Project description:We sorted Eomes-negative NK cells (CD3- CD56+ CXCR6- CD16-) and Eomes-positive NK cells (CD3- CD56+ CXCR6+) from total leukocytes isolated from the perfusion fluid of five healthy human livers destined for transplantation. Total RNA was extracted from sorted cells, cDNA generated and RNASeq performed.
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: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: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: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:Classical NK cells are the prototypical type 1 innate lymphocytes mounting cytolytic and IFNg responses to intracellular pathogens and tumors. A similar but distinct population termed ILC1 was recently described, differing from NK cells in multiple though relatively subtle ways, including tissue residency vs recirculation, and levels of NK receptors. Whether the differences reflect distinct lineages or mere variations in program expression is not fully understood. Here, using transcription factor reporter mice and cell transfers of bone marrow precursors, we found that, upon transfer in vivo, Eomes-expressing, lineage-negative, NK receptor-negative cells acquired properties typically associated with classical NK cells including the capacity to prevent metatstatic disease. In contrast, as previously reported, PLZFhigh cells mostly generated ILC1, ILC2, or ILC3. These findings identify the Eomes-expressing NK precursor as the long-elusive bone marrow precursor to classical NK cells, and demonstrate that the NK and ILC1 lineages diverge early during development.
Project description:This study shows that liver Eomes- NK cells are not precursors of classical Eomes+ NK cells but rather constitute a distinct lineage of innate lymphoid cells. Gene profile analyses show that Eomes- NK cells share part of their transcriptional program with NKT cells that includes genes involved in liver homing, NK cell receptors, and several cytokines and cytokine receptors. Eomes- NK cells, Eomes+ Nk cells and NKT cells were sorted by flow cytometry from Eomes-GFP reporter mice. Total RNA was extracted and hybridized to Affymetrix microarrays.
