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.

Compelling evidence suggests that Foxp3-expressing CD25 + CD4 + regulatory T cells (Treg) are generated within the thymus as a separate lineage.

Foxp3 + CD4 + CD25 + regulatory T (T reg ) cells are essential for the prevention of autoimmunity 1,2 . T reg cells have an attenuated cytokine response to T-cell receptor stimulation, and can suppress the proliferation and effector function of neighbouring T cells 3,4 . The forkhead transcription factor Foxp3 (forkhead box P3) is selectively expressed in T reg cells, is required for T reg development and function, and is sufficient to induce a T reg phenotype in conventional CD4 + CD25 − T cells [5][6][7][8] . Mutations in Foxp3 cause severe, multi-organ autoimmunity in both human and mouse 9-11 . FOXP3 can cooperate in a DNA-binding complex with NFAT (nuclear factor of activated T cells) to regulate the transcription of several known target genes 12 . However, the global set of genes regulated directly by Foxp3 is not known and consequently, how this transcription factor controls the gene expression programme for T reg function is not understood. Here we identify Foxp3 target genes and report that many of these are key modulators of T-cell activation and function. Remarkably, the predominant, although not exclusive, effect of Foxp3 occupancy is to suppress

Regulatory T (T(Reg)) cells constitute a unique T-cell lineage that has a crucial role in immunological tolerance. Several years ago, forkhead box P3 (FOXP3) was identified as the transcription factor that was responsible for determining the development and function of these cells. However, the underlying mechanisms that are involved in the regulation of the FOXP3 gene remain unclear and therefore preclude accurate identification and manipulation of T(Reg) cells. In this Progress article, we summarize recent advances in understanding how FOXP3 expression is controlled and highlight evidence suggesting that epigenetic regulation of the FOXP3 locus contributes to its role as a lineage-specification factor.

Stable expression of Foxp3 in regulatory T cells (Tregs) depends on DNA demethylation at the Treg-specific demethylated region (TSDR), a conserved, CpG-rich region within the Foxp3 locus. The TSDR is selectively demethylated in ex vivo Tregs purified from secondary lymphoid organs, but it is unclear at which stage of Treg development demethylation takes place. In this study, we show that commitment to a stable lineage occurred during early stages of murine thymic Treg development by engraving of lineage-specific epigenetic marks in parallel with establishment of a Treg-specific gene expression profile. TSDR demethylation was achieved through an active mechanism and involved enzymes of the ten-eleven-translocation family and hydroxylation of methylated cytosines, a modification that is implicated as an initiating step of mitosis-independent DNA demethylation pathways and has not yet been observed at specific loci during immune cell differentiation. Together, our results demonstrate that initiating TSDR demethylation during early stages of thymic Treg development commences stabilization of Foxp3 expression and guarantees full functionality and long-term lineage stability of Tregs.