Transcriptional profiling of Th1, Th2, Th9, and Th17 T cell clones
ABSTRACT: 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 previously reported that CCR4+CCR6+Th17 cells are permissive to HIV, while CXCR3+CCR6- Th1 cells are relatively resistant. To identify molecular mechanisms underlying these differences, we performed a genome-wide transcriptional profiling in CXCR3+CCR6-Th1, CCR4+CCR6-Th2, CCR4+CCR6+Th17, and CXCR3+CCR6+Th1Th17 upon TCR triggering. Transcriptional differences between Th17 and Th1 were the most remarkable. HIV-DNA integration was highly efficient in Th17 versus Th1 upon exposure to both wild-type and VSV-G-pseudotyped HIV, indicative that post-entry mechanisms contribute to HIV permissiveness. 4 cell populations from up to 5 donors for a total of 19 samples.
Project description:We wish to show that Th2A cells have a distinct gene expression profile compared to Th2 cells in allergic subjects Overall design: We sorted conventional Th1 cells (CXCR3+ CCR4- CCR6-), conventional Th17 cells (CCR6+), Th2A cells (CRTH2+ CD161+ CD49d+ CD27- CD45RB-) and conventional Th2+ cells (CRTH2+ CD161-) from the PBMC of 3 subject pools. Each pool consisted of cells from 2-3 allergic donors. Sorted TH subsets were stimulated for 6 hours with anti-CD3/CD28 beads (Life Technologies, STIM samples) or left unstimulated (CTRL samples) prior to extraction of RNA (RNeasy Mini kit; Qiagen).
Project description:The aim of this study was to identify differentially-expressed genes in CCR4hi/CXCR3- and CCR4lo CXCR3+ CCR6+ human Th17 cell subsets Human CD45RO+ memory T cells isolated from the peripheral blood of healthy adult donors were sorted into 4 predominant CCR7lo CD25- effector memory subsets: (1) Th1 - CCR6- CCR4lo CXCR3+; (2) Th2 - CCR6- CCR4hi CXCR3+; (3) Th17 - CCR6+ CCR4hi CXCR3-; (4) Th17.1 - CCR6+ CCR4lo CXCR3-. Sorted cells were cultured in media and activated via anti-CD3/anti-CD28 beads for 36 hours. All subsets were then harvested and used for RNA extraction and microarray experiments. Th1 vs Th2; Th1 vs Th17; Th1 vs Th17.1; Th2 vs Th17; Th2 vs Th17.1; Th17 vs Th17.1
Project description:In latent tuberculosis infection (LTBI) spread of the bacteria is contained by a persistent immune response, which includes CD4+ T cells as important contributors. Here we show that TB-specific CD4+ T cells have a characteristic chemokine expression signature (CXCR3+CCR6+CCR4-; Th* cells), and that the overall number of Th* cells is significantly increased in LTBI donors. We have comprehensively characterized the transcriptional signature of Th* cells and find significant differences to conventional Th1, Th17 and Th2 cells, but no major changes between healthy and LTBI donors. Th* cells display linage-specific signatures of both Th1 and Th17 cells, but also have a unique gene expression program including genes associated with susceptibility to TB, enhanced T cell activation, enhanced cell survival, and induction of a cytotoxic program akin to CTL cells. Overall, the gene expression signature of Th* reveals characteristics of cells important in controlling latent infections. Overall design: Examination of gene expression in 5 human cell types in healthy and Latent TB infected individuals . The 'Differentially_expressed_genes.xlsx' contains genes that are differentially expressed in pair-wise comparisons of different cell types and disease groups. The data format/content description is provided in the 'readme.txt'
Project description:Human IL-10– and IL-10+ TH17 clones maintained their pro- or anti-inflammatory characteristics after long-term culture. There were similarities between human IL-10– vs. IL-10+ TH17 clones and mouse pathogenic vs. non-pathogenic TH17 cells. Overall design: From healthy donors (n=5), CD4+ T cells clones were established from CCR6+CCR4+CXCR3– memory CD4+ T cells that were enriched for TH17 cells 14, 18 and screened for clones producing IL-17 with or without co-secretion of IL-10.
Project description:Th17 cells act as the first line of defense against pathogens at mucosal surfaces. Their paucity during HIV infection causes disease progression. Here, we reveal the existence of two new CCR6+CD161+ subsets, CCR4-CXCR3- (DN; double negative) and CCR4+CXCR3+ (DP; double positive), expressing typical Th17 transcripts (e.g., RORγt, RORα, PTPN13, ARNTL), C. albicans specificity, and exhibiting Th17-lineage commitment versus flexibility. 4 cell populations from up to 6 donors for a total of 20 samples.
Project description:Human T-cells that express CXCR5 and/or CCR6 provide help to naive B-cells for IgG production. To understand the molecular pathways that are shared or unique to individual B helper T-cell subsets in human peripheral blood, CD4+IL-7R+CD25-/lo helper T-cells were purified according to the expression of CXCR5, CCR6, CXCR3, CD161 and CCR5 as follows: 1) TFH17(CD161-): CXCR5+CXCR6+CXCR3-CD161- 2) Th17: CXCR5-CCR6+CXCR3-, CD161+ or CCR5+ 3) CCR6”SP”: CXCR5-CCR6+CXCR3-CD161-CCR5- RNA-seq was performed with FACS-purified T-cell subsets from 3 healthy individuals.
Project description:Purified CD4 T cells isolated from the cord blood were divided in subsets according to IL2R, IL7R and chemokine receptors (CCR6, CXCR3 and CCR4) and compared by whole genome microarray analysis. CD4 T cell memory subsets and Naive T cell collected from 3 donors (#480, #487, #488)
Project description:TL1A contributes to the pathogenesis of several chronic inflammatory diseases, including Inflammatory Bowel Diseases by enhancing TH1, TH17, and TH2 responses. TL1A mediates a strong co-stimulation of these TH subsets particularly of mucosal CCR9+ T cells. However, the signaling pathways that TL1A induces in different TH subsets are incompletely understood. Here, we investigated the function of TL1A on human TH17 cells. TL1A together with TGF- IL-6, and IL-23 enhanced the secretion of IL-17 and IFN- from human CD4+ memory T cells. TL1A induced the expression of the transcription factors BATF and T-bet that correlated with the secretion of IL-17 and IFN-. In contrast, TL1A alone induced high levels of IL-22 in memory CD4+ T cells and committed TH17 cells. However, TL1A did not enhance expression of IL-17A in TH17 cells. Expression of the transcription factor aryl hydrocarbon receptor that regulates expression of IL-22 was not affected by TL1A. We performed transcriptome analysis of TH17 cells to determine genes that are transcriptionally regulated by TL1A. transcriptome analysis revealed increased expression of IL-9 in response to TL1A. Overall design: Total RNA was obtained from human TH17 cells (sorted by flow cytometry for CD4+ CD45RO+ CCR6+) that were untreated (control) or treated with TL1A (TL1A) for 24, 48, or 72 h. Total RNA was extracted from 5 healthy donors (5 independent experiments).