ABSTRACT: Gene expression studies comparing IFNg+ Tregs versus IFNg- Tregs from human peripheral blood Ex vivo sorted Tregs (CD25highCD127neg) were stimulated for 4 hours and IFNg-secreting cells were detected by a IFNg-capture kit. The samples were resorted based on IFNg expression.
Project description:Gene expression studies comparing IFNg+ Tregs versus IFNg- Tregs from human peripheral blood Overall design: Ex vivo sorted Tregs (CD25highCD127neg) were stimulated for 4 hours and IFNg-secreting cells were detected by a IFNg-capture kit. The samples were resorted based on IFNg expression.
Project description:Microarray used to detail the global gene transcription underlying sorted IFNg+ and IFNg- Tregs (CD4+CD25+CD127lo) and Tconv (CD4+CD25-CD127+) for fresh (unexpanded) and 14 day expanded cells from human blood. Treg and Tconv were FACS isolated from five healthy subjects (median age of 26, range 22-30). Sorted cells were separated into two groups: the first group was stimulated for 4 hours with PMA/ionomycin and labeled with the IFNg cytokine capture kit (Miltenyi Biotech) followed by FACS isolation of IFNg- and IFNg+ populations. The second set was expanded to day 14 prior to reactivation and cytokine cell capture. For each IFNg sorted population, cells were pelleted and flash frozen before RNA isolation and processing.
Project description:CD70TG mice are a model for sterile chronic immune activation and develop Anemia of Inflammation, which is dependent on the production of Ifng by effector CD4 and CD8 T cells. We used microarrays to identify Ifng-dependent differentially expressed genes that could account for the erythropoietic defect. CD71+ cells were MACS-enriched from the bone marrow of WT, CD70TG, IFNg-/- and CD70TG*IFNg-/- male mice of 10-12 weeks of age (3 mice per genotype group).
Project description:Production of reactive oxygen species (ROS) is one of the important antimicrobial mechanisms of phagocytic cells. Enhanced oxidative burst requires these cells to be primed with agents such as IFNg and LPS with a synergistic effect of these agents on the level of the burst. However, excessive ROS generation will lead to tissue damage and has been implicated in a variety of inflammatory and autoimmune disease. Therefore, this process needs to be tightly regulated. In order to understand the genes regulating this process, we will treat bone marrow derived macrophages with above mentioned priming agents and study the gene expression. We used microarrays to determine the changes in gene expression that occur in bone marrow derived macrophages after treatment with IFNg, LPS, or a combination of IFNg and LPS. Four condition experiment; Biological replicates: four replicates per condition
Project description:Regulatory T cells (Tregs) are essential for maintaining proper immune homeostasis. Extracellular signals (e.g. TCR, CD28, IL-2R) are necessary for the generation and maintenance of Tregs, but how these signals are integrated to control the gene expression patterns of Tregs is less clear. Here we show that the epigenetic regulator, Ezh2, was induced by CD28 costimulation and Ezh2 activity was elevated in Tregs as compared to conventional CD4+ T cells. Deletion of Ezh2 in mouse Tregs led to a progressive autoimmune disease because Tregs were compromised after activation, losing proper control of essential Treg lineage genes and adopting a gene expression pattern similar to Foxp3-deficient ‘Tregs.’ Lineage-tracing of Ezh2-deficient Tregs in vivo confirmed that the cells were destabilized selectively in activated Treg populations, which led to a significant loss of Tregs in non-lymphoid tissues. These studies reveal an essential role for Ezh2 in the maintenance of Treg “identity” during cellular activation and differentiation. RNAseq of sorted populations of CD62Lhi or CD62Llo Tregs for both Ezh2-HET (Foxp3YFP-Cre/Foxp3WT;Ezh2fl/+ female mice) and Ezh2-KO (Foxp3YFP-Cre/Foxp3WT;Ezh2fl/fl female mice) were generated, in triplicate for each condition, using Illumina HiSeq 2500 single-end 50bp sequencing platform.
Project description:Human responder T-cells (Tresp) and 2 lineages of regulatory T cells (Tregs), namely G2 and G3, were isolated by flow sorting based on their immunoreacitivities against specific cell surface markers and their gene expression profiles were interrogated using Affymetrix Exon 1.0 ST Arrays Comparison of gene expression profiles between responder T-cells, G2 Tregs and G3 Tregs
Project description:This experiment was designed to identify IFNg-regulated, IRF1-dependent genes during infection with the intracellular pathogen Shigella flexneri. WT and Irf1-/- MEFs were stimulated for 18 hours with IFNg or left unstimulated; all of the cells were subsequently infected for 6 hours with S. flexneri prior to harvest of total RNA.
Project description:In order to fully characterize emodin's effects on macrophage activation, peritoneal macrophages were stimulated with LPS+IFNg with or without emodin and gene expression was analyzed using a whole genome microarray. Emodin significantly attenuated the IFNg/LPS induced changes in a large percentage of responsive genes (31%) through inhibiting multiple signaling pathways. RT-qPCR was used to confirm the results in several genes associated with M1 macrophage activation including: TNF, IL6, IL1b, iNOS, MMP2, and MMP9. Three-condition, one-color experiment: Vehicle control, LPS-IFNg or LPS-IFNg-Emodin treated periferal WBC PMN samples: 4 biological replicates each.
Project description:MBP TCR Tg mouse (1B3) generates only iTregs when crossed onto RAGKO background. Tregs from 1B3.RAG mouse were used for gene expression analsysis to determine genes selectively expressed in peripherally generated iTregs iTregs from 1B3.RAG mice (>90% Foxp3+) were run against total Tregs from 1B3 and WT NOD mice. Foxp3- CD4+T cells were used as control from all strains as Tconv. (1B3 denotes MBP-TCR Tg mouse).
Project description:Naturally occurring FoxP3+CD4+CD25+high regulatory T cells (Tregs) play an important role in dominant tolerance, suppressing auto-reactive CD4+CD25- T cell activity. Although Tregs from T1D subjects are functionally deficient, there is little knowledge of the molecular mechanisms that orchestrate this loss of Treg function. We observed increased apoptosis (by a novel YOPRO-1/7AAD dual staining protocol) and decreased suppression in polyclonal Tregs in the periphery from high at-risk and T1D subjects. We hypothesize that prior to and during the onset of disease, Tregs lack pro-survival signals and are caught up in a relatively deficient cytokine milieu whose effects may be detectable in the periphery. Microarray analysis was performed on un-stimulated Tregs from 12 subjects with newly diagnosed T1D and 15 healthy controls. Experiment Overall Design: Recent-onset T1D subjects were T1D patients diagnosed within 1 year. At the time of each visit, the following clinical measurements were taken: HbA1c, glucose level, height, weight and BMI. The presence of auto-antibodies was measured from peripheral blood. Peripheral blood mononuclear cells (PBMCs) were collected and Tregs were isolated using FACS isolation. RNA was isolated, amplified and cRNA was fragmented and hybridized to Affymetrix whole genome U133Plus2.0 arrays having 54675 probe-sets. Bayesian Hierarchical analysis (BGX) was used to analyze the data.