Project description:To investigate miRNA expressions of miRNA upon activation. In vitro-differentiated human NK cells were freshly incubated in the presence of IL15 after 24h deprivation of IL-15. The cells were harvested at the times (0h, resting-Sample 1 and 2; 6h, activated-Sample 3 and 4) and analyzed by microarray.
Project description:Natural killer (NK) cells are innate lymphocytes important for early host defense against infectious pathogens and surveillance against malignant transformation. Resting murine NK cells regulate the translation of effector molecule mRNAs (e.g. granzyme B, GzmB) through unclear molecular mechanisms. MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate the translation of their mRNA targets, and are therefore candidates mediating this control process. While the expression and importance of miRNAs in T and B lymphocytes has been established, little is known about miRNAs in NK cells. Here, we utilized two next-generation sequencing (NGS) platforms to define the miRNA transcriptomes of resting and cytokine-activated primary murine NK cells, with confirmation by RT-qPCR and microarrays. We delineate a bioinformatics analysis pipeline that identified 302 known and 28 novel mature miRNAs from sequences obtained from NK cell small RNA libraries. These miRNAs are expressed over a broad range, exhibit isomiR complexity, and a subset is differentially expressed following cytokine-activation. Using this miRNA NGS data, miR-223 was identified as a mature miRNA present in resting NK cells with decreased expression following cytokine-activation. Further, we demonstrate that miR-223 specifically targets the 3’UTR of murine GzmB in vitro, indicating that this miRNA may contribute to control of GzmB translation in resting NK cells. Thus, the sequenced NK cell miRNA transcriptome provides a valuable framework for further elucidation of miRNA expression and function in NK cell biology. Illumina GA (SRR036363, SRR036364) and SOLiD (SRR036206, SRR036210) sequencing data have been submitted to the NCBI Sequence Read Archive (SRA). The study uses a custome made array to characterize miRNA of activated and resting murine splenic natural killer cells
Project description:<p>Natural killer (NK) cells are forced to cope with different oxygen environments even under resting conditions. The adaptation to low oxygen is regulated by oxygen-sensitive transcription factors, the hypoxia-inducible factors (HIFs). The function of HIFs for NK cell activation and metabolic rewiring remains controversial. Activated NK cells are predominantly glycolytic, but the metabolic programs that ensure the maintenance of resting NK cells are enigmatic. By combining <em>in situ</em> metabolomic and transcriptomic analyses in resting murine NK cells, our study defines HIF-1a as a regulator of tryptophan metabolism and cellular nicotinamide adenine dinucleotide (NAD+) levels. The HIF-1a/NAD+ axis prevents ROS production during oxidative phosphorylation (OxPhos) and thereby blocks DNA damage and NK cell apoptosis under steadystate conditions. In contrast, in activated NK cells under hypoxia, HIF-1a is required for glycolysis, and forced HIF-1a expression boosts glycolysis and NK cell performance <em>in vitro</em> and <em>in vivo</em>. Our data highlight two distinct pathways by which HIF-1a interferes with NK cell metabolism. While HIF-1a-driven glycolysis is essential for NK cell activation, resting NK cell homeostasis relies on HIF-1a-dependent tryptophan/NAD+ metabolism.</p><p><br></p><p><strong>Linked cross omic data sets:</strong></p><p>RNA-seq data associated with this study are available in ArrayExpress (BioStudies): accession <a href='https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-12082' rel='noopener noreferrer' target='_blank'>E-MTAB-12082</a>.</p>
Project description:Natural killer (NK) cells are innate lymphocytes important for early host defense against infectious pathogens and surveillance against malignant transformation. Resting murine NK cells regulate the translation of effector molecule mRNAs (e.g. granzyme B, GzmB) through unclear molecular mechanisms. MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate the translation of their mRNA targets, and are therefore candidates mediating this control process. While the expression and importance of miRNAs in T and B lymphocytes has been established, little is known about miRNAs in NK cells. Here, we utilized two next-generation sequencing (NGS) platforms to define the miRNA transcriptomes of resting and cytokine-activated primary murine NK cells, with confirmation by RT-qPCR and microarrays. We delineate a bioinformatics analysis pipeline that identified 302 known and 28 novel mature miRNAs from sequences obtained from NK cell small RNA libraries. These miRNAs are expressed over a broad range, exhibit isomiR complexity, and a subset is differentially expressed following cytokine-activation. Using this miRNA NGS data, miR-223 was identified as a mature miRNA present in resting NK cells with decreased expression following cytokine-activation. Further, we demonstrate that miR-223 specifically targets the 3’UTR of murine GzmB in vitro, indicating that this miRNA may contribute to control of GzmB translation in resting NK cells. Thus, the sequenced NK cell miRNA transcriptome provides a valuable framework for further elucidation of miRNA expression and function in NK cell biology. Illumina GA (SRR036363, SRR036364) and SOLiD (SRR036206, SRR036210) sequencing data have been submitted to the NCBI Sequence Read Archive (SRA).
Project description:Activated but not resting Tregs affect NK cell differentiation. We used microarray analysis to study differences of gene expression of NK cell differentiation in the presence of resting or activated Tregs.
Project description:Resting NK cells are notoriously difficult to transduce, especially using the VSVG lentivirus pseudotype. When NK cells are expanded and activated using the NKAES system, there is a modest improvement of transduction rates compared to their resting NK counterparts. Still, the transduction efficiency remains poor for robust development of NK cell-based cancer immunotherapy. We envisaged to have a genome-wide expression profile of resting and activated NK (NKAES) cells in order to improve currrent transduction protocols .
Project description:In this study we have compared the proteomic profile of extracellular vesicles (EVs) prepared from primary, human NK cells or the human NK cell lines NK-92 and KHYG-1 cultured for 48hrs in serum-free conditions. EVs were harvested from cells either under resting conditions (culture in IL-15) or upon activation (combination of IL-12, IL-15, and IL-18). In addition, primary NK cells were activated in the presence of anti-CD16-coated beads, and EVs harvested after 48hrs. The aim was to compare their ability to target and kill a variety of tumor cell line-derived spheroids
Project description:NGS-based assesement of miRNA expression and post-transcriptional modification kinetics in human primary resting and activated natural killer (NK) cells and their released small EVs