Project description:Pooled purified peripheral blood derived CD56dimCD16+ NK, CD56brightCD16- NK and in vitro activated CD56+CD16+ NK subsets obtained from 9 healthy donors were analyzed for gene expression pattern. Each pooled NK subset sample was hybridized in replicates (A and B). Keywords: other
Project description:It is known that natural killer (NK) cells are a heterogeneous population of functionally distinct NK cell subsets. Here we report on different genomic, phenotypic and functional properties of human NK cell subsets derived from peripheral blood, thymus and bone marrow. NK cell subpopulations were defined via expression of CD56 and CD16.
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:The aim of the study was to investigate the activation of human NK cells by IL2 through analyzing the global gene expression at different time points (0, 2, 8 and 24 hours) after culture with the cytokine IL2 at 100 IU/ml. NK cells with the CD56+/CD16+ and CD3- phenotype were negatively selected by immunomagnetic beads and re-examined by flow-cytometry to ensure greater than 90% purity . Keywords: resting and IL2 activated NK cells(time series)
Project description:Human NK cells from the decidua basalis of gravid uteri (dNK) and from cycling endometrium (eNK) of women undergoing hysterectomy were isolated and compared by gene expression profiling using Affymetrix microarrays with probes representing ~47,400 transcripts. Substantial differences indicate that these two types of NK cells represent distinct subsets. Freshly isolated NK cells were obtained by FACS sorting. 4 dNK and 5 eNK samples were obtained form independent donors. dNK cells were isolated from the decidua basalis of first trimester placentas and sorted as CD3-, CD16-, CD56+ cells. eNK cells were obtained from non-affected regions of cycling endometrium of donor women undergoing hysterectomy and were sorted as CD45+, CD56+, CD3- cells . The preliminary patient diagnoses included genital prolapse, fibroids, cervical dysplasia, or menorrhagia. All cycling endometrium samples were from the secretory phase of the cycle with exception of sample eNK_S6 that was from the proliferative phase.
Project description:There is limited knowledge on the origin and development of the ample spectrum of human NK cells, particularly of specialized NK subsets. Here, we characterized the NK cell progeny of CD34+DNAM-1bright CXCR4+ precursors that reside in healthy bone marrow and circulate in the peripheral blood (PB) of patients with chronic infections/inflammation. including HIV, HCV or HCMV reactivation after HSC transplantation. Unlike conventional CD34+ precursors they rapidly differentiated in vitro into cytotoxic, IFNγ-secreting CD94/NKG2C+KIR+CD57+ maturing NK cell progenies with HCMV-inhibiting activity. Progeny characterization led also to identification of an additional new PB Lin-CD56-CD16+ precursor giving rise to the same CD94/NKG2C+KIR+CD57+ maturing NK cell progenies. Microarray analysis of NK cell progenies revealed a signature compatible with maturing adaptive NK cells. In vivo circulation of multiple common lymphocyte precursors with rapid development to NKG2C+ NK cell progeny is steadily occurring and may thus be a crucial resource for the prompt control of HCMV. We used microarray to compare the transcriptional profiles of human NKG2C+ NK cells derived from i) CD34+DNAM1brightCXCR4+ precursors, ii) Lin-CD34-CD16+CD56- precursors, iii) peripheral blood.
Project description:Identification of genes differentially expressed between human CD14+CD16- and CD16+ monocyte-derived macrophages generated in the presence of either GM-CSF (termed GM14 and GM16, respectively) or M-CSF (termed M14 and M16, respectively) Human peripheral CD14+CD16- and CD16+ blood monocytes from three independent healthy donors (D1, D2 and D3) were isolated by positive selection from peripheral blood mononuclear cells (PBMC) using magnetic separation systems (MACS, Miltenyi Biotec). Briefly, PBMC were first incubated with MACS anti-CD56 antibody conjugated to paramagnetic microbeads in order to eliminate the NK (CD16+) cell fraction. NK-depleted PBMC were further incubated with MACS anti-CD16 antibody to isolate CD16+ monocytes. CD56-CD16- PBMC were finally incubated with MACS anti-CD14 antibody to obtain the CD14+CD16- monocyte fraction. Monocytes were cultured for 7 days in medium containing either GM-CSF or M-CSF. Total RNA from each condition was extracted using the RNeasy kit (Qiagen) and hybridized to an Agilent Human Whole Genome (4x44) Oligo Microarray. All experimental procedures were performed following manufacturer instructions.
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: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:Natural killer cells are cytotoxic innate lymphoid cells that play an important role for early host defenses against infectious pathogens and surveillance against tumor growth and metastasis. In humans, NK cells may be divided in various subsets on the basis of the relative expression of the CD56 molecule and of the low-affinity FcγRIIIA CD16. In particular, the two main NK cell subsets are represented by the CD56bright CD16-/dull and the CD56dull CD16bright NK cells. A number of experimental evidences indicate that CD56bright and CD56dull NK cells represent different maturative stages of the NK cell developmental pathway. In an effort to identify NK cell miRNA signatures that may contribute to the NK cell-specific maturation program, we determined the miRNA profile of human NK cell subpopulations derived from peripheral blood samples of healthy donors. We identified multiple miRNAs that are differentially expressed in CD56bright CD16- and CD56dull CD16bright NK cells. Among these, we found a few miRNAs with a consistent differential expression in the two NK cell subsets, and with an intermediate expression in the CD56brightCD16dull NK cell subset, representing a transitional step of maturation of NK cells. Our data have been confirmed by both quantitative real-time PCR and multivariate analysis. These analyses allowed us to establish the existence of a miRNA signature able to discriminate the two main NK cell subsets, regardless of their surface phenotype. In addition, by analyzing the putative targets of representative miRNAs we show that miR-146a-5p, that displays a significant up-regulation in CD56bright as compared to CD56dull NK cells, may be involved in the regulation of killer Ig-like receptor (KIR) expression. These findings may contribute to a better understanding of the physiologic significance of miRNAs in the regulation of NK cell development/function. In addition, our results suggest that miR-146a-5p targeting, resulting in KIR down-regulation, may be exploited as a tool to generate/increment the effect of NK KIR-mismatching against HLA class I+ tumor cells and thus to improve the NK-mediated anti-tumor activity.