) populations were lysed in Trizol, and provided to the Genomics Core Facility of the Memorial Sloan-Kettering Cancer Center (MSKCC) for quality control, quantification, reverse transcription, labeling and hybridization to MOE430A 2.0 microarray chips (Affymetrix). Arrays were scanned per the manufacturer’s specifications for the Affymetrix MOE430v2 chip.

2011-08-09 | E-GEOD-31082 | biostudies-arrayexpress

Characterization of young and aged lung tissue-resident influenza nucleoprotein-specific CD8 memory T cells

Project description:To identify the disparaties of D^b-nucleoprotein specific resident memory CD8 T cells between young and aged mice, we pooled n=11 aged or n=18 young lungs together after intraveounously labeling T cells and sorted ivCD90- CD8a+ CD44hi CD69+ D^b-NP tetramer (PE+ & APC+) cells. By scRNA-seq, we found the aged population was missing a cluster in the young population that resembled typical functional resident memory CD8 T cells indicating that aged resident memory CD8 T cells of this specificity would have poor recall function.

2020-10-06 | GSE159065 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data