Transcriptional profiles of CCR7lo effector memory human T cell subsets
ABSTRACT: 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: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 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 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:In this study, we examined differential gene expression in naïve human CD4+ T cells, as well as in effector Th1, Th17-negative and Th17-enriched CD4- T cell subsets. We observed a marked enrichment for increased gene expression in effector CD4+ T cells compared to naive CD4+ among immune-mediated disease oci genes. Within effector T cells, expression of disease-associated genes was increased in Th17-enriched compared to Th17-negative cells. We used microarray to examine the gene expresssion profile and level of human naïve, Th1 and effector T cell subsets. Human PBMCs were isolated and sorted to naïve, CD161-CCR6- and CD161+CCR6+ memory T cells. Naïve T cells were differentiatied to Th1 cells, and CD161-CCR6- and CD161+CCR6+ memory T cells were in vitro expanded for Th17-negative and Th17-enriched effector T cells. The gene profile was compared among naive, Th1, Th17-negative, and Th17-enriched cell subsets.
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:In this study, we examined differential gene expression in naïve human CD4+ T cells, as well as in effector Th1, Th17-negative and Th17-enriched CD4- T cell subsets. We observed a marked enrichment for increased gene expression in effector CD4+ T cells compared to naive CD4+ among immune-mediated disease oci genes. Within effector T cells, expression of disease-associated genes was increased in Th17-enriched compared to Th17-negative cells. We used microarray to examine the gene expresssion profile and level of human naïve, Th1 and effector T cell subsets. Overall design: Human PBMCs were isolated and sorted to naïve, CD161-CCR6- and CD161+CCR6+ memory T cells. Naïve T cells were differentiatied to Th1 cells, and CD161-CCR6- and CD161+CCR6+ memory T cells were in vitro expanded for Th17-negative and Th17-enriched effector T cells. The gene profile was compared among naive, Th1, Th17-negative, and Th17-enriched cell subsets.
Project description:The aim of this study was to identify differential gene expression in lung T cell subsets upon germline Serpinb1a ablation. CD8-CD19- lymphocytes enriched by negative selection from digested lungs of naïve, 9-12 week old wild-type 129S6 mice or Serpinb1a homozygous null mice were FACS sorted into CD4+ CCR6+ effector/ memory T cells, gd T cells, or CD4+ CD25hiCD127low regulatory T cells. FACS sorted cells were collected into RLT buffer (Qiagen) directly, lysed, and total mRNA was isolated using Quiagen's RNeasy mini kit. Wild-type vs. Serpinb1a-/- gd+ T cells, CD4+ CCR6+ effector/ memory T cells, or CD4+ CD25hiCD127low regulatory T cells
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:We previously demonstrated that Th1Th17 cells are highly permissive to HIV-1, whereas Th1 cells are relatively resistant. Here, we investigated molecular mechanisms underlying these differences. Superior HIV replication in Th1Th17 vs. Th1 cells was regulated by entry and post-entry mechanisms. We used microarrays to detail the gene expression signatures caracterizing Th1 cells from Th1Th17. Primary human Th1 (CXCR3+CCR6- phenotype) and Th1Th17 (CXCR3+CCR6- phenotype) CD4+ T-cells were isolated by flow cytometry from HIV-uninfected, healthy donors. Cells were stimulated via CD3/CD28 for 3 days. The RNA was extracted and hybridized on the GeneChip® Human Genome U133 Plus 2.0 Array (Affymetrix).
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'