Project description:We compared the methylated and non-methylated regions in the genome of ex vivo-isolated naive CD4+ T cells, Th1 cells, Th17 cells and regulatory T cells by methyl-CpG binding domain protein sequencing (MBD-seq). Naive T cells and Th1 cells share more methylated regions than naive T cells and Th17 cells or Th1 and Th17 cells. However, analysis of the non-methylated regions revealed the highest similarity between Th1 and Th17 cells. Another aim was the analysis of the Th17 lineage on the basis of the methylome. We searched for regions absent in the methylome of Th17 but present in naive T cells, Th1 cells and regulatory T cells. Here, we identified differential methylation in the loci of Il17a, Chn2, Dpp4 and Dclk1. CD4+ T effector cells were prepared ex vivo, stimulated with PMA/Ionomycin, subjected to a comercially available cytokine secretion kit (IL-17A and IFNg), stained by adding fluorescence-labeled antibodies against CD3, CD4 and CD45RB and sorted by flow cytometry. We sorted naive CD4+ T cells (CD3+CD4+CD45RB_high), Th1 cells (CD3+CD4+CD45RB_low_IFNg+IL17A-), Th17 cells (CD3+CD4+CD45RB_low_IFNg-IL17A+) and regulatory T cells (CD3+CD4+CD25++).
Project description:We compared the methylated and non-methylated regions in the genome of ex vivo-isolated naive CD4+ T cells, Th1 cells, Th17 cells and regulatory T cells by methyl-CpG binding domain protein sequencing (MBD-seq). Naive T cells and Th1 cells share more methylated regions than naive T cells and Th17 cells or Th1 and Th17 cells. However, analysis of the non-methylated regions revealed the highest similarity between Th1 and Th17 cells. Another aim was the analysis of the Th17 lineage on the basis of the methylome. We searched for regions absent in the methylome of Th17 but present in naive T cells, Th1 cells and regulatory T cells. Here, we identified differential methylation in the loci of Il17a, Chn2, Dpp4 and Dclk1.
Project description:Functionally distinct CD4+ helper T (Th) cell subsets, such as Th1, Th2, Th17, and regulatory T cells (Treg), play a pivotal role in the host-defense against pathogen invasion and the pathogenesis of inflammatory disorders. In this project, DIA-MS-based proteome analysis was performed on naïve CD4+ T, Th0, Th1, Th2, Th17 and iTreg cells using Q Exactive HF-X (Thermo Fisher Scientific) to search for proteins that differ among the cell subsets.
Project description:CD4+ T cells are critical components in the human immune system. They produce cytokines to fight against pathogens and abnormal cells and stimulate other cells, such as B cells, macrophages, and neutrophils, to generate an immune response.
Naive CD4+ T cells are precursor cells that can differentiate into T helper - 1, - 2, - 17 (Th1, Th2, Th17) and regulatory T cells (Tregs) subtypes based on the type of pathogens or disease. The naive CD4+ T cell model consists of 5179 reactions, 3153 metabolites, and 1055 genes. Together with Th1, Th2, and Th17 models, the naive CD4+ T cell model helped identify drug targets and repurposable drugs against autoimmune diseases.
Project description:In this study we determined whole genome gene expression of murine naive CD4+ T cells, in vitro differentiated Th17 cells, and CD4+ T cells isolated from experimental autoimmune encephalitomyelitis (EAE)-affected animals either after adoptive transfer of Th17 cells or after immunization with MOG35-55-peptide. The overall goal was to identify candidate genes involved in T cell pathology, encephalitogenicity and plasticity. These findings could then be correlated to multiple sclerosis pathology. Naive CD4+ T cells were isolated from B6.2d2 transgenic mice with MOG-specific T cell receptors and differentiated in vitro into Th17 cells. These Th17 cells were adoptively transferred into lymphopenic RAG1-/- mice to induce EAE. Further, EAE was induced by immunizing wild-type C57BL/6 mice with MOG35-55 peptide. RNA was extracted from naive CD4+ T cells, Th17 cells, and from CD4+ T cells isolated from the CNS of EAE-affected mice for gene expression analysis. Replicates from three independent experiments were analyzed.
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:Naive murine CD4+ T cells from GREAT/SMART-17A mice were cultured under Th1 or Tfh(1 ng/ml TGF-β)-polarizing conditions in 96-well plates coated with anti-CD3/anti-CD28 for 3.5 days; sorted by flow cytometry on IFNg+ (Th1), or CXCR5-IL17A+ (Th17) and CXCR5+IL17A- (Tfh); and subjected to bulk RNA-seq.
Project description:To analyse the influence of pentanoate on the differentiation of naive CD4 T cells under Th17 inducing conditions, murine CD4 T cells were isolated from spleen and lymphnodes of FIR/TIGER mice (IL-10 (GFP) - Foxp3 (RFP) - Reporter mice). The T cells were kept for 3 days under Th17 inducing conditions either with 4 mM pentanoate or without pentanoate (control).
Project description:FACS-sorted CR2+ and CR2- naive and memory CD4 T cells, plus CR2+ naive CD4 T cells activated with CD3/CD28 beads, were analyzed ex-vivo on a NanoString Immune Expression Profiling panel.