FOXP3 Promoter Demethylation Reveals the Committed Treg Population in Humans

Abstract

BackgroundNaturally occurring thymus derived regulatory T cells (Tregs) are central in the maintenance of self-tolerance. The transcription factor FOXP3 is crucial for the suppressive activity of Tregs and is considered the most specific marker for this population. However, human non regulatory T cells upregulate FOXP3 transiently upon activation which calls for other means to identify the Treg population. Since epigenetic mechanisms are involved in the establishment of stable gene expression patterns during cell differentiation, we hypothesized that the methylation profile of the FOXP3 promoter would allow the distinction of truly committed Tregs.Methodology/Principal FindingsHuman CD4+CD25hi Tregs displayed a demethylated FOXP3 promoter (1.4%±0.95% SEM methylated) in contrast to CD4+CD25lo T cells which were partially methylated (27.9%±7.1%). Furthermore, stimulated CD4+CD25lo T cells transiently expressed FOXP3 but remained partially methylated, suggesting promoter methylation as a mechanism for regulation of stable FOXP3 expression and Treg commitment. In addition, transient FOXP3 expressing cells exhibited suppressive abilities that correlate to the methylation status of the FOXP3 promoter. As an alternative to bisulphite sequencing, we present a restriction enzyme based screening method for the identification of committed Tregs and apply this method to evaluate the effect of various culturing conditions. We show that a partial demethylation occurs in long-term cultures after activation, whereas the addition of TGF-β and/or IL-10 does not induce any additional change in methylation level.Conclusions/SignificanceThe unique FOXP3 promoter methylation profile in Tregs suggests that a demethylated pattern is a prerequisite for stable FOXP3 expression and suppressive phenotype. Presently, FOXP3 is used to identify Tregs in several human diseases and there are future implications for adoptive Treg transfer in immunotherapy. In these settings there is a need to distinguish true Tregs from transiently FOXP3+ activated T cells. The screening method we present allows this distinction and enables the identification of cells suitable for in vitro expansions and clinical use.

Human antigen‐specific CD4+CD25+CD134+CD39+ T cells are enriched for regulatory T cells and comprise a substantial proportion of recall responses

“…2A). A similar trend was also observed in the relative presence of the Treg-cell-specific demethylated region (TSDR), an epigenetic mark within the foxp3 gene proposed to identify stable, tTreg cells [27]. We analysed two healthy male donors and found that the average TSDR expression in SEB-specific CD4 + CD25 + CD134 + CD39 + T cells was 28.80%, which corresponds to approximately five times the average expression in SEB-specific CD4 + CD25 + CD134 + CD39 − T cells (6.19%).…”

Molecular mechanisms underlying the regulation and functional plasticity of FOXP3+ regulatory T cells

“…These findings suggest that the epigenetic modification of the Foxp3 gene is critical for the stable expression and suppressive function of Treg cells. These observations have also been confirmed in cord blood Treg cells 74 and peripheral Treg cells 75 in humans, where the latter transient expressers of FOXP3 bear a partially methylated TSDR. 75 Consistent with this FOXP3 þ regulatory T cell regulation and plasticity Y Gao et al notion, a TcR response element was found in the first intron of the Foxp3 gene that is located within this CNS region 76 and also Est-1 binding sites, which are important for positively regulating Foxp3 transcription.…”

“…These observations have also been confirmed in cord blood Treg cells 74 and peripheral Treg cells 75 in humans, where the latter transient expressers of FOXP3 bear a partially methylated TSDR. 75 Consistent with this FOXP3 þ regulatory T cell regulation and plasticity Y Gao et al notion, a TcR response element was found in the first intron of the Foxp3 gene that is located within this CNS region 76 and also Est-1 binding sites, which are important for positively regulating Foxp3 transcription. 77,78 The TSDR also overlaps with the binding site for the transcription factor cyclic AMP (cAMP) response element-binding, where an increase in TSDR methylation negatively correlates with cAMP response elementbinding and Foxp3 expression.…”

Enhancement of antigen-specific Treg vaccination in vivo

“…2) because cell cycling increases the expression of certain methylating enzymes such as DNA methyltransferase 1 (DNMT1), which could interfere with stable Foxp3 expression (13,22,23). Complete demethylation of CpG motifs at the foxp3 locus was shown to correlate with a stable human (24,25) and mouse Treg phenotype (13). Pharmacologic inhibition of the DNMT1 activity and knocking down or ablating DNMT1 gene markedly increased the efficacy of induction and stability of Foxp3 expression (13,22,23).…”