Project description:For the purpose of the Gene Regulatory Network validation we have performed chromatin-immunoprecipitation sequencing (ChIP-Seq) experiment for three transcription factors; namely GATA3, MAF and MYB. ChIP-Seq of GATA3, MAF and MYB in human naïve CD4+ T-cells differentiated toward Th1 and Th2 until day 6. Matched INPUT samples were also sequenced for each condition.
Project description:A generic genome-scale metabolic model (GEMs) of human CD4+ T-cells. Several cell-specific GEMs for CD4+ T-cell subsets such as Th1, Th2, Th17 and iTreg cells derived from "HTimmR" are included as additional files. The model formats are compatible with RAVEN v.2.0 toolbox.
Project description:The involvement of thousands of genes complicates the identification of clinically relevant candidate genes in common diseases. We hypothesized that genes co-regulated with a key gene in allergy, IL13, would form a module that could help to discover novel candidate genes. We identified a Th2 cell module by siRNA mediated knock down of 25 putative IL13-regulating transcription factors (TFs) followed by expression profiling. Human CD4+ T cells (hBP CD4+ T cells, 2W-200, Lonza, Vallensbak Strand, Denkmark) were nucleofected either with nucleofection buffer, 1 µM human on target plus SMART pool siRNA against ELK1, GATA3, NFATC3, MAF, NFKB1, JUN, STAT3 (Dharmacon, Lafayette, CO) or non-targeting siRNA using the AMAXA nucleofection program U-014. Six hours after then nucleofection cells were washed, activated and polarized towards Th2. The CD4+ cells were activated with plate bound anti-CD3 (500ng / ml for coating of the plate), with soluble anti-CD28 (500ng / ml) and with IL-2 (17ng /ml, all purchased from R&D). Th2 polarization was induced with anti-IL-12 (5µg/ml) and IL-4 (10ng / ml). For RT-PCR and microarray analyisis the cells were harvested 12 hours of polarization and total RNA was extracted.
Project description:Differentiation of CD4+T-cells into effector subsets is a critical component of the adaptive immune system and an incorrect response can lead to autoimmunity or immune deficiency. Cellular differentiation including T-cell differentiation is accompanied by large-scale epigenetic remodeling, including changes in DNA methylation at key regulators of T-cell differentiation. The TET family of enzymes were recently shown to be able to catalyse methylated cytosine (5mC) into 5-hydroxymethylcytosine (5hmC) enabling a pathway of active removal of DNA methylation. Here, we characterize 5hmC, 5mC and transcriptional dynamics during human CD4+T-cell polarisation in a time series approach and relate these changes to profiles in ex-vivo CD4+memory subsets. We observed large-scale remodelling during early CD4+T-cell differentiation which was predictive of subsequent changes during late time points, these changes were also related to disease associated regions which we show can act as functional regulatory elements. This dataset was designed to assess how gene expression changes over time during human CD4+T-cell polarization towards Th1 and Th2. DNA methylation was assessed in relationship to 5hmC levels and changes (see data series), we observed that regions gaining 5hmC early was highly predictive of regions losing DNA methylation during late time points. This submission contains data from the DNA methylation by array profiling of human CD4+T-cells in-vitro polarized towards Th1 and Th2 time-series. It is part of series containing 5hmC and DNA methylation profiling of the same samples. See related experiments E-MTAB-4685, E-MTAB-4686, E-MTAB-4687, E-MTAB-4689.
Project description:There remains a need for analysis of CD4 helper T cells differentiation in vivo. To this end ovalbumin (OVA)-specific CD4 (OTII) T cells transferred into congenic mice were studied. Live attenuated OVA-expressing Salmonella (SalOVA) induce T-bet and IFN-g in OTII cells, while alum-precipitated OVA (alumOVA) induces GATA-3 and IL-4. Although 70% of alumOVA-responding OTII cells express GATA-3, only 7% produce IL-4. Thus Th2-polarization defined solely by IL-4 production does not recognize the diversity of GATA-3-expressing effectors. Low-density arrays were designed to assess the expression of 384 genes by real-time RT-PCR. Extensive early diversification occurred in both responses. SalOVA selectively induced many chemokines and pro-inflammatory cytokines, while alumOVA induced few Th2-associated cytokines. Several cytokines and molecules associated with Th17 cells and follicular helper cells were also induced by both antigens. The transcription factor Helios was exclusively induced in alumOVA-responding OTII cells, and critically not in standard in vitro Th2-polarization systems. Early synchronous up-regulation of Helios and GATA-3 mRNA is paralleled at protein level with largely coincident localization in specific nuclear foci of OTII cells responding to alumOVA. This appears to be consistent with a key role for both transcription regulators in the direction of Th2 responses in vivo. Keywords: In vivo T cell polarization Ovalbumin (OVA)-specific CD4 (OTII) T cells were transferred into C57BL/6 mice that were immunized either with live attenuated OVA-expressing Salmonella (Sal) or with alum-precipitated OVA (alum), or not (Naïve). Gene expression assay was performed on FACS sorted OTII cells (Naïve, Sal, Alum). OTII cells were purified from three independent groups of ten naïve, or SalOVA-immunized or alumOVA-immunized mice.
Project description:Differentiation of CD4+T-cells into effector subsets is a critical component of the adaptive immune system and an incorrect response can lead to autoimmunity or immune deficiency. Cellular differentiation including T-cell differentiation is accompanied by large-scale epigenetic remodeling, including changes in DNA methylation at key regulators of T-cell differentiation. The TET family of enzymes were recently shown to be able to catalyse methylated cytosine (5mC) into 5-hydroxymethylcytosine (5hmC) enabling a pathway of active removal of DNA methylation. Here, we characterize 5hmC, 5mC and transcriptional dynamics during human CD4+T-cell polarisation in a time series approach and relate these changes to profiles in ex-vivo CD4+memory subsets. We observed large-scale remodelling during early CD4+T-cell differentiation which was predictive of subsequent changes during late time points, these changes were also related to disease associated regions which we show can act as functional regulatory elements. This dataset was designed to assess how gene expression changes over time during human CD4+T-cell polarization towards Th1 and Th2. Gene expression was assessed in relationship to 5hmC and DNA methylation levels and changes (see data series), we observed characteristic gene expression for the specific time points and stimuli or cell type and the expression was correlated with gene body 5hmC as well as anticorrelated with promoter DNA methylation levels. This submission contains data from transcription profiling by array of human CD4+T-cells, Th1/Th2 polarized time-series and primary memory subsets. It is part of series containing 5hmC and DNA methylation profiling of the same samples. See related experiments E-MTAB-4685, E-MTAB-4686, E-MTAB-4688, E-MTAB-4689.
Project description:This study looks at the ATAC+RNA profiles of human CD4 T cells under bioreactor-like conditions as ACT. Cells were stimulated toward Th1,Th2,Treg and Th17 under anti-CD3/28 activation, and profiled on day 5.