Epigenetic Control of the foxp3 Locus in Regulatory T Cells

Abstract

Compelling evidence suggests that the transcription factor Foxp3 acts as a master switch governing the development and function of CD4+ regulatory T cells (Tregs). However, whether transcriptional control of Foxp3 expression itself contributes to the development of a stable Treg lineage has thus far not been investigated. We here identified an evolutionarily conserved region within the foxp3 locus upstream of exon-1 possessing transcriptional activity. Bisulphite sequencing and chromatin immunoprecipitation revealed complete demethylation of CpG motifs as well as histone modifications within the conserved region in ex vivo isolated Foxp3+CD25+CD4+ Tregs, but not in naïve CD25−CD4+ T cells. Partial DNA demethylation is already found within developing Foxp3+ thymocytes; however, Tregs induced by TGF-β in vitro display only incomplete demethylation despite high Foxp3 expression. In contrast to natural Tregs, these TGF-β–induced Foxp3+ Tregs lose both Foxp3 expression and suppressive activity upon restimulation in the absence of TGF-β. Our data suggest that expression of Foxp3 must be stabilized by epigenetic modification to allow the development of a permanent suppressor cell lineage, a finding of significant importance for therapeutic applications involving induction or transfer of Tregs and for the understanding of long-term cell lineage decisions.

Is FOXP3 a bona fide marker for human regulatory T cells?

“…It is therefore possible that, depending on the cell subset and/ or the stage of T cell differentiation, FOXP3 expression can exert different functions and act as a negative or positive regulator. This is consistent with the fact that FOXP3 is a gene highly subjected to epigenetic modifications [22], which may contribute to diversifying its function.…”

Unravelling the molecular basis for regulatory T‐cell plasticity and loss of function in disease

“…The modifiable mediators of this regulation include methylation,37, 38, 39, 40, 41, 42, 43, 44 acetylation45, 46, 47 and microRNA‐mediated control of transcript translation 28, 48, 49, 50, 51, 52, 53, 54, 55. The global regulation of FOXP3 is influenced by chromatin organises, including SATB1 acts both during development and in mature cells 48, 58.…”

Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation

“…We recently identified a highly conserved region within the murine as well as the human FOXP3 gene locus that is completely [17,18]. We therefore analyzed the DNA methylation status of CD127 À Treg directly after isolation from PBMC to explore whether they contain significant numbers of in vivo induced Treg.…”

“…Lastly, the possibility had to be considered that natural Treg do not maintain FOXP3 expression indefinitely, but have the capacity to down-regulate FOXP3 and to convert to cells with variable phenotypic and functional characteristics under certain circumstances such as chronic stimulation. Here, we aimed to clarify these issues with the help of two recent advances in the field: First, we recently described that natural Treg differ from other T-cell populations in their DNA methylation status of a defined region within the FOXP3 locus termed Treg-specific demethylation region (TSDR): Demethylation of this region is exclusively observed in natural Treg, but neither in activated Tconv cells nor in TGF-b-induced Treg [17,18]. Second, the groups of Fazekas de Saint Groth and Bluestone reported that human CD4 1 CD25 1 Treg can be distinguished from activated (and thus also CD25 1 ) Tconv cells by their different surface expression levels of CD127 (IL-7 receptor a-chain, IL-7Ra): whereas activated Tconv cells are strongly positive for CD127, Treg show only low or no expression of this marker [19,20].…”

“…Any reduction in FOXP3 expression is therefore likely to interfere with correct assembly of such complexes and ultimately leads to loss of their repressive activity [31]. Interestingly, we and others recently identified putative binding sites for several transcription factors, including the transcriptional activator CREB/activating transcription factor (ATF) within the TSDR of the foxp3 locus [18,33]. Thus, DNA methylation in the TSDR, as seen in expanded CD45RA À or CD127 À Treg, could indeed result in reduced FOXP3 expression, as it was shown in murine cells that methylation of this region inversely correlated not only with CREB binding, but also with TCR-induced FOXP3 expression [33].…”