DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3⁺ conventional T cells

Abstract

The transcription factor FOXP3 is critical for development and function of regulatory T cells (Treg). Their number and functioning appears to be crucial in the prevention of autoimmunity and allergy, but also to be a negative prognostic marker for various solid tumors. Although expression of the transcription factor FOXP3 currently constitutes the best-known marker for Treg, in humans, transient expression is also observed in activated non-Treg. Extending our recent findings for the murine foxp3 locus, we observed epigenetic modification of several regions in the human FOXP3 locus exclusively occurring in Treg. Importantly, activated conventional CD4 + T cells and TGF-b-treated cells displayed no FOXP3 DNA demethylation despite expression of FOXP3, whereas subsets of Treg stable even upon extended in vitro expansion remained demethylated. To investigate whether a whole set of genes might be epigenetically imprinted in the Treg lineage, we conducted a genome-wide differential methylation hybridization analysis. Several genes were found displaying differential methylation between Treg and conventional Tcells, but none beside FOXP3 turned out to be entirely specific toTreg when tested on a broad panel of cells and tissues. We conclude that FOXP3 DNA demethylation constitutes the most reliable criterion for natural Treg available at present.

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

“…Second, when we analyzed the DNA methylation status of the TSDR within the foxp3 locus, we found complete demethylation of that region in all freshly sorted Treg populations. Based on our earlier studies, in which we showed that only natural, but not induced, Treg (as obtained after in vitro stimulation in the presence of TGF-b) display such a pattern [17,18], this clearly indicates that none of the starting populations contained significant numbers of induced Treg that rapidly re-convert to their effector phenotype upon in vitro culture. These findings suggest that natural Treg down-regulate FOXP3 expression upon repetitive re-stimulation in vitro and develop into cells with a nonregulatory phenotype.…”

“…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].…”

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.…”

“…Occupancy by specific transcription factors at each of these loci is now characterised, and this includes AP1 and NFAT at CNS1,59 Runx1 and CBFβ at CNS264 and cREl at CNS3 63. The observation that naïve human CD4+ T cells induce FOXP3 upon activation,65, 66, 67 and that in the presence of TGFβ and all‐trans retinoic acid (ATRA)63 the expression of FOXP3 is stabilised to some degree, was defined transcriptionally by the finding that the activation‐induced expression of FOXP3 results from partial but not complete demethylation of the FOXP3 locus in iTreg 37. This suggests that the relative methylation state of the FOXP3 regulatory elements encoded by CNS1, 2 and 3 are an axis for Treg plasticity.…”