Down-regulation of Tet2 prevents TSDR demethylation in IL2 deficient regulatory T cells

doi:10.1016/j.bbrc.2014.06.110

Biochemical and Biophysical Research Communications

Volume 450, Issue 1, 18 July 2014, Pages 918-924

https://doi.org/10.1016/j.bbrc.2014.06.110 Get rights and content

Highlights

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TSDR was not demethylated in IL2 deficient Treg cells.
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Tet2 was down-regulated in IL2 deficient Treg cells.
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Over-expression of Tet2 restored TSDR demethylation in IL2 deficient Treg cells.
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TSDR in Tet2 deficient Treg cells was not demethylated.

Abstract

Stable expression of Foxp3 in regulatory T (Treg) cells is dependent on both intrinsic factors like epigenetic changes (demethylation) of Treg cell specific demethylation region (TSDR) and environmental cues like inflammations. Interleukin-2 (IL2) was reported to be one of the cytokines that give signals to Foxp3 stability but the underlying mechanism is still elusive. Here we show that IL2 and epigenetic changes in foxp3 locus are closely connected through tet methylcytosine dioxygenase 2 (Tet2) and, together help Treg cells to express Foxp3 stably. TSDR in foxp3 locus was not demethylated and Foxp3 expression was labile in IL2 deficient Treg cells, which was not restored by recombinant IL2, but correlated with the down-regulation of Tet2. Tet2 was up-regulated by TCR signaling and IL2 had a minimal effect. Rather, IL2 seemed to maintain the high level of Tet2 indirectly. Furthermore, over-expression of Tet2 restored TSDR demethylation in IL2 deficient Treg precursors. Collectively, our results suggest that up-regulation of Tet2 is required for Foxp3 stability and IL2 is required to maintain the high level of Tet2 during the thymic Treg development.

Introduction

Forkhead box P3⁺ (Foxp3⁺) Treg cells are a dedicated cell population that maintains immune tolerance and prevents autoimmune diseases [1], [2]. Since Treg cells regulate immune responses usually in a Foxp3-dependent manner [3], [4], the stability of Foxp3 is critical. Indeed, T cells whose Foxp3 was shut down have been reported to play roles in the development of some inflammatory diseases like diabetes [5] and lethal infection [6]. Stable Foxp3 expression is related to the epigenetic mechanisms, namely DNA demethylation of the conserved non-coding regions in foxp3 locus, called Treg cell specific demethylation region (TSDR) [7], [8]. In Treg cells with demethylated TSDR, Foxp3 protein binds to the demethylated TSDR and enhances its own expression, which induces the positive feedback loop [9]. However, the detail mechanism of TSDR demethylation is unknown.

Interleukin-2 (IL2) plays dual and often opposing roles in immune responses, contributing to both the generation of effector T cells and the maintenance of Foxp3⁺ Treg cells [10], [11]. Many studies indicate that IL2 provides essential signals for Tregs in at least 3 different levels: thymic development [12], [13], competitive fitness [14], [15] and Foxp3 stability [16], [17], [18], [19]. Here, we studied TSDR demethylation in IL2 deficient Treg cells and found that IL2 and epigenetic changes in foxp3 locus are closely related, which, as a result, helps Treg cells to express Foxp3 stably.

Section snippets

Mice

CD45.1 congenic (B6.SJL-Ptprc^a Pepc^b/BoyJ), IL2 deficient (B6.129P2-Il2^tm1Hor/J, Il2^−/−), Foxp3-GFP transgenic (Foxp3 bicistronic reporter mice expressing EGFP: B6.Cg-Foxp3^tm2Tch/J), Bcl2 transgenic (B6.Cg-Tg(BCL2)25Wehi/J, hBcl-2^TG), Lck-Cre transgenic (B6.Cg-Tg(Lck-cre)548Jxm/J), and floxed Tet2 transgenic (B6;129S-Tet2^tm1.1Iaai/J, Tet2^fl/fl) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Il2^−/− mice were crossed to Foxp3-GFP transgenic mice to identify Treg cells (GFP⁺) and

IL2 plays essential roles in TSDR demethylation

While the suppressive function of Il2^−/− peripheral Treg (pTreg) cells [22] was examined, we accidentally found that many Il2^−/− pTreg cells lost Foxp3 expression (data not shown). These results led us to study the role of IL2 in Foxp3 stability and TSDR demethylation. WT pTreg cells (GFP⁺CD25⁺ and GFP⁺CD25⁻) and naïve T cells (GFP⁻CD25⁻) were FACS-sorted and used for the analysis of TSDR. The purified cells were also cultured with recombinant IL2 (rIL2) and anti-CD3 plus anti-CD28 mAbs

Disclosures

The authors have no conflicting financial interests.

Acknowledgments

We thank Guoliang Xu for Tet2 expression vector; Hyun-Ah Lee (Central laboratory Kangwon National University, Korea) for cell sorting; Eunbyeol Cheon for genotyping; Divya Chandrasekharan Nair for editing. This work was supported by the National Research Foundation of Korea (NRF-2007-0052254, NRF-2013R1A1A2059821) and Hallym University (HRF-201211-016).

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Down-regulation of Tet2 prevents TSDR demethylation in IL2 deficient regulatory T cells

Highlights

Abstract

Introduction

Section snippets

Mice

IL2 plays essential roles in TSDR demethylation

Disclosures

Acknowledgments

Immunity

Immunity

Immunity

Immunity

Immunity

Trends Immunol.

Immunity

Cancer Cell

Immunity

Regulatory T cells: mechanisms of differentiation and function

Annu. Rev. Immunol.

Control of regulatory T cell development by the transcription factor Foxp3

Science

Foxp3 programs the development and function of CD4+CD25+ regulatory T cells

Nat. Immunol.

Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo

Nat. Immunol.