Project description:This dataset includes whole genome sequence data for ChIPmentation assays (18 H3K4me3, 20 H3K27ac and 3 input samples) of human stimulated and cultured CD4+ Treg cells.
Project description:Goals and objectives of this study: to identify genes of the Treg signature induced by consitutive expression of GARP or FOXP3 in antigen-specific Th cells with potential role for stabililization & maintenance of the regulatory program. Keywords: T-cell receptor stimulation, gene-regulation, comparative gene expression profiling, cell type comparison, human, regulatory T cells, FOXP3, GARP Human alloantigen-specific Treg cells (THU) and Th cells (CD4-39), established and described recently (Ocklenburg et al.. Lab Invest.2006; 86: 724-737), were sitmulated for 3 days with cognate antigen (EBV B cells) and IL2 as described (Ocklenburg et al.. Lab Invest.2006; 86: 724-737) and analyzed using human Affymetrix U133 2.0 in monoplicate. Th cells had been transduced with a retroviral vector containing human GARP (LRRC32) or FOXP3 and an IRES-driven GFP as marker or empty GFP control, sorted for GFP+, expanded as described recently (Ocklenburg et al.. Lab Invest.2006; 86: 724-737), and characterized functially, phenotypically, and genetically as described (WO/2007/113301). Abbreviations: Treg THU d3 = alloantigen-specific Treg cells; GFP d3 = GFP-transduced alloantigen-specific Th cells (CD4-39); FoxP3 d3 = FOXP-transduced alloantigen-specific Th cells (CD4-39); Garp d3 = GARP-transduced alloantigen-specific Th cells (CD4-39); all stimulated for 3 days (=d3) with EBV B cells and IL2.
Project description:The CD4+ regulatory T (Treg) cell lineage comprises thymus-derived (t)Treg cells and peripherally induced (p)Treg cells. As a model for Treg cells, studies employ TGF-β-induced (i)Treg cells generated from CD4+ conventional T (Tconv) cells in vitro. Here, we describe the relationship of iTreg cells to tTreg and Tconv cells. Proteomic analysis revealed that iTreg, tTreg and Tconv cell populations each have a unique protein expression pattern. iTreg cells had very limited overlap in protein expression with tTreg cells, regardless of cell activation status and instead shared signaling and metabolic proteins with Tconv cells. tTreg cells had a uniquely modest response to CD3/CD28-mediated stimulation. As a benchmark, we used a previously defined proteomic signature that sets ex vivo naïve and effector phenotype Treg cells apart from Tconv cells and includes unique Treg cell properties (Cuadrado et al., Immunity, 2018). This Treg cell core signature was largely absent in iTreg cells. We also used a proteomic signature that distinguishes ex vivo effector Treg cells from Tconv cells and naïve Treg cells. This effector Treg cell signature was partially present in iTreg cells. In conclusion, iTreg cells are distinct from tTreg cells and share limited features with ex vivo Treg cells at the proteomic level.
Project description:This dataset maps gene expression regulation in human primary regulatory CD4+ T cells (Tregs) stimulated using PMA and ionomycin or cultured without stimulus for 18 hours. It includes whole genome sequence data for ChM-seq (18 H3K4me3, 20 H3K27ac and 3 inputs), ATAC-seq (42 samples) and whole transcriptome (39 samples). All individuals were genotyped using coreExome Illumina SNP chip array (included in EGAS00001003516).
Project description:Foxp3+ regulatory T cells (Treg) play a central role for tolerance against self and innocuous environmental antigens. However, the role of antigen-specificity for Treg-mediated tolerance is only incompletely understood. Here we show by direct ex vivo characterization of human CD4+ T cells, that the response against innocuous airborne antigens, such as plant pollen or fungal spores, is dominated by memory-like antigen-specific Treg. Surprisingly, breakdown of tolerance in atopic donors was not accompanied by a quantitatively or qualitatively altered Treg response, but instead correlated with a striking dichotomy of Treg versus Th2 target specificity. Allergenic proteins, are selectively targeted by Th2 cells, but not Treg. Thus human Treg specific for airborne antigens maintain tolerance at mucosal sites and the failure to generate specific Treg against a subgroup of antigens provides a window of opportunity for allergy development. PBMCs from sex and age matched birch pollen allergic patients and healthy controls, were stimulated (7h) with airborne fungal (A. fumigatus) or birch pollen antigen (birch) and sorted into antigen specific conventional and regulatory T cells according to their expression of CD154+ and CD137+ on CD4+ T cells, respectively. Number of samples per group in parentheses: Healthy controls stimulated with A. fumigatus (n=5), allergic patients stimulated with A. fumigatus (n=6), healthy controls stimulated with birch (n=6), allergic patients stimulated with birch (n=4).
Project description:Foxp3+ regulatory T (Treg) cells prevent inflammatory disease but the mechanistic basis of suppression is not understood completely . Gene silencing by RNA interference can act in a cell-autonomous and non-cell-autonomous manner, providing mechanisms of inter-cellular regulation. Here, we demonstrate that non-cell-autonomous gene silencing, mediated by miRNA-containing exosomes, is a mechanism employed by Treg cells to suppress T cell-mediated disease. Treg cells transferred microRNAs (miRNA) to various immune cells, including T helper 1 (Th1) cells, suppressing Th1 cell proliferation and cytokine secretion. Use of Dicer-deficient or Rab27a and Rab27b double-deficient Treg cells to disrupt miRNA-biogenesis or the exosomal pathway, respectively, established a requirement for miRNAs and exosomes for Treg cell-mediated suppression. Transcriptional analysis and miRNA inhibitor studies showed that exosome-mediated transfer of Let-7d from Treg cell to Th1 cells contributed to suppression and prevention of systemic disease. These studies reveal a mechanism of Treg cell-mediated suppression mediated by miRNA-containing exosomes. Regulatory T cells (CD4+CD25hiFoxp3rfp+, Treg) were isolated from naive mice. RNA as extracted form some Treg cells, while others were cultured in complete IMDM media for 3 days, stimulated with anti-CD3 anti-CD3 (1ug/ml) and anti-CD28 (10ug/ml). Exosomes were recovered from Treg cell supernatant, as described, and RNA was extracted form the purified exosomes. To identify which miRNAs were transferred to Dicer-deficient (KO) cells from Treg cells, we cultured Dicer KO cells alone, or co-cultured Dicer KO cells with Treg cells. RNA was extracted form Dicer KO cells cultured alone or from Dicer KO cells cultured in the presence of Treg cells. 3 x biological replicates were used. Each biological replicate was derived from a pool of 3-5 samples.
Project description:This study evaluated changes in gene expression upon IL-7 treatment in human naive and memory Treg. CD4+CD25+CD127low naïve and memory Treg were isolated from fresh PBMC and separately stimulated with ?CD3?/CD28 coupled beads (Invitrogen-Dynal) at a 1:10 bead/T cell ratio, treated with or without 10 ng/ml of rhIL-7 (R&D Systems) for 16 hours. qPCR gene expression profiling of CD4+CD25+CD127low naïve and memory Treg obtained from 2 separate donors. Cell lysates were prepared separately from the 2 donors and pooled prior to RNA extraction.
Project description:Using a CRISPR/Cas9-based approach we engineer primary CD4+ T cells in which the CD6 protein was tagged with an affinity Twin-Strep-tag (OST) with the purpose of determining by quantitative mass spectrometry the composition and dynamics of the signalosome assembling around the tagged protein prior to and following T cell activation. Affinity purification of the OST tagged protein was performed using Streptactin beads, from T cells that were non-stimulated, or stimulated for 30s or 120s with anti-CD3 and anti-CD4 antibodies, or 300s with pervanadate. Each AP-MS purification is associated with a corresponding control (purification from non edited WT CD4+ T cells, cultured and stimulated in the same conditions). The number of replicate biological experiments was n=5 (cells non-stimulated or stimulated for 30s with anti-CD3/CD4), n=2 (cells stimulated for120s with antiCD3/CD4), or n=3 (cells stimulated 300s with pervanadate), and each sample was analyzed in duplicate or triplicate by LC-MS, resulting in 78 raw files.
Project description:Analysis of CD4+ cells activated with anti-CD3 and cultured in the presence of IL2. The effects of TGFb and IL6 on Treg conversion were analyzed to discover methods of inhibiting Treg conversion and/or Treg functional inhibition. CD4+ T-cells were enriched from lymph nodes of Foxp3-GFP mice and cultured under the under the following conditions: 1) anti-CD3+IL2; 2) anti-CD3+IL2+TGFβ; 3) anti-CD3+IL2+TGFβ+IL6; and 4) anti-CD3+IL2+IL6. On day 4 cells were collected, stained with anti-CD4-PE, and sorted into CD4+FOXP3- and CD4+FOXP3+ populations (>95% purity). For each treatment condition T-effector cells and Tregs were labeled independently (either cy3 or cy5) and hybridized together for a two-color array experiment. Each treatment condition had dye swapped replicates, and a minimum of 3 replicate in total.