Project description:Transcriptional profiling of mouse Th2, Th9, and iTreg cells. Transcriptomes were compared with that of naïve CD4 T cells. Goal was to screen subset-specific genes.
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: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:Microarray analyses were performed to compare gene expression in cultured mouse Th9, Th2 and Treg cells and resting versus activated Th9 cells.
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.
Project description:we performed proteome analysis of Th9 cells to understand the involvement of proteins that might be crucial for the anti-tumor functions of Th9 cells. Here we show for the first time a comprehensive proteomic analysis of murine Th0 and Th9 cells, which identified 1451 total proteins among which 1367 proteins were common. Further analysis revealed that 118 proteins were upregulated while 81 proteins were downregulated in Th9 cells. Pathway analysis suggested an abundance of phosphoproteins in the proteome of Th9 cells. Among upregulated phosphoproteins which showed to be involved in immune 34 system, Ppp2ca (catalytic subunit of protein phosphatase, PP2A) was found to be highly expressed in Th9 cells. Although the role of PP2A has been shown to regulate the differentiation and functions of Th1,Th2, Th17 and Tregs, its role in differentiation and functions of Th9 cells is not identified yet. Our results show for the first time that PP2A is required for the differentiation and anti-tumor functions of Th9 cells. PP2A inhibition leads to the suppression of IL-9 induction and the expression of key transcription factors of Th9 cells.
Project description:Although the master transcription factors (TFs) are the key to the development of specific T cell subsets, whether additional transcriptional regulators are induced by the same stimuli that dominantly repress development of other T cell lineages has not been fully elucidated. Using Transforming growth factor-b (TGF-b) induced regulatory T cell (iTreg) system, we identify the TF Musculin (MSC) as critical for iTreg development by repression of TH2 transcriptional program. Loss of MSC reduces Foxp3 expression and induces TH2 differentiation even under TGF-b induced iTreg differentiation conditions. MSC mediates this effect by interrupting binding of GATA3 to TH2 locus and reducing intrachromosomal interactions within the Th2 locus. MSC-deficient iTregs are not able to suppress TH2 responses and the Msc–/– mice spontaneously develop gut and lung inflammation with age. Our data indicate that MSC enforces Foxp3 expression and promotes unidirectional induction of iTregs by repressing development of the TH2 developmental program.
Project description:Transcriptional profiling of mouse induced Treg (iTreg) cells comparing control (MIEG3) vector-transduced iTreg cells with iTreg cells transduced with YY1-overexpression vector. Tranduced cells were sorted by GFP expression. Goal was to determine the effects of YY1 on global iTreg gene expression.
Project description:T helper 9 cells (Th9) are interleukin 9 (IL-9)–producing cells that have diverse functions ranging from anti-tumor immune responses to driving allergic inflammation. Th9 cells differentiate from naïve CD4+ T cells in the presence of IL-4 and transforming growth factor-beta (TGF-β). In this reports, we have found that suppression of fatty acid biosynthesis increased IL-9 production in murine and human Th9 cells. This dataset include a set of data showing the effects of suppression of fatty acid synthesis on gene expression, chromatin remodelling and histone modifications in Th9 cells as analyzed by RNA-seq, ATAC-seq and ChIP-seq, respectively.