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      Hypomethylation at the Regulatory T Cell–Specific Demethylated Region in CD25 hi T Cells Is Decoupled from FOXP3 Expression at the Inflamed Site in Childhood Arthritis

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          Abstract

          The maintenance of FOXP3 expression in CD25 hi regulatory T cells (Tregs) is crucial to the control of inflammation and essential for successful Treg transfer therapies. Coexpression of CD25 and FOXP3 in combination with a hypomethylated region within the FOXP3 gene, called the Treg-specific demethylated region (TSDR), is considered the hallmark of stable Tregs. The TSDR is an epigenetic motif that is important for stable FOXP3 expression and is used as a biomarker to measure Treg lineage commitment. In this study, we report that, unlike in peripheral blood, CD4 + T cell expression of CD25 and FOXP3 is frequently dissociated at the inflamed site in patients with juvenile idiopathic arthritis, which led us to question the stability of human Tregs in chronic inflammatory environments. We describe a novel CD4 +CD127 loCD25 hi human T cell population that exhibits extensive TSDR and promoter demethylation in the absence of stable FOXP3 expression. This population expresses high levels of CTLA-4 and can suppress T conventional cell proliferation in vitro. These data collectively suggest that this population may represent a chronically activated FOXP3 lo Treg population. We show that these cells have defects in IL-2 signaling and reduced expression of a deubiquitinase important for FOXP3 stability. Clinically, the proportions of these cells within the CD25 hi T cell subset are increased in patients with the more severe courses of disease. Our study demonstrates, therefore, that hypomethylation at the TSDR can be decoupled from FOXP3 expression in human T cells and that environment-specific breakdown in FOXP3 stability may compromise the resolution of inflammation in juvenile idiopathic arthritis.

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          IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo.

          IL-2 plays a critical role in the maintenance of CD4+CD25+ FOXP3(+) regulatory T cells (Tregs) in vivo. We examined the effects of IL-2 signaling in human Tregs. In vitro, IL-2 selectively up-regulated the expression of FOXP3 in purified CD4+CD25+ T cells but not in CD4+CD25- cells. This regulation involved the binding of STAT3 and STAT5 proteins to a highly conserved STAT-binding site located in the first intron of the FOXP3 gene. We also examined the effects of low-dose IL-2 treatment in 12 patients with metastatic cancer and 9 patients with chronic myelogenous leukemia after allogeneic hematopoietic stem cell transplantation. Overall, IL-2 treatment resulted in a 1.9 median fold increase in the frequency of CD4+CD25+ cells in peripheral blood as well as a 9.7 median fold increase in FOXP3 expression in CD3+ T cells. CD56+CD3- natural killer (NK) cells also expanded during IL-2 therapy but did not express FOXP3. In vitro treatment of NK cells with 5-aza-2'-deoxycytidine restored the IL-2 signaling pathway leading to FOXP3 expression, suggesting that this gene was constitutively repressed by DNA methylation in these cells. Our findings support the clinical evaluation of low-dose IL-2 to selectively modulate CD4+CD25+ Tregs and increase expression of FOXP3 in vivo.
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            Stabilization of the transcription factor Foxp3 by the deubiquitinase USP7 increases Treg-cell-suppressive capacity.

            Stable Foxp3 expression is required for the development of functional regulatory T (Treg) cells. Here, we demonstrate that the expression of the transcription factor Foxp3 can be regulated through the polyubiquitination of multiple lysine residues, resulting in proteasome-mediated degradation. Expression of the deubiquitinase (DUB) USP7 was found to be upregulated and active in Treg cells, being associated with Foxp3 in the nucleus. Ectopic expression of USP7 decreased Foxp3 polyubiquitination and increased Foxp3 expression. Conversely, either treatment with DUB inhibitor or USP7 knockdown decreased endogenous Foxp3 protein expression and decreased Treg-cell-mediated suppression in vitro. Furthermore, in a murine adoptive-transfer-induced colitis model, either inhibition of DUB activity or USP7 knockdown in Treg cells abrogated their ability to resolve inflammation in vivo. Our data reveal a molecular mechanism in which rapid temporal control of Foxp3 expression in Treg cells can be regulated by USP7, thereby modulating Treg cell numbers and function. Copyright © 2013 Elsevier Inc. All rights reserved.
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              Quantitative DNA methylation analysis of FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue.

              Regulatory T-cells (Treg) have been the focus of immunologic research due to their role in establishing tolerance for harmless antigens versus allowing immune responses against foes. Increased Treg frequencies measured by mRNA expression or protein synthesis of the Treg marker FOXP3 were found in various cancers, indicating that dysregulation of Treg levels contributes to tumor establishment. Furthermore, they constitute a key target of immunomodulatory therapies in cancer as well as transplantation settings. One core obstacle for understanding the role of Treg, thus far, is the inability of FOXP3 mRNA or protein detection methods to differentiate between Treg and activated T cells. These difficulties are aggravated by the technical demands of sample logistics and processing. Based on Treg-specific DNA demethylation within the FOXP3 locus, we present a novel method for monitoring Treg in human peripheral blood and solid tissues. We found that Treg numbers are significantly increased in the peripheral blood of patients with interleukin 2-treated melanoma and in formalin-fixed tissue from patients with lung and colon carcinomas. Conversely, we show that immunosuppressive therapy including therapeutic antibodies leads to a significant reduction of Treg from the peripheral blood of transplantation patients. In addition, Treg numbers are predictively elevated in the peripheral blood of patients with various solid tumors. Although our data generally correspond to data obtained with gene expression and protein-based methods, the results are less fluctuating and more specific to Treg. The assay presented here measures Treg robustly in blood and solid tissues regardless of conservation levels, promising fast screening of Treg in various clinical settings.
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                Author and article information

                Journal
                J Immunol
                J. Immunol
                jimmunol
                jimmunol
                JI
                The Journal of Immunology Author Choice
                AAI
                0022-1767
                1550-6606
                15 September 2014
                04 August 2014
                : 193
                : 6
                : 2699-2708
                Affiliations
                [* ]Infection, Inflammation and Rheumatology Section, University College London Institute of Child Health, London WC1N 1EH, United Kingdom; and
                []Medical School, Imperial College London, London SW7 2AZ, United Kingdom
                Author notes
                [1]

                Current address: Department of Surgery, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.

                Address correspondence and reprint requests to Dr. David Bending, University College London Institute of Child Health, 30 Guilford Street, London WC1N 1EH, U.K. E-mail address: d.bending@ 123456ucl.ac.uk
                Author information
                http://orcid.org/0000-0003-0071-1163
                Article
                ji_1400599
                10.4049/jimmunol.1400599
                4157061
                25092890
                09b5fd51-e234-40a4-a9dc-ce99f20307e7
                Copyright © 2014 The Authors

                This is an open-access article distributed under the terms of the CC-BY 3.0 Unported license .

                History
                : 06 March 2014
                : 02 July 2014
                Page count
                Pages: 10
                Categories
                Clinical and Human Immunology

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