Predominant Requirement of Mitochondrial Transcriptional Factor A in Tissue-resident Regulatory T cell Maintenance and Function
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE131883 | GEO | 2020/04/13
REPOSITORIES: GEO
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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:We investigated the role of DNMT1 in immune homeostasis by generating mice lacking DNMT1 in Foxp3+ T-regulatory (Treg) cells. These mice showed decreased peripheral Foxp3+ Tregs, complete loss of Foxp3+ Treg suppressive functions in vitro and in vivo, and died from autoimmunity by 3-4 weeks unless they received perinatal transfer of wild-type Tregs that prolonged their survival. Methylation of CpG-sites in the TSDR region of Foxp3 was unaffected by DNMT1 deletion, but microarray revealed more >500 proinflammatory and other genes were upregulated in DNMT1-/- Tregs. CD4-Cre-mediated DNMT1 deletion showed inability of conventional T cells to convert to Foxp3+ Treg under appropriate polarizing conditions. Hence, DNMT1 is absolutely necessary for maintenance of the gene program required for normal Treg development and function. RNA from three independent samples of magnetically separated CD4+CD25+ Treg of fl-DNMT1/Foxp3cre mice, compared to wild type (C57BL6) control
2013-04-21 | E-GEOD-27434 | biostudies-arrayexpress
Project description:Regulatory T cells (Tregs) play crucial role in maintenance of peripheral tolerance. Numerous clinical trials confirmed safety and efficacy of Treg treatment of for deleterious immune responses. However, Tregs lose their characteristic phenotype and suppressive potential during expansion ex vivo. In our experiment we demonstrate that mild hypothermia of 33C induces robust proliferation of human Tregs, preserves expression of FoxP3, CD25 and Helios, and prevents TSDR methylation during culture in vitro. Tregs expanded at 33C showed stronger immunosuppressive potential and anti-inflammatory phenotype. We show that a simple change in temperature can preserve Treg stability, function and accelerate their proliferation in vitro.
2017-10-04 | E-MTAB-5322 | biostudies-arrayexpress
Project description:We investigated the role of DNMT1 in immune homeostasis by generating mice lacking DNMT1 in Foxp3+ T-regulatory (Treg) cells. These mice showed decreased peripheral Foxp3+ Tregs, complete loss of Foxp3+ Treg suppressive functions in vitro and in vivo, and died from autoimmunity by 3-4 weeks unless they received perinatal transfer of wild-type Tregs that prolonged their survival. Methylation of CpG-sites in the TSDR region of Foxp3 was unaffected by DNMT1 deletion, but microarray revealed more >500 proinflammatory and other genes were upregulated in DNMT1-/- Tregs. CD4-Cre-mediated DNMT1 deletion showed inability of conventional T cells to convert to Foxp3+ Treg under appropriate polarizing conditions. Hence, DNMT1 is absolutely necessary for maintenance of the gene program required for normal Treg development and function.
2013-04-21 | GSE27434 | GEO
Project description:CD4+ regulatory T cells (Tregs) are key mediators of immunological tolerance and promising effector cells for immuno-suppressive adoptive cellular therapy to fight autoimmunity and chronic inflammation. Their functional stability is critical for their clinical utility and has been correlated to the demethylated state of the TSDR/CNS2 enhancer element in the Treg lineage transcription factor FOXP3. However, proof for a causal contribution of the TSDR de-methylation to FOXP3 stability and Treg induction is so far lacking. We here established a powerful transient-transfection CRISPR-Cas9-based epigenetic-editing method for the selective de-methylation of the TSDR within the endogenous chromatin environment of a living cell. The induced de-methylated state was stable over weeks in clonal T cell proliferation cultures even after expression of the editing complex had ceased. Epigenetic editing of the TSDR resulted in FOXP3 expression, even in its physiological isoform distribution, proving a causal role for the de-methylated TSDR in FOXP3 regulation. However, successful FOXP3 induction was not associated with a switch towards a functional Treg phenotype, in contrast to what has been reported from FOXP3 overexpression approaches. Thus, TSDR de-methylation is required, but not sufficient for a stable Treg phenotype induction. Therefore, targeted demethylation of the TSDR may be a critical addition to published in vitro Treg induction protocols which so far lack FOXP3 stability.
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Project description:The epigenetic regulation of transcription factor genes is critical for T cell lineage specification. A specific methylation pattern within a conserved region of the lineage specifying transcription factor gene FOXP3, the Treg-specific demethylated region (TSDR), is restricted to regulatory T (Treg) cells and required for stable expression of FOXP3 and suppressive function. We analyzed the impact of hypomethylating agents 5-Aza-2`-deoxycytidine and Epigallocatechin-3-gallate (EGCG) on human CD4+CD25- T for generating Treg cell specific DNA methylation pattern within FOXP3-TSDR and inducing functional Treg cells. Gene expression, including lineage specifying transcription factors of the major T cell lineages and their leading cytokines, functional properties and global transcriptome changes were analyzed. 5-Aza-2`-deoxycytidine induced FOXP3-TSDR methylation and expression of Treg cell specific genes FOXP3 and LRRC32. Proliferation of 5-Aza-2´deoxycytidine treated cells was reduced, but they did not show suppressive function. Hypomethylation was not restricted to FOXP3-TSDR and expression of master transcription factors and leading cytokines of Th1 and Th17 cells were induced. EGCG induced global DNA hypomethylation to a lower degree than 5-Aza-2´deoxycitidine, but no relevant hypomethylation within FOXP3-TSDR or expression of Treg cell specific genes. Both DNMT inhibitors did not induce full functional human Treg cells. Although 5-Aza-2`-deoxycytidine treated cells phenotypically appeared to be Treg cells, they did not suppress proliferation of responder cells, which is an essential capability to be used in Treg cell transfer therapy.
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Project description:The epigenetic regulation of transcription factor genes is critical for T cell lineage specification. A specific methylation pattern within a conserved region of the lineage specifying transcription factor gene FOXP3, the Treg-specific demethylated region (TSDR), is restricted to regulatory T (Treg) cells and required for stable expression of FOXP3 and suppressive function. We analyzed the impact of hypomethylating agents 5-Aza-2`-deoxycytidine and Epigallocatechin-3-gallate (EGCG) on human CD4+CD25- T for generating Treg cell specific DNA methylation pattern within FOXP3-TSDR and inducing functional Treg cells. Gene expression, including lineage specifying transcription factors of the major T cell lineages and their leading cytokines, functional properties and global transcriptome changes were analyzed. 5-Aza-2`-deoxycytidine induced FOXP3-TSDR methylation and expression of Treg cell specific genes FOXP3 and LRRC32. Proliferation of 5-Aza-2´deoxycytidine treated cells was reduced, but they did not show suppressive function. Hypomethylation was not restricted to FOXP3-TSDR and expression of master transcription factors and leading cytokines of Th1 and Th17 cells were induced. EGCG induced global DNA hypomethylation to a lower degree than 5-Aza-2´deoxycitidine, but no relevant hypomethylation within FOXP3-TSDR or expression of Treg cell specific genes. Both DNMT inhibitors did not induce full functional human Treg cells. Although 5-Aza-2`-deoxycytidine treated cells phenotypically appeared to be Treg cells, they did not suppress proliferation of responder cells, which is an essential capability to be used in Treg cell transfer therapy. In this study we analyze the potency of the two hypomethylating agents 5-Aza-2`-deoxycytidine (5-Aza-dC) and Epigallocatechin-3-gallate (EGCG) for in vitro induction of functional Treg cell cells through generation of a specific methylation pattern within FOXP3-TSDR. We analyzed the expression of Treg cell specific genes and for their functional properties from CD4+CD25- T cells. 5-Aza-dC is a derivative of 5-Azacytidine. Both substances are inhibitors of DNA methyltransferases (DNMTs) and used for therapy of patients with myelodysplastic syndrome and acute myeloid leukaemia. In these patients, 5-Azacytidine has been reported to augment regulatory T cell expansion in blood. EGCG is the most abundant catechin of green tea and has been reported to have cardio protective, anti-cancer, anti-infective properties and protective effects on autoimmune diseases. EGCG has also been described as a potent inhibitor of DNMTs and to induce Foxp3 in Jurkat T cell line.
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