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CRISPR screen identifies genes that sensitize AML cells to double negative T cell therapy [RNA-Seq]

ABSTRACT: Here, we used a targeted CRISPR/Cas9 screen to identify genes that confer susceptibility of AML cells to DNT cell therapy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE157617 | GEO | 2020/12/14

REPOSITORIES: GEO

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CRISPR screen identifies genes that sensitize AML cells to double negative T cell therapy [guide RNA]

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CRISPR screen identifies genes that sensitize AML cells to double negative T cell therapy

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Double-negative T cell therapy alone or in combination with oral UDCA alleviates primary biliary cholangitis in mice

Project description:Methods: The DNT cells incubation with anti-CD3/CD28 antibodies were stimulated with UDCA (60uM) for 48 hours, then transcriptome sequencing studies were performed on DNT cells RNA.Transcriptome sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and sequenced on an Illumina Hiseq platform (Illumina, San Diego, CA). Sequences were aligned to the reference genome with TopHat and processed with Cufflinks, which quantifies each transcript in each sample using reference annotations produced by the University of California Santa Cruz UCSC. Differentially expressed genes with a fold change of >=1.41 and pvalue <= 0.05 between UDCA treated and control DNT cells were submitted to GO and KEGG enrichment analysis, which uses unbiased methods to assess pathway enrichment.

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Transcriptome landscape of double negative T cells by single-cell RNA sequencing

Project description:CD4 and CD8 coreceptor double-negative TCRαβ+ T (DNT) cells are increasingly being recognized for their critical and diverse roles in the immune system. However, their molecular and functional signatures remain poorly understood and controversial. We performed single-cell RNA sequencing on 28,835 single immune cells isolated from mixed splenocytes of mice using strict fluorescence-activated cell sorting. In this study, our analysis revealed five transcriptionally distinct naïve DNT clusters, which expressed unique sets of genes and mainly performed T helper, cytotoxic and innate immune functions. Anti-CD3/CD28 activation enhanced their T helper and cytotoxic functions. Moreover, by comparing with CD4+, CD8+ T cells and NK cells, we found Ly6c2 and Ikzf2 were highly expressed by naïve DNT cells. Upon activation, cytotoxic DNT subpopulation, had significantly higher Ikzf2 expression and protein expression of Ly-6C. Our results presented a comprehensive view of the transcriptional states of individual functional subset of DNT cells, highlighting the importance of DNT cells in immune surveillance, defense, and homeostasis.

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OX40 regulates double-negative regulatory T cells dependently of its effects on conversion and function

Project description:Methods: To better understand the role of OX40 in the regulation of double-negative regulatory T cells (DNT), OX40 deficiency and wild-type DNT converted from naïve CD4 T cells were compared in a transcriptome sequencing study. Total RNA was isolated from FACS-separated DNT. Transcriptome sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and sequenced on an Illumina Hiseq platform (Illumina, San Diego, CA). Sequences were aligned to the reference genome with TopHat and processed with Cufflinks, which quantifies each transcript in each sample using reference annotations produced by the University of California Santa Cruz UCSC. Differentially expressed genes with a fold change of >=2.0 and padj < 0.05 between OX40 deficiency and control DNT were submitted to GO and KEGG enrichment analysis, which uses unbiased methods to assess pathway enrichment. Results: Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that OX40 knockout are involved in regulatory function of DNT; including suppressive function, apoptosis, proliferation, and so on. Compared to control DNT, OX40 knockout DNT exhibited up-regulation of 52 genes and down-regulated of 46 genes. The most enriched GO term were Signal Transduction, Protein Binding, and Banding.

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Project description:Methods: The DNT cells incubation with CD3/CD28 antibodies were stimulated with IL-10 (50 ng/ml) for 24 hours, then transcriptome sequencing studies were performed on DNT cells RNA. Results: The distributions of up- (107) and downregulated (114) genes (≥ 1.5-fold difference, padj < 0.05) compared with without IL-10-treated DNT were depicted in a volcano plot. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that changed genes from IL-10-stimulated monocytes were involved in the immune response; inflammatory response; regulation of apoptotic and proliferation; cytolysis; cytokine-cytokine receptor interaction; and T cell receptor signaling pathway, among others. Compared with control DNT cells, IL-10-treated DNT exhibited downregulated levels of cell survival and activation genes (Bcl-xl, Cytc, Epcam, and Spi1), cell cytotoxicity genes (Nkp46, Klrg1, Slamf6, Cd107a, Cd161, and Cd226), immune responses genes (Lta, Cd127, H2-Oa, and H2-Dma), and upregulated cell exhaustion genes (Pd-1, Ctla4, and Tim3).In this study, we found IL-10 stimulated DNT cells highly expressed PI3K-AKT signaling genes, such as Jak3, Pik3ap1, Pik3cb, and Sgk1, and MAPK pathway genes, such as Stmn1, Fos, Kras, Nras, and Map3k5, which indicated that IL-10 upregulated NKG2A expression in DNT cells may through these pathways.

2020-11-11 | GSE134346 | GEO

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