DNA methylation differences between human regulatory T cells and conventional T cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE49667 | GEO | 2013/09/09
SECONDARY ACCESSION(S): PRJNA214687
REPOSITORIES: GEO
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Project description:Regulatory T cells (Treg) prevent the emergence of autoimmune disease. Prototypic natural Treg (nTreg) are programmed by Forkhead-box P3 (FOXP3) and can be reliably identified by demethylation at the FOXP3 locus. To explore the nTreg methylation landscape we performed genome-wide methylation studies on human naïve resting nTreg (rTreg) and conventional naïve CD4+ T cells (Naïve). We detected 2,315 differentially methylated CpGs between these two cell types, many of which clustered into 127 regions of differential methylation (RDMs). T cell activation induced changes in 466 individual CpGs and 18 RDMs in naïve CD4+ T cells, but did not alter DNA methylation in rTreg. Expression of mRNA for TIGIT, an immune suppressive receptor demethylated in rTreg, was upregulated in nTreg and reduced in peripheral blood mononuclear cells of individuals at risk for autoimmune (type 1) diabetes. Gene-set testing of the 127 RDMs revealed enrichment of common Treg signature genes, FOXP3 bound genes and genes directly upregulated by FOXP3, which was primarily driven by the subset of demethylated RDMs. A putative Forkhead-binding motif overrepresented in promoter-associated RDMs suggests methylation regulates gene expression by influencing FOXP3 binding. Our findings provide new insights into epigenetic regulation of human nTreg and the potential to exploit differential methylation as an immune biomarker in human diseases. Naïve and rTreg cells were sorted from buffy coats of 3 healthy male donors (M28, 29, 30) and then activated for 6 days with anti-CD3 and anti-CD28 antibodies, supplemented with IL-2 at day 4. DNA was harvested and bisulfite converted for methylation analysis on illumina HM450 array from 2-3 biological replicates of each cell type: rTreg (M28, M30), naïve (M28, M29, M30), Act-naïve (M28, M29, M30) and Act-rTreg (M29, M30).
2013-09-09 | E-GEOD-49667 | biostudies-arrayexpress
Project description:The relative contribution of induced and natural Foxp3+ regulatory T cells (iTreg and nTreg cells, respectively) to the maintenance of tolerance is unknown. We examined their respective roles by in vivo adoptive transfer immunotherapy of newborn Foxp3-deficient BALB/c mice. Survival, weight gain, tissue infiltration, T cell activation, and the concentration of proinflammatory cytokines were used as outcome measurements. Treatment with iTreg cells alone was not successful. While effective in preventing death, treatment with nTreg cells alone was associated with chronic inflammation and autoimmunity. Outcomes markedly improved when conventional T (Tconv) cells were transferred together with the nTreg cells, where 10% of the peripheral Treg cell pool was derived by in-situ conversion. This enhancement depended upon the capacity of Tconv cells to express Foxp3. The gene expression profile of in vivo derived iTreg cells was similar to the established nTreg cell genetic signature. These results identify iTreg cells as an essential regulatory subset that supplements tolerance maintained by nTreg cells. Purified cells sorted by flow cytometry from 4-8 treated mice(pooled EGFP+ Thy1.1+ iTreg cells with that of EGFP+ Thy1.1+ nTreg cells sorted from the spleens and lymph nodes of treated mice) were used to generate total RNA for each iTreg and nTreg array set, which was labeled and hybridized to Affymetrix 430 2.0 GeneChips in accordance to the manufacturer’s protocol. Three sets of arrays were performed, and the results were averaged. Both iTreg and nTreg array sets were compared to a) naïve CD4+EGFP– Tconv cells from Foxp3EGFP mice and b) in vitro derived iTreg cells 72 hours after Foxp3 induction, generated earlier. The subset of probe sets whose expression increased or decreased by twofold or more relative to Tconv cells as a common standard was identified and used for further analysis. Naïve CD4+EGFP– Tconv cells from Foxp3EGFP mice and in vitro derived iTreg cells 72 hours after Foxp3 induction datasets obtained from GSE14415: Naïve CD4+EGFP– Tconv cells from Foxp3EGFP mice: GSM360171 - GSM360173 In vitro derived iTreg cells 72 hours after Foxp3 induction: GSM360147 - GSM360151
2011-06-30 | E-GEOD-19512 | biostudies-arrayexpress
Project description:The proposed use of Foxp3+ T-regulatory (Treg) cells as potential cellular therapy in patients with autoimmune diseases, or post-hemopoietic stem cell or organ transplantation, requires a sound understanding of the transcriptional regulation of Foxp3 expression. Conserved CpG dinucleotides in the Treg-specific demethylated region (TSDR) upstream of Foxp3 are demethylated only in stable, thymic-derived Foxp3+ Tregs. Since methyl-binding domain (Mbd) proteins recruit histone-modifying and chromatin-remodeling complexes to methylated sites, we tested whether targeting of Mbd2 might promote demethylation of Foxp3 and thereby promote Treg numbers or function. Surprisingly, while ChIP analysis showed Mbd2 binding to the Foxp3-associated TSDR site in Tregs, Mbd2 targeting by homologous recombination or siRNA decreased Treg numbers and impaired Treg suppressive function in vitro and in vivo. Moreover, we found complete TSDR demethylation in WT Tregs but >75% methylation in Mbd2-/- Tregs, whereas re-introduction of Mbd2 into Mbd2-null Tregs restored TSDR demethylation, Foxp3 gene expression and Treg suppressive function. Lastly, Mbd2-/- Tregs had markedly binding of the DNA demethylase enzyme, Tet2, in the TSDR region. These data show that Mbd2 has a key role in promoting TSDR demethylation, Foxp3 expression and Treg suppressive function. RNA from three independent samples from magnetically separated CD4+CD25+ Treg of MBD2–/– mice, compared to wild type control (all Balb/c background).
2013-09-01 | E-GEOD-48653 | biostudies-arrayexpress
Project description:The proposed use of Foxp3+ T-regulatory (Treg) cells as potential cellular therapy in patients with autoimmune diseases, or post-hemopoietic stem cell or organ transplantation, requires a sound understanding of the transcriptional regulation of Foxp3 expression. Conserved CpG dinucleotides in the Treg-specific demethylated region (TSDR) upstream of Foxp3 are demethylated only in stable, thymic-derived Foxp3+ Tregs. Since methyl-binding domain (Mbd) proteins recruit histone-modifying and chromatin-remodeling complexes to methylated sites, we tested whether targeting of Mbd2 might promote demethylation of Foxp3 and thereby promote Treg numbers or function. Surprisingly, while ChIP analysis showed Mbd2 binding to the Foxp3-associated TSDR site in Tregs, Mbd2 targeting by homologous recombination or siRNA decreased Treg numbers and impaired Treg suppressive function in vitro and in vivo. Moreover, we found complete TSDR demethylation in WT Tregs but >75% methylation in Mbd2-/- Tregs, whereas re-introduction of Mbd2 into Mbd2-null Tregs restored TSDR demethylation, Foxp3 gene expression and Treg suppressive function. Lastly, Mbd2-/- Tregs had markedly binding of the DNA demethylase enzyme, Tet2, in the TSDR region. These data show that Mbd2 has a key role in promoting TSDR demethylation, Foxp3 expression and Treg suppressive function.
2013-09-01 | GSE48653 | GEO
Project description:CD4+CD25+FOXP3+ regulatory T cells (Treg) are pivotal for peripheral self-tolerance. They prevent immune responses to auto- and alloantigens and are thus under close scrutiny as cellular therapeutics for autoimmune diseases and the prevention or treatment of alloresponses after organ or stem cell transplantation. We previously showed that human Treg cells with a memory cell phenotype, but not those with a naïve phenotype, rapidly down-regulate expression of the lineage-defining transcription factor forkhead box P3 (FOXP3) upon in vitro expansion. We now compared the transcriptomes of stable FOXP3+ Treg and converted FOXP3- 'ex-Treg' cells by applying a newly developed intranuclear staining protocol that permits the isolation of intact mRNA from fixed, permeabilized and FACS-purified cell populations. Whole genome microarray analysis revealed strong and selective upregulation of Th2 signature genes, including GATA-3, IL-4, IL-5 and IL-13, upon downregulation of FOXP3. Th2 differentiation of converted, FOXP3- ex-Treg cells occurred even under non-polarizing conditions and could not be prevented by IL-4 signaling blockade. Thus, our studies identify Th2 differentiation as the default developmental program of human Treg cells after downregulation of FOXP3. RNA preparations from FOXP3 stained in vitro expanded CD4+CD25highCD45RA- “memory” Treg cells from five independent donors were analyzed using Whole Human Genome Oligo Microarrays (Agilent). An adapted FOXP3 staining procedure to stain FOXP3 in human regulatory T cells to isolate intact RNA for microarray hybridization was developed (see extract protocol).
2011-12-14 | E-GEOD-26190 | biostudies-arrayexpress
Project description:In this screen we compared cDNA from rTreg (AICD resistent TReg cells) against cDNA derived from all CD4+CD25hi Treg for further molecular characterization of rTreg Experiment Overall Design: Single comparison of rTReg vs. TReg. We pooled rTreg-cDNA derived from FACS-sorted CD4+CD25hi Treg of eight healthy blood donors and performed gene chip microarray analysis.
2008-06-12 | E-GEOD-4243 | biostudies-arrayexpress
Project description:The forkhead transcription factor, Foxp3, is pivotal to the development and function of CD4+CD25+ T regulatory (Treg) cells that limit autoimmunity and maintain immune homeostasis. Previous data indicated that many of the functions of Foxp3 are controlled by the acetylation of several lysines within the forkhead domain. We now show that mutation of each of two lysines within the forkhead domain of Foxp3, lysine at position 382 (K17) and lysine at position 393 (K18), impaired Treg suppressive function in vivo and in vitro. Lysine mutations also decreased Treg expression of multiple functionally important Foxp3-regulated genes, and inhibited the promoter remodeling of target genes (CTLA-4 and IL-2) without affecting Foxp3 expression level. These data point to the need for a further understanding of the effects of various post-translational modifications on Foxp3 function. Our studies also provide a rationale for developing small molecule inhibitors of such post-translational modifications so as to regulate Foxp3+ Treg function clinically. RNA from three independent samples from magnetically separated CD4+CD25- T-effector cells transduced with EV, WT-Fopx3, Foxp3-K17R or Foxp3-K18R
2011-02-05 | E-GEOD-27082 | biostudies-arrayexpress
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.
2020-05-26 | PXD018764 | Pride
Project description:Introduction: Expansion of antigen (Ag)-specific natural occurring regulatory T cells (nTregs) is required to obtain sufficient numbers of cells for cellular immunotherapy. In this study, different allogeneic stimuli were studied for their capacity to generate functional alloAg-specific nTregs. Methods: A highly enriched nTreg-fraction (CD4+CD25brightCD127- T cells) was alloAg-specific expanded using HLA-mismatched immature, mature monocyte-derived dendritic cells (moDC) or peripheral blood mononuclear cells (PBMC). The allogeneic mature moDC-expanded nTregs were fully characterized by analysis of the demethylation status within the TSDR of the FOXP3 gene and the expression of both protein and mRNA of FOXP3, HELIOS, CTLA4 and cytokines. In addition, the antigen-specific suppressive capacity of these expanded nTregs was tested. Results: Allogeneic mature moDC and skin-derived DC were superior in inducing nTreg-expansion compared to immature moDC or PBMC in an HLA-DR and CD80/CD86-dependent way. Remarkably, the presence of exogenous IL-15 without IL-2, could facilitate optimal mature moDC-induced nTreg-expansion. Allogeneic mature moDC-expanded nTregs were at low ratios (<1:320), potent suppressors of alloAg-induced proliferation without significant suppression of completely HLA-mismatched-Ag-induced proliferation. Mature moDC-expanded nTregs were highly demethylated at the TSDR within the FOXP3 gene and highly expressed of FOXP3, HELIOS and CTLA4. A minority of the expanded nTregs produced IL-10, IL-2, IFN-g and TNF-a but very few IL-17 producing nTregs were found. Next generation sequencing of mRNA of moDC-expanded nTregs revealed a strong induction of Treg-associated mRNAs. Conclusions: Human allogeneic mature moDC are highly efficient stimulator cells, in presence of exogenous IL-15, for expansion of stable alloAg-specific nTregs with superior suppressive function. Four different batches of highly pure regulatory T cells (all from the same donor) were expanded in two different ways, and compared to non-expanded samples.
2015-04-20 | E-GEOD-66385 | biostudies-arrayexpress
Project description:Foxp3+ regulatory T cells (Treg), playing a crucial role in the maintenance of immune tolerance and prevention of autoimmune diseases, consist of thymus-derived naturally-occurring CD4+Foxp3+ Treg cells (nTreg) and another CD4+Foxp3+ Treg cells that can be induced ex vivo with TGF-β (iTreg). Although both Treg subsets share similar phenotypes and functional characteristics, they also have potential biologic differences on their biology. However, the role of iTreg in regulating B cells of lupus disease mice remains unclear so far. The lupus-prone New Zealand Mixed 2328 (NZM2328) mouse, a recombinant inbred strain that originated from the crosses among New Zealand Black and New Zealand White mice and their progenies also develops Lupus Glomerulonephritis. NZM2328 mice develop autoantibodies and glomerulonephritis with female predominance similarly to humans with systemic lupus erythematosus. The lupus disease onset is usually around 3-4 months age. In our experiments, iTreg induced from NZM2328 lupus-prone mice (10-12 weeks old) and nTreg sorted from the thymus of 10-12 weeks old NZM2328 lupus-prone mice were adoptively transferred into old NZM2328 mice (>4 months age) with established lupus for 32 days. Totally, there were three groups including NZM2328 mice as model (receive PBS), NZM2328 mice received iTreg, and NZM2328 received nTreg. The serum IgG and IgM autoantibody were detected by autoantibodies microarrays (UTSW Medical Center Autoantigen Array) at days 0, 14, 32 after cell transfer. Our results demonstrated that adoptive transfer of iTreg had a superior effect than nTreg subset on suppressing lupus B cell responses in vivo.
2017-01-26 | E-MTAB-4441 | biostudies-arrayexpress