Project description:CD4+CD25+FOXP3+ human regulatory T cells (Treg) are essential for self-tolerance and immune homeostasis. Here, we generated genome-wide maps of poised and active enhancer elements marked by histone H3 lysine 4 monomethylation and histone H3 lysine 27 acetylation for CD4+CD25highCD45RA+ naive and CD4+CD25highCD45RA- memory Treg and their CD25- conventional T cell (Tconv) counterparts after in vitro expansion . In addition we generated genome-wide maps of the transcription factors STAT5, FOXP3, RUNX1 and ETS1 in expanded CD4+CD25highCD45RA+ Treg- and CD4+CD25- Tconv to elucidate their role in cell type-specific gene regulation. ChIP-seq of 2 histone marks and transcription factors ETS1, STAT5, FOXP3 and RUNX1 in expanded T cell subpopulations
Project description:Understanding human regulatory T cells (Tregs) heterogeneity may identify markers of disease pathogenesis and facilitate the development of optimized cellular therapeutics. To better elucidate human Treg subsets, we conducted direct transcriptional profiling of CD4+FOXP3+Helios+ thymic-derived Treg (tTreg) and CD4+FOXP3+Helios- peripherally-induced Treg (pTreg), followed by comparison to CD4+FOXP3-Helios- T conventional (Tconv) cells. This analysis revealed that the coinhibitory receptor T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) was highly expressed on tTreg. In this study CD4 T cells were stained for the Treg-associated transcription factors FOXP3 and Helios, and subsequently FACS sorted to yield three populations: tTreg (CD4+FOXP3+Helios+), pTreg (CD4+FOXP3+Helios–) and the reference population Tconv (CD4+FOXP3–Helios–). A direct transcriptional profile was obtained from the recovered RNA from the populations defined as tTreg, pTreg, and Tconv.
Project description:Several clinical trials have shown anti-CD3 treatment to be a promising therapy for autoimmune diabetes, but its mechanism of action remains unclear. Foxp3+ regulatory T (Treg) cells are likely to be involved, and we have shown a strong effect of anti-CD3 on homeostatic control of CD4+ FoxP3+ regulatory T (Treg) cells. To analyze the early consequences of anti-CD3 treatment, we sorted and profiled Treg and conventional CD4+ T (Tconv) cells in the first hours and days after anti-CD3 treatment of NOD mice. In practice, NOD mice carrying the Foxp3-GFP reporter were treated with anti-CD3 mAb KT3 (50 ug iv) and CD4+ T cells were sorted from pooled spleen and lymph nodes after 2, 8, 24 and 72 hrs, separating Treg and Tconv cells on the basis of GFP expression. Anti-CD3 treatment led to a transient transcriptional response, terminating faster than most antigen-induced responses. Most transcripts were similarly induced in Treg and Tconv cells, but several were differential, in particular those encoding the IL7 receptor (IL7R) and transcription factors Id2/3 and Gfi1, upregulated in Treg but repressed in Tconv cells. In parallel experiments, we tested the effect of soluble anti-CD3 added to cultures of fresh splenocytes, sorting Treg and Tconv cells at the same time points. Many of the anti-CD3 elicited changes, and of the differential response observed in vivo, were also observed in vitro. Two independent replicate series; Treg and Tconv samples abbreviated TR and TC, respectively. Keywords: Transcriptional activation, TCR All gene expression profiles were obtained from highly purified T cell populations sorted by flow cytometry. RNA from 5 x 104 cells was amplified, labeled, and hybridized to Affymetrix ST1.0 Gene arrays. Raw data were preprocessed with the RMA algorithm in GenePattern, and averaged expression values were used for analysis.
Project description:CD4+CD25+FOXP3+ human regulatory T cells (Treg) are essential for self-tolerance and immune homeostasis. Here, we generated genome-wide maps of poised and active enhancer elements marked by histone H3 lysine 4 monomethylation and histone H3 lysine 27 acetylation for CD4+CD25highCD45RA+ naive and CD4+CD25highCD45RA- memory Treg and their CD25- conventional T cell (Tconv) counterparts after in vitro expansion . In addition we generated genome-wide maps of the transcription factors STAT5, FOXP3, RUNX1 and ETS1 in expanded CD4+CD25highCD45RA+ Treg- and CD4+CD25- Tconv to elucidate their role in cell type-specific gene regulation.
Project description:Several clinical trials have shown anti-CD3 treatment to be a promising therapy for autoimmune diabetes, but its mechanism of action remains unclear. Foxp3+ regulatory T (Treg) cells are likely to be involved, and we have shown a strong effect of anti-CD3 on homeostatic control of CD4+ FoxP3+ regulatory T (Treg) cells. To analyze the early consequences of anti-CD3 treatment, we sorted and profiled Treg and conventional CD4+ T (Tconv) cells in the first hours and days after anti-CD3 treatment of NOD mice. In practice, NOD mice carrying the Foxp3-GFP reporter were treated with anti-CD3 mAb KT3 (50 ug iv) and CD4+ T cells were sorted from pooled spleen and lymph nodes after 2, 8, 24 and 72 hrs, separating Treg and Tconv cells on the basis of GFP expression. Anti-CD3 treatment led to a transient transcriptional response, terminating faster than most antigen-induced responses. Most transcripts were similarly induced in Treg and Tconv cells, but several were differential, in particular those encoding the IL7 receptor (IL7R) and transcription factors Id2/3 and Gfi1, upregulated in Treg but repressed in Tconv cells. In parallel experiments, we tested the effect of soluble anti-CD3 added to cultures of fresh splenocytes, sorting Treg and Tconv cells at the same time points. Many of the anti-CD3 elicited changes, and of the differential response observed in vivo, were also observed in vitro. Two independent replicate series; Treg and Tconv samples abbreviated TR and TC, respectively. Keywords: Transcriptional activation, TCR
Project description:In this study, we compared the proteomes of mouse CD4+Foxp3+ regulatory T cells (Treg) and CD4+Foxp3- conventional T cells (Tconv) in order to build a data set of proteins differentially regulated in these two cell populations. The data set contains mass spectrometry results from the analysis of 7 biological replicates of Treg/Tconv cell samples purified by flow cytometry, each experiment performed from a pool of 4-5 mice. Global proteomic analysis of each sample was performed by single-run nanoLC-MS/MS, using chromatographic separation of peptides on 50cm C18 reverse-phase columns, with either a 480min gradient on LTQ-Velos orbitrap mass spectrometer (replicates 1 and 2) or a 300min gradient on Q-Exactive orbitrap mass spectrometer (replicates 3-7). Several MS injection replicates were performed for some experiments, leading to 27 raw files composing the data set. The detailed description of each analysis (file name, sample type, biological replicate number, MS technical replicate number, MS instrument used, sample name in MaxQuant ouput) is given in the table “Files list.txt”.
Project description:Regulatory T (Treg) cells are important regulators of the immune system and have versatile functions for the homeostasis and repair of tissues. They express the forkhead box transcription factor Foxp3 as a lineage-defining protein. In mature Treg cells, the Foxp3 core promoter is unmethylated indicating that this area could harbor a transcription factor complex to initiate or repress gene expression, respectively. We used an unbiased method to identify Foxp3-promoter-binding transcription factors (TFs) by inverted chromatin immunoprecipitation (IP) followed by quantitative mass spectrometry. We identified several candidate factors which showed Foxp3-promoter suppressive capacity, one of which was T-cell factor 1 (Tcf1). Using viral overexpression and CRISPR/Cas knockout studies, we identified Tcf1 as a repressor of Foxp3 expression in primary conventional CD4 T cells (Tconv). In Tcf1-deficient animals, increased levels of Foxp3intermediateCD25negative T cells were identified in secondary lymphoid tissues, implicating that Tcf1 protects Foxp3-negative T cells from inadvertent Foxp3 expression.
Project description:Understanding human regulatory T cells (Tregs) heterogeneity may identify markers of disease pathogenesis and facilitate the development of optimized cellular therapeutics. To better elucidate human Treg subsets, we conducted direct transcriptional profiling of CD4+FOXP3+Helios+ thymic-derived Treg (tTreg) and CD4+FOXP3+Helios- peripherally-induced Treg (pTreg), followed by comparison to CD4+FOXP3-Helios- T conventional (Tconv) cells. This analysis revealed that the coinhibitory receptor T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) was highly expressed on tTreg.
Project description:Platelets are a rich source of many cytokines and chemokines including transforming growth factor β-1 (TGFβ1). TGFβ1 is required to convert conventional CD4+ T (Tconv) cells into induced regulatory T (iTreg) cells that express the transcription factor Foxp3. To explore whether other platelet contents will affect the properties of TGFβ induced Treg cell, we used platelet lysate that contain many other cytokines and chemokines besides TGFβ1 (pltTGFβ) to induce Foxp3 expression (pltTGFb-iTreg) from conventional CD4+ T (Tconv) cells. We used purified TGFβ1 to induce Treg (purTGFβ-iTreg) cells as a control. Gene expression profiles in iTreg cells were analyzed by microarray asay.