Project description:ATAC-seq was performed on peripheral blood NK cells that were CRISPR-edited to knock-out expression of T-BET and EOMES. The following samples were analyzed to elucidate the role of T-BET and EOMES in regulating mature human NK cell chromatin accessibility.
Project description:Peripheral blood NK cells that were CRISPR-edited to knock-out expression of T-BET or EOMES were prepared for scRNA-sequencing(10X Genomics). The following samples were analyzed to elucidate the role of T-BET and EOMES in regulating mature human NK cell transcriptional profiles.
Project description:T-BET and EOMES are key transcription factors in the development of mature NK cells in mice. However, the role of these transcription factors during human NK cell development is less well understood. Therefore, we overexpressed T-BET or EOMES 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 mature stage 4 (CD56+CD94+CD16-) and stage 5 (CD56+CD94+CD16+) NK cells from T-BET or EOMES overexpression and control cultures were sorted, whereafter mRNA was isolated and transcriptome analysis was performed by RNA sequencing. Evaluation of the transcriptome in mature NK cells with T-BET or EOMES overexpression could reveal the molecular mechanisms of how T-BET and EOMES play a role in terminal NK cell maturation.
Project description:This project aims at understanding the role of T-bet and Eomes in the differentiation of Natural Killer (NK) cells. NK cells are innate cytotoxic lymphocytes that play an important role in the early control of viral infections and in immune surveillance of cancers. T-bet and Eomes are related T-box factors that are co-expressed by NK cells and are supposed to bind to similar DNA motifs. Their role in NK cell differentiation are not very well understood, in part because their direct target genes are unknown. This study is designed to identify T-bet and Eomes target genes in NK cells. It is based on two complementary mouse strains that we generated in the lab that express tagged (HA) forms of T-bet or Eomes. The tag sequences were inserted in the C-terminus region of both factors, by homomogous recombination in ES cells, allowing endogenous regulation of T-bet and Eomes.
Project description:T-BET and EOMES are key transcription factors in the development of mature NK cells in mice. However, the role of these transcription factors during human NK cell development is less well understood. Therefore, we overexpressed T-BET or EOMES 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 mature stage 4 (CD56+CD94+CD16-) and stage 5 (CD56+CD94+CD16+) NK cells from T-BET or EOMES overexpression and control cultures were sorted, whereafter genomic DNA was isolated and the chromatin accessibility landscape was determined by assay for transposase-accessible chromatin (ATAC) sequencing. Profiling of the epigenetic changes during T-BET or EOMES overexpression in mature NK cells revealed new insights in the regulatory role of T-BET and EOMES during terminal NK cell maturation.
Project description:T-BET and EOMES are key transcription factors in the development of mature Natural Killer (NK) cells in mice. However, the role of these transcription factors during human NK cell development is less well understood. Therefore, we overexpressed T-BET or EOMES in human umbilical cord blood-derived HPC and cultured them in vitro in an NK cell differentiation model. To evaluate the effect of early overexpression of T-BET and EOMES in HPC, transcriptome profiling was performed on T-BET and EOMES overexpressing HPC and compared to control transduced HPC by RNA sequencing on day 0 of culture.
Project description:T-BET and EOMES are key transcription factors in the development of mature Natural Killer (NK) cells in mice. However, the role of these transcription factors during human NK cell development is less well understood. Therefore, we overexpressed T-BET or EOMES in human umbilical cord blood-derived HPC and cultured them in vitro in an NK cell differentiation model. To evaluate the effect of early overexpression of T-BET and EOMES in HPC, the chromatin landscape was uncovered using assay for transposase-accessible chromatin (ATAC) sequencing. In this way, the regulatory effect of T-BET and EOMES overexpression on the epigenome of HPC was evaluated.
Project description:T-bet and Eomes are related T-box transcription factors that control NK cell development. This study was designed to understand the specific roles of Eomes and T-bet in regulating gene expression. RNAseq data were generated for immature (CD11b- CD27+) and mature (CD11b+ CD27-) NK cells from T-bet KO (Tbet Ho) or control mice (Tbet WT) or from Eomes KO (Eomes Ho) or control mice (NK-Cre). Three samples were generated for each condition (Tri 1, 2, 3).