Project description:Research has already gained much insight into the roles of various members of the RUNX family of transcription factors in the development of human T- and B-cells, however the importance in human NK cell development remains unclear. We performed a ChIP-Seq analysis with a RUNX2-specific antibody on sorted human peripheral blood NK cells (CD3- CD19- CD56+) to shed more light onto the genes that are under control of RUNX2 in this cell type.
Project description:Research has already gained much insight into the roles of various members of the RUNX family of transcription factors in the development of human T- and B-cells, however the importance in human NK cell development remains unclear. Therefore, we transduced a shRNA that specifically binds to all known RUNX2 isoforms in human umbilical cord blood-derived hematopoietic progenitor cells (HPC) and cultured them in vitro in an NK cell differentiation model. On day 21 of culture (CD56+CD94+) NK cells from shRNA and control cultures were sorted, after which mRNA was isolated and transcriptome analysis was performed by RNA sequencing. Evaluation of the transcriptome in these NK cells could reveal the molecular mechanisms of RUNX2.
Project description:Fragmented RNA cocktails from FACS sorted Human decidual NK cell, and peripheral blood CD56Bright and CD56Dim NK cells, previously hybridization to HGU95AV2 chips (Koopman et al J Exp Med. 2003 Oct 20;198(8):1201-1), were stored long term at -80C, thawed and hybridized to HG-U133A arrays. Transcriptome analysis of Human decidual NK cells and NK cells from peripheral blood using Affymetrix UGU133A arrays.
Project description:Fragmented RNA cocktails from FACS sorted Human decidual NK cell, and peripheral blood CD56Bright and CD56Dim NK cells, previously hybridization to HGU95AV2 chips (Koopman et al J Exp Med. 2003 Oct 20;198(8):1201-1), were stored long term at -80C, thawed and hybridized to HG-U133B arrays. Transcriptome analysis of Human decidual NK cells and NK cells from peripheral blood using Affymetrix UGU133B arrays.
Project description:Research has already gained much insight into the roles of various members of the RUNX family of transcription factors in the development of human T- and B-cells, however the importance in human NK cell development remains unclear. In this study we discovered that NK cells predominantly express the shorter of the two principal isoforms of RUNX2, also referred to as RUNX2-I. Therefore, we overexpressed the RUNX2-I isoform in human umbilical cord blood-derived hematopoietic progenitor cells (HPC) and cultured them in vitro in an NK cell differentiation model. On day 14 of culture (CD56+CD94+) NK cells from RUNX2-I overexpression and control cultures were sorted, after which mRNA was isolated and transcriptome analysis was performed by RNA sequencing. Evaluation of the transcriptome in these NK cells could reveal the molecular mechanisms of the RUNX2-I isoform.
Project description:Human peripheral blood NK cell popiulations were seperated on the basis of CD56 expression with flowcytometry. Sorted CD56-bright (CD56Br) and CD56-dim (CD56Dim) NK cells were subjected to H3K4Me2 ChIPmentation to obtain epigenome profiles for downstream analyses.
Project description:Fragmented RNA cocktails from FACS sorted Human decidual NK cell, and peripheral blood CD56Bright and CD56Dim NK cells, previously hybridization to HGU95AV2 chips (Koopman et al J Exp Med. 2003 Oct 20;198(8):1201-1), were stored long term at -80C, thawed and hybridized to HG-U133B arrays.
Project description:Fragmented RNA cocktails from FACS sorted Human decidual NK cell, and peripheral blood CD56Bright and CD56Dim NK cells, previously hybridization to HGU95AV2 chips (Koopman et al J Exp Med. 2003 Oct 20;198(8):1201-1), were stored long term at -80C, thawed and hybridized to HG-U133A arrays.
Project description:Human Natural Killer (NK) cells in peripheral blood perform many functions but classification of specific subsets has been a long-standing problem. Here, we report single-cell RNA sequencing of NK cells from healthy CMV-negative donors, comparing gene expression in unstimulated and IL-2 activated cells. Unsupervised clustering identified seven NK cell subsets. Three resembled well-described populations, CD56brightCD16-, CD56dimCD16+CD57- and CD56dimCD16+CD57+. CD56dimCD16+CD57- cells sub-divided to include a population with higher chemokine mRNA and increased frequency of KIR expression. Three novel human blood NK cell populations were identified as: a population of type I interferon responding NK cells which were CD56neg; a population exhibiting an in-vivo cytokine-induced memory-like phenotype, including increased granzyme B mRNA in response to IL-2, and finally, a small population, with low ribosomal expression, down-regulation of oxidative phosphorylation and high levels of immediate early response genes indicative of cellular activation. Human cytomegalovirus positive donors also included a higher frequency of adaptive NK cells. Together, these data establish an unexpected diversity in blood NK cells and provide a new framework for analysing NK cell responses in health and disease.