ATAC-seq of in vitro differentiated naïve, Th1, and Th9 cells
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ABSTRACT: To investigate changes in chromatin accessibility over time, we differentiated Th9 cells in vitro from naïve T cells from human and murine sources, and followed them over an extended time course, during which they gained and then lost capacity for IL-9 production
Project description:To investigate changes in gene expression over time, we differentiated Th9 cells in vitro from naïve T cells from human and murine sources, and followed them over an extended time course, during which they gained and then lost capacity for IL-9 production We then performed gene expression profiling analysis using data obtained from RNA-seq in cells from 2 different human donors and from 2 mice at 4 time points.
Project description:To investigate changes in chromatin accessibility over time, we differentiated Th9 cells in vitro from naïve T cells from human and murine sources, and followed them over an extended time course, during which they gained and then lost capacity for IL-9 production We then performed Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone modifications H3K4me1, K3K4me3, and H3K27Ac in cells from 2 different human donors and from 2 mice at 4 time points.
Project description:We assessed the global transcriptional changes driven by STING activation with the cyclic dinucleotide 2'3'-cGAMP TH1 and TH9 cells polarized in vitro for 16h and 48h. Our data indicated that STING activation led to an upregulation of Type I Interferon, Interferon Stimulated Genes and pro-inflammatory molecule production that was maintained over time. Furthermore, not only STING activation enhanced the transcription of TH1 and TH9 main effector cytokines (respectively Ifng and Il9) but they also also globally enhanced their differentiation programs.
Project description:To test whether human in vitro primed Th9 cells recapitulate the core pathogenic Th2 cell phenotype, we differentiated naïve T cells into Th1 (IL-12), Th2 (IL-4), Th9 (IL-4+TGF-β), and iTreg (TGF-β). After 7 days transcriptomic profiling by bulk RNA-seq was performed.
Project description:We report that the Th9 differenciation program is boosted in presence of Il-1beta Examination of the expression profile of Th9 CD4+ T cells after 1 hour and 3 days of differentiation and after in vivo injection
Project description:Microarray analyses were performed to compare gene expression in cultured mouse Th9, Th2 and Treg cells and resting versus activated Th9 cells. Three replicates were analyzed for each culture condition; Th9 unstim, Th2 unstim, Treg unstim, Th9 stim
Project description:We next sought to identify the transcriptional program that differentiates IL-9+Th2 cells from “conventional” Th2 cells. To this end, we selected representative Th1, Th17, Th2, and IL-9+Th2 clones (Figure 5A) and determined their transcriptome in the resting state and at different time points after activation using RNAseq. Peripheral Blood Mononuclear Cells (PBMC) were isolated by Ficoll-Plaque Plus (GE Healthcare, UK) density gradient centrifugation. Human CD4+ T cells were isolated from PBMC by EasySep positive selection kit (Stemcell Technologies) according to manufacturer’s instruction. Positively selected CD4+ T cells were washed with PBS and stained for subsequent Th cell subset sorting. Memory Th cell subsets were sorted to over 90% purity according to their expression of chemokine receptors from CD45RA-CD25-CD8-CD3+ cells: Th1(CXCR3+CCR8-CCR6-CCR4-), Th2 (CXCR3-CCR8-CCR6-CCR4+), Th17 (CXCR3-CCR8-CCR6+CCR4+), Th9 (CXCR3-CCR8+CCR6-CCR4+). Single cell Th subset clones were directly sorted into 96well plate according to their expression of chemokine receptors (see above). Single cell clones were expanded and maintained by periodic restimulation with PHA (phytohaemaglutinine, 1 µg/ml, Sigma-Chemicals) and irradiated allogenic feeder cells (5x104/well) in culture medium. T cells were polyclonally activated using beads coated with antibodies against CD3, CD2, and CD28 (T cell/bead = 2:1, human T cell activation/expansion Kit, Miltenyi). Cell cultures were sampled before activation (time 0h) and 2, 4, 6, 9, 12, 24, and 48 hours after activation.