RNA sequencing facilitates quantitative analysis of 28ζ CAR, BBζ CAR and aTCR T cell transcriptomes
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE189932 | GEO | 2022/04/30
REPOSITORIES: GEO
Similar Datasets
Project description:Purpose: Next-generation sequencing (NGS) has been utilized for systems-based analysis of all liver samples. The goals of this study are to use NGS-derived mouse CAR and human CAR initiated transcriptome profiling (RNA-seq) and find out similarity and difference drug processing gene (DPG) pattern after CAR activation in different genotype include WT (C57BL/6 and human CAR transgenic mice with C57BL/6 background) Methods: Liver mRNA profiles of wild-type (WT) and human CAR knockin (hCAR-TG) mice at the age of day 5 and day 60 treated with mouse CAR activator (TCPOBOP) and human CAR activator (CITCO) respectively were generated by deep sequencing, in triplicate, using HiSeq 2000 sequencer. The sequence reads that passed quality filters were analyzed at the transcript level with followed method: HISAT followed by Cufflinks. Results: Using an optimized data analysis workflow,RNA-Seq generated approximately 47 to 68 million reads per sample, among which approximately 40 to 60 million reads were uniquely mapped to the mouse reference genome (NCBI GRCm/38/mm10). And we identified 393 drug processing genes in the livers of WT and hCAR-TG with with HISAT workflow. RNA-seq data confirmed that among all the 393 DPGs with known important functions in xenobiotic biotransformation, 90 DPGs were not expressed in livers of any groups (threshold: average FPKM < 1 in all treatment groups); whereas a total of 303 genes were expressed in livers of at least one groups, among which 258 DPGs were differentially regulated by mCAR or hCAR activation in either Day 5 or Day 60 (FDR-BH<0.05), and 45 genes were stably expressed among all treatment groups. Conclusions: Our study represents the first detailed analysis of drug processing genes, with 3 biologic replicates, generated by RNA-seq technology. The optimized data analysis reported here should provide a framework for comparative investigations of expression profiles by mouse CAR activation and human CAR activation. Our results show that NGS offers a comprehensive and accurate quantitative and qualitative evaluation of mRNA content within tissues.
2017-05-09 | GSE98666 | GEO
Project description:The study aimed to elucidate the transcriptional changes introduced by genetic engineering of CAR T cells to stably express the homeostatic chemokine receptor CCR7. In vitro killing assays hinted to a higher cytolytic capacity that could be tied to an altered cytoskeletal rearrangement. Human CAR and CAR.CCR7 T cells were generated by retroviral transduction and enriched by FACS at the end of the expansion phase (day 10), either without or with a previous over-night co-culture with target Jeko1 tumor cells that express the CAR target antigen CXCR5. Enriched cells were immediately processed for bulk mRNA Seq
2023-06-30 | E-MTAB-12778 | biostudies-arrayexpress
Project description:Adoptive transfer of chimeric antigen receptor (CAR)-T cells is expected to become the first line of treatment for multiple malignancies, following the enormous success of anti-CD19 therapies. However, their mechanism of action is not fully understood, and clear guidelines for the design of safe and efficient receptors are missing. We hereby describe a systematic analysis of the CAR “signalosome” in human primary T cells. Two CAR designs were compared: a second-generation (PSCA2) and a third-generation (PSCA3) anti-PSCA CAR. Phosphorylation events triggered by CAR-mediated recognition of target cells were quantified by mass spectrometry.
2019-11-07 | PXD007086 | Pride
Project description:CAR-T cell therapy against MM currently shows promising results, but usually with serious toxicities. CAR-NK cells may exert less toxicity when redirected against resistant myeloma cells. CARs can be designed through the use of receptors, such as NKG2D, which recognizes a wide range of ligands to provide broad target specificity. Here, we test this approach by analyzing the anti-tumor activity of activated and expanded NK cells (NKAE) and CD45RA- T cells from MM patients that were engineered to express an NKG2D-based CAR. NKAE cells were cultured with irradiated Clone9.mbIL21 cells. Then, cells were transduced with an NKG2D-4-1BB-CD3z-CAR. CAR-NKAE cells exhibited no evidence of genetic abnormalities. Although memory T cells were more stably transduced, CAR-NKAE cells exhibited greater in vitro cytotoxicity against MM cells, while showing minimal activity against healthy cells. In vivo, CAR-NKAE cells mediated highly efficient abrogation of MM growth, and two of the treated mice remained disease-free. Overall, these results demonstrate that it is feasible to modify autologous NKAE cells from MM patients to safely express a NKG2D-CAR. Additionally, autologous CAR-NKAE cells display enhanced anti-myeloma activity demonstrating that they could be an effective strategy against MM supporting the development of NKG2D-CAR NK cell therapy for MM.
2021-08-16 | GSE179408 | GEO
Project description:This dataset is a four-ligand x three-genotype Affymetrix microarray analysis of the regulation of liver genes in the mouse by the constitutive androstane receptor (CAR). 24 female mice of mixed background (C57BL/6x129Sv) were divided into three groups: wild-type (contains only mouse CAR; mCAR), CAR.KO = knockout mice (mice ablated for mCAR gene; mCAR -/-), and CAR.AH= contains human CAR transgene under the control of the mouse albumin promoter in the mCAR -/- background. Each of the three groups underwent four different treatment regimens: CO = corn oil vehicle control, PB = phenobarbitol (100 mg/kg/day), an anti-convulsant agent which can translocate both mCAR and hCAR into the nucleus to turn on target gene expression, TC = TCPOBOP, a potent non-metabolized ligand of mCAR (3 mg/kg), CITCO = a hCAR specific ligand (30 mg/kg/day). Two mice were used per treatment group and each mouse RNA was used for one chip.
2012-08-14 | E-GEOD-40120 | biostudies-arrayexpress
Project description:Adoptive transfer of chimeric antigen receptor (CAR)-T cells is expected to become the first line of treatment for multiple malignancies, following the enormous success of anti-CD19 therapies. However, their mechanism of action is not fully understood, and clear guidelines for the design of safe and efficient receptors are missing. We hereby describe a systematic analysis of the CAR “interactome” in human primary T cells, which allowed us to identify molecular traits that influence CAR-T cell efficacy. Interactome analysis was based on immunoprecipitation of CARs followed by protein identification by mass spectrometry.
2019-11-07 | PXD007085 | Pride
Project description:Purpose: The 10x Genomics Visium platform allows us to define the spatial topography of gene expression and provides detailed molecular maps that overcome limitations associated with sn/scRNA-seq and microscopy-based spatial transcriptomics methods. The goals of this study are to compare and identify unique transcriptome profiling (RNA-seq) signature between unfavorable and favorable Wilms Tumors and against human fetal kidney. Methods: Human fetal kidney and Wilms tumor spatial topography of gene expression were generated using the 10X Visium platform Results: Using an optimized data analysis workflow, we mapped the reads to the hg38 genome build and grouped the spots into 9 clusters based on gene expression profiles. Conclusion: Our study represents the first implement of Visium technology in human fetal kidney and Wilms Tumor tissue, providing a number of important functional insights about the spatial and molecular definitions of cell populations across human fetal kidney and different subtypes of Wilms Tumor through analyzing gene expression within the intact spatial organization of the human samples.
2023-07-01 | GSE178349 | GEO
Project description: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.
2021-10-01 | GSE184895 | GEO
Project description:To characterize transfer of molecules from target cells into CAR T cells via trogocytosis we cultured NALM-6 leukemia cell line expressing a CD19-mCherry fusion protein with CAR T cells. NALM6-CD19-mCherry were loaded with heavy amino acid and cocultured with CAR T cells for 1 hour. CAR T cells were next sorted into two fractions, mCherry-positive (TrogPos), and -negative (TrogNeg). Proteomics analysis revealed the presence of targeted antigen (CD19) in the TrogPos only.
2019-03-29 | PXD012833 | Pride
Project description:An obstacle with continued clinical development of CAR T cells is the limited understanding of CAR T cell biology and its mechanisms of anti-tumor immunity. We and others have shown that CARs with a CD28 co-stimulatory domain drive high levels of T cell activation that also lead to exhaustion and shortened persistence. This led us to hypothesize that by incorporating null mutations of CD28 subdomains (YMNM, PRRP, or PYAP) we could optimize CAR T cell signaling and reduce exhaustion. In vivo we found that mice given CAR T cells with a PYAP CD28 endodomain had a significant survival advantage. We also observed that mutant CAR T cells had significantly less expression of PD1 and were increased in the bone marrow and spleen. In addition, these mutant CAR T cells had optimized signaling resulting in a reduction of exhaustion related transcription factors and genes. Our results demonstrate that CAR T cells with a mutant CD28 endodomain have better survival and decreased exhaustion and is the result of decreased CAR dependent NFAT/NR4A1 transcription factors. This work allows for development of enhanced CAR T cell therapies by optimizing CAR T cell signaling.
2020-12-16 | GSE142506 | GEO