Project description:Human T cell clones were derived according to standard protocols, once assessed for their functional profile were studied for gene expression. The purpose of this study is to analyze human classic Th1, non-classic Th1 and Th17 clones with and without stimulation with anti-CD2+anti-CD3+anti-CD28 coated beads to measure differences in gene expression.
Project description:Human T cell clones were derived according to standard protocols using different mix of cytokines, once assessed for their functional profile were studied for gene expression. The purpose of this study is to analyze human Th1 and Th17 clones with and without stimulation with PMA and Ionomycin to measure differences in gene expression that can distinguish the two lineages.
Project description:Naive CD4+ T cells are highly plastic cells that - upon activation - can differentiate into various T helper (Th) cell fates characterized by the expression of specific transcription factors and effector cytokines. Their expression can be stabilized by epigenetic mechanisms including DNA methylation. So far, our knowledge about the link between DNA methylation and T helper cell differentiation processes is fragmentary. In this study, we generated methylomes of CD4+ T helper cells by performing a whole-genome bisulfite sequencing analysis of ex vivo isolated human naïve T cells and selected clones of Th1, non-classic Th1 and Th17 cells. The pairwise comparison of the methylomes identified differentially methylated regions (DMRs) mainly located within introns, followed by promoter regions and exons. In accordance with literature, several DMRs were identified within lineage-specific loci like IFNG, TBX21, IL-17A and RORC. Thus, the identified DMRs are suitable to further characterize the T helper lineages and might be helpful to define lineage-specific regulatory modules involved in human T helper cell differentiation.
Project description:Human T cell clones were derived according to standard protocols using different mix of cytokines, once assessed for their functional profile were studied for gene expression. The purpose of this study is to analyze human Th1 and Th17 clones with and without stimulation with PMA and Ionomycin to measure differences in gene expression that can distinguish the two lineages. Human T cell clones were derived according to standard protocols using different mix of cytokines, once assessed for their functional profile were studied for gene expression
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:Genome-wide DNA methylation profiling of human CD4+ T helper cells define epigenetic signatures for Th1, Th17 and non-classic Th1 cells
Project description:Th17 cells have better stemness and differentiation potential, and under certain circumstances, they can be stimulated to differentiate into other subcelltypes including Th1 and Treg. Here, we report that compared to traditional Th1 and Th17 cells, Th17-derived Th1 cells had greater anti-tumor activity. Both C57 mice ACT therapy and CAR-T therapy models were used to validate it. Mechanism: Th17-derived Th1 cells are able to express Th1 effector molecules as well as retain a lower level of exhaustion than Th1 cells. Additionally, it has a higher amount of type 1 interferon response and a stronger capacity for migration. Our research offers a fresh perspective on how ACT therapy and CAR-T therapy might develop in the future.
Project description:There is evidence that microglia interact with infiltrating Th1 and Th17 cells and this interaction results in mutual activation. However, the potential of a distinct cytokine milieu generated by these effector T cell subsets to activate microglia is poorly understood. In this study, we tested the ability of factors secreted by Th1 and Th17 cells to induce microglial activation. Interestingly, we found that only Th1-associated factors had the potential to activate microglia while the Th17-associated factors as well as direct contact of Th17 cells with microglia only had a minimal effect. Further Th1-derived factors triggered a proinflammatory M1-type gene expression profile in microglia Microglia harvested from mixed glial cultures were treated with supernatants from Th1- or Th17 cultures. Microglia cultured in medium was used as controls. At 16h post treatment RNA was isolated from the microglia and probed on Agilent´s murine 4x44k microarrays. RNA isolated from four independent experiments were used for the gene expression profiling. Microglia, Th1, Th17
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.