Dynamic Imprinting of the Treg Cell-Specific Epigenetic Signature in Developing Thymic Regulatory T Cells.

Susanne Herppich,Yohko Kitagawa,Shohei Hori,Aras Toker,Stefan Floess,Shimon Sakaguchi,Naganari Ohkura,Takahisa Miyao,Jochen Huehn,Beate Pietzsch

doi:10.3389/fimmu.2019.02382

Abstract

Regulatory T (Treg) cells mainly develop within the thymus and arise from CD25+Foxp3− (CD25+ TregP) or CD25−Foxp3+ (Foxp3+ TregP) Treg cell precursors resulting in Treg cells harboring distinct transcriptomic profiles and complementary T cell receptor repertoires. The stable and long-term expression of Foxp3 in Treg cells and their stable suppressive phenotype are controlled by the demethylation of Treg cell-specific epigenetic signature genes including an evolutionarily conserved CpG-rich element within the Foxp3 locus, the Treg-specific demethylated region (TSDR). Here we analyzed the dynamics of the imprinting of the Treg cell-specific epigenetic signature genes in thymic Treg cells. We could demonstrate that CD25+Foxp3+ Treg cells show a progressive demethylation of most signature genes during maturation within the thymus. Interestingly, a partial demethylation of several Treg cell-specific epigenetic signature genes was already observed in Foxp3+ TregP but not in CD25+ TregP. Furthermore, Foxp3+ TregP were very transient in nature and arose at a more mature developmental stage when compared to CD25+ TregP. When the two Treg cell precursors were cultured in presence of IL-2, a factor known to be critical for thymic Treg cell development, we observed a major impact of IL-2 on the demethylation of the TSDR with a more pronounced effect on Foxp3+ TregP. Together, these results suggest that the establishment of the Treg cell-specific hypomethylation pattern is a continuous process throughout thymic Treg cell development and that the two known Treg cell precursors display distinct dynamics for the imprinting of the Treg cell-specific epigenetic signature genes.

Highlights

CD4+ regulatory T (Treg) cells are crucial for the maintenance of self-tolerance
We had previously demonstrated that thymus-derived Treg (tTreg) cell maturation, which manifests as a progressive demethylation of the Treg-specific demethylated region (TSDR), is a continuous process that progresses after up-regulation of Foxp3 expression [12]
In order to study the dynamics of this maturation process in more detail and in a more precise system, we here made use of transgenic Foxp3hCD2xRag1GFP reporter mice, which express green fluorescent protein (GFP) under control of the recombination-activating gene 1 (Rag1) promoter [25]

Summary

Introduction

The continuous expression of the lineage-specification factor, Foxp, endows these immunoregulatory cells with long-term stability and suppressive activity [1, 2]. In vivo disruption of the Foxp gene by green fluorescent protein (GFP; Foxp3gfpko mice) resulted in Foxp3−GFP+ cells still expressing several Treg cell-specific signature genes [8]. To this end, it was shown that the CpG DNA demethylation at a set of Treg cell-specific epigenetic signature genes essentially but independently complements Foxp expression for entire Treg cell functionality and long-term lineage stability [9,10,11,12]. Significant progress has been made in understanding the importance of epigenetic imprinting on generating stable Treg cells, factors that initiate and drive this imprinting process are still incompletely understood

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Results

Conclusion