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: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:Natural killer (NK) cells represent one of three lymphoid lineages and play a vital role in controlling viral infections and cancer. In contrast to B and T lymphopoiesis where cellular and regulatory pathways have been extensively characterized, the cellular stages of early human NK-cell commitment remain poorly understood Here we described a novel NK-lineage restricted progenitor (NKP) in fetal development, umbilical cord blood and adult bone marrow. We used microarrays to detail the global programme of gene expression genes of this new progenitor. Global gene expression analysis was performed on the NKP, multipotent progenitors LMPP, common lymphoid progenitor candidate and mature NK cells purified from Cord blood CD34+ cells (3-4 replicates, 2 experiments)
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:Natural Killer cells (NK), a major constituent of innate immune system, have the ability to kill the transformed and infected cells without prior sensitization; can be put to immunotherapeutic use against various malignancies. NK cells discriminate between normal cells and transformed cells via a balance of inhibitory and activating signals induced by interactions between NK cell receptors and target cell ligands. Present study investigates whether expansion of NK cells could augment their anti-myeloma (MM) activity. For NK cell expansion, peripheral blood mononuclear cells from healthy donors and myeloma patients were co-cultured with irradiated K562 cells transfected with 4-1BBL and membrane-bound IL15 (K562-mb15-41BBL). A genome-wide profiling approach was performed to identify gene expression signature in expanded NK (ENK) cells and non-expanded NK cells isolated from healthy donors and myeloma patients. A specific set of genes involved in proliferation, migration, adhesion, cytotoxicity, and activation were up regulated post expansion, also confirmed by flow cytometry. Exp-NK cells killed both allogeneic and autologous primary MM cells more avidly than non-exp-NK cells in vitro. Multiple receptors, particularly NKG2D, natural cytotoxicity receptors, and DNAM-1 contributed to target lysis, via a perforin mediated mechanism. In summary, vigorous expansion and high anti-MM activity both in vitro and in vivo provide the rationale for testing exp-NK cells in a clinical trial for high risk MM. Differential gene expression profile in expanded natural killer (ENK) cells and non-expanded natural killer (NK) cells from healthy donors and myeloma patients Eight healthy donor and eight myeloma patients were used in the study. Non-expanded natural killer (NK) cells were isolated from PBMCs of healthy donors and myeloma patients. Expanded natural killer (ENK) cells were generated from same set of samples as mentioned in expansion protocol. All ENK and NK cells were used for gene expression profiling.
Project description:Natural killer (NK) cells represent one of three lymphoid lineages and play a vital role in controlling viral infections and cancer. In contrast to B and T lymphopoiesis where cellular and regulatory pathways have been extensively characterized, the cellular stages of early human NK-cell commitment remain poorly understood Here we described a novel NK-lineage restricted progenitor (NKP) in fetal development, umbilical cord blood and adult bone marrow. We used microarrays to detail the global programme of gene expression genes of this new progenitor.
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