Gene expression profiling of NK cells isolated from the pancreas of BDC TCR expressing NOD mice before or after Treg ablation by DTR. Also, NK cells cultured in vitro with activating cytokines IL12 and IL-18 with or without regulatory TGFb.
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ABSTRACT: Following Treg ablation in the BDC/NOD.Foxp3-DTR strain, NK cells produce IFNg and accumulate to higher percentage and number. We explored the signature pathways responsible for this phenomenon using microarray prolifing and comparison to other activation signatures. All microarray populations were obtained from highly purified (double sorted) NK cell populations from the pancreas or the spleen following Treg ablation. Triplicates were generated for all ex vivo data. Raw data were preprocessed with the RMA algorithm in GenePattern and averaged expression values were used for analysis.
Project description:Natural Killer Gene Complex (NKC)-encoded immunomodulatory C-type lectin-like receptors (CTLR) include members of the NKRP1 and CLEC2 gene families, which constitute genetically linked receptor-ligand pairs and are thought to allow for NK cell-mediated immunosurveillance of stressed or infected tissues. The mouse CTLR Nkrp1g was previously shown to form several receptor-ligands pairs with the CLEC2 proteins Clr-d, Clr-f, and Clr-g, respectively. Recently, we demonstrated a gut-restricted expression of Clr-f on intestinal epithelial cells that is spatially matched by Nkrp1g on subsets of intraepithelial lymphocytes (Leibelt et al., 2015). We now investigated expression and ligand interaction of Nkrp1g in the splenic compartment, and found an exclusive expression of Nkrp1g on a small subset of NK cells that upregulates Nkrp1g after cytokine exposure. To further characterize NKrp1g+ NK cells, we performed microarray analysis of resting Nkrp1g+ versus Nkrp1g- splenic NK cells that were purified by FACsorting. Total RNA isolation was followed by an Affymetrix microarray analysis.
Project description:Natural killer (NK) cells can be grouped into distinct subsets that are localized to different organs and exhibit different capacity to secrete cytokines and mediate cytotoxicity. Despite these hallmarks that reflect tissue-specific specialization in NK cells, little is known about the factors that control the development of these distinct subsets. The basic leucine zipper transcription factor nuclear factor interleukin 3 (Nfil3; E4bp4) is essential for bone marrow-derived NK cell development but it is not clear whether Nfil3 is equally important for all NK cell subsets nor how it induces NK lineage commitment. Here we show that Nfil3 is required for the formation of Eomesodermin (Eomes)-expressing NK cells, including conventional medullary and thymic NK cells, whereas TRAIL+ Eomes- NK cells develop independent of Nfil3. Loss of Nfil3 during the development of bone marrow-derived NK cells resulted in reduced expression of Eomes and, conversely, restoration of Eomes expression in Nfil3-/- progenitors rescued NK cell development and maturation. Collectively, these findings demonstrate that Nfil3 drives the formation of mature NK cell by inducing Eomes expression and reveal the differential requirements of NK cell subsets for Nfil3. RNA-sequencing of natural killer (NK) cell subsets
Project description:It is known that NK cells are a heterogeneous population of functionally distinct NK cell subsets. Here we report on different genomic, phenotypic and functional properties of four murine NK cell subsets distinguished by CD117 (c-kit), CD27 and CD11b expression. Gene expression was measured in NK cell subsets freshly sorted from murine C57Bl/6 splenocytes. Two to three different batches were analysed.
Project description:NK cells are innate immune cells that recognize and kill foreign, virally-infected and tumor cells without the need for prior immunization. NK expansion following viral infection is IL-2 or IL-15-dependent. To identify Runx3 regulated genes, NK cells were isolated from spleen of IL15/Ra injected WT and Runx3-/- mice and sorted to obtain 3 subpopulations of immature (CD27) and mature (DP and CD11c) NK cells.
Project description:Human NK cells were sorted into CD56dim and CD56bright NK cell subpopulations. In order to define characteristics of both populations gene profiling was performed using Affymetrix arrays U133a and U133B.
Project description:Somatic STAT5B gain-of-function mutations have been frequently found in patients with T- and NK-cell neoplasms. STAT5BN642H represents the most frequently occuring STAT5B mutation. To investigate the molecular mechanism of STAT5BN642H-driven NK-cell leukemia, we performed RNA-Seq of liver derived FACS-sorted diseased N642HNK/NK and aged non-diseased control (Cre neg, GFPNK/NK), STAT5BNK/NK, N642HNK/NK NK cells.
Project description:ChIP-seq was conducted using freshly isolated (resting) splenic WT NK cells with anti-Runx3 antibody (Ab), anti-H3K4me1 Ab and non-immune serum (NIS) as control. Runx3 and H3K4me1 IP from splenic NK cells isolated by negative selection using NK cell isolation kit (R&D) followed by sorting of NKp46+ cells.
Project description:ChIP-seq was conducted using splenic WT NK cells cultured for 7 days with IL-2 using anti-Runx3 antibodies (Ab), anti-H3K4me1 Ab and non-immune serum (NIS) as control. Two biological Runx3, one H3K4me1 and two NIS IP repeats from splenic NK cells isolated from individual mice by negative selection using NK cell isolation kit (R&D) cultured with IL-2.
Project description:ChIP-seq was conducted using freshly isolated splenic WT NK cells from IL-15/Ra treated mice with anti-Runx3 antibody (Ab) and non-immune serum (NIS) as control. Runx3 and NIS IP from splenic NK cells of IL-15/Ra treated WT mice, isolated by negative selection using NK cell isolation kit (R&D) followed by sorting of NKp46+ cells.
Project description:NK cells are innate immune cells that recognize and kill foreign, virally-infected and tumor cells without the need for prior immunization. NK expansion following viral infection is IL-2 or IL-15-dependent. To identify Runx3 responsive genes, NK cells were isolated from spleen of WT and Runx3-/- mice . Six samples (3 WT and 3 Runx3-/-) of freshly isolated NK cells (resting) were separately obtained from individual mice.