RNAseq of Tregs before and after treatment with Multipep
Ontology highlight
ABSTRACT: Intent: to investigate whether treatment of an individual with peptide immunotherapy using MultiPepT1De results in a change in the transcriptional profile of CD4+ regulatory T cells. Regulatory T cells (CD4+ CD25+ CD127lo) were isolated by flow cytometry cell sorting from cryopreserved PBMC from subjects before and after treatment with MultiPepT1De. RNA was extracted using the AllPrep micro kit (Qiagen) and RNAseq libraries prepared from extracted RNA (91-140 ng)
Project description:Regulatory CD4+ T cells (Tregs) are functionally distinct from conventional CD4+ T cells (Tconvs). To understand Treg identity, we compared by proteomics and transcriptomics human naïve (n) and effector (e)Tregs, Tconvs and transitional FOXP3+ cells. Only 12% of 422 differentially expressed proteins was identified as such at the mRNA level, demonstrating the importance of direct proteome measurement. Fifty-one proteins discriminated Tregs from Tconvs. This common Treg protein signature indicates altered signaling by TCR-, TNF receptor-, NFB-, PI3 kinase/mTOR-, NFAT- and STAT pathways, and unique cell biological and metabolic features. Another protein signature uniquely identified eTregs and revealed active cell division, apoptosis sensitivity and suppression of NFB- and STAT signaling. eTreg fate appears consolidated by FOXP3 outnumbering its partner transcription factors. These features explain why eTregs cannot produce inflammatory cytokines, whereas transitional FOXP3+ cells can. Collectively, our data reveal that Treg identity is defined and protected by uniquely “wired” signaling pathways.
Project description:RNA sequencing of the chromatin associated RNA and nucleoplasm associated RNA of Naive CD4+ T cells to identify novel chromatin associated RNAs containing TEs. RNA sequencing of Naive CD4+ T cells or Activated Naive CD4+ T cells treated with Scr or LINE1 antisense oligonucleotides (ASO).
Project description:Generation of transcriptomes to provide in-depth expression profiles of human Treg and Tconv from the Naive (CD45RA+CD45RO-) and Memory (CD45RA-CD45RO+) compartments
Project description:Generation of transcriptomes to provide in-depth expression profiles of tTreg and tTconv and to obtain the differential expression signature defining thymic Tregs in humans.
Project description:Peripheral Blood Mononuclear Cells (PBMCs) were isolated from a buffy coat (Australian Blood Bank) using Ficoll methodology. CD4+ T cells were isolated using Dynal Beads kit. Pure CD4+ T cells were then stained using a cocktail of monoclonal antobodies (mAbs), including: anti-CD4PE, CD45RO ECD, CD62L APC-Cy7, CD25 APC, CD127 Pacific Blue. After incubation, cells were washed twice in PBS/FCS (0.2%), and sorted into five different cell subsets: CD4+CD25+CD127low CD62L+CD45RO- (naive regulatory T cells), CD4+CD25+CD127low CD62L+/- CD45RO+ (activated regulatory T cells), CD4+CD25+CD127hi CD62L+/- CD45RO+ (memory T cells), CD4+CD25-CD127low CD62L+/- CD45RO+ (effector T cells) and CD4+CD25-CD127hi CD62L+ CD45RO- (naive T cells).
Project description:To find specific transcription factors (TFs) interact with Altre to regulate target gene expression by Cis- or Trans- action on chromatin.
Project description:Single-cell transcriptome of >55,000 cells multiplexed into 4 channels obtained from peripheral blood and synovial fluid of two patients with HLA-B27+ ankylosing spondylitis,.
Project description:The aim of the project is to do proteomic analysis of total cell lysate from rest and activated mouse Tregs. The proteomic data were mapped to the Gene Ontology Cellular Component (GOCC)database to obtain the list of membrane proteins on activated Tregs. The set of membrane proteins identified were compared with the potential Siglec-1 counter-receptors on activated Tregs.
Project description:The therapeutic use of regulatory T cells (Tregs) in patients with autoimmune disorders has been hampered by the biological variability of memory Treg populations in the peripheral blood. In this study, we reveal through a combination of quantitative proteomic, multiparametric flow cytometry, RNA-seq data analysis and functional assays, that CD49f is heterogeneously expressed among human Tregs and impacts their immunomodulatory function. High expression of CD49f defines a subset of dysfunctional Tregs in the human blood characterized by a Th17-like phenotype and impaired suppressive capacity. CD49f is similarly distributed between naïve and memory Tregs and impacts the expression of CD39, CTLA-4, FoxP3 and CCR6 specifically in the memory compartment. Accumulation of CD49f high memory Tregs in the blood of ulcerative colitis patients correlates with disease severity. Our results highlight important considerations for Treg immunotherapy design in patients with inflammatory bowel disease which could possibly extend to other autoimmune disorders.