Jochen Huehn

Epigenetic Control of the foxp3 Locus in Regulatory T Cells

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.

Selective depletion of Foxp3+ regulatory T cells induces a scurfy-like disease

The scurfy mutant mouse strain suffers from a fatal lymphoproliferative disease leading to early death within 3–4 wk of age. A frame-shift mutation of the forkhead box transcription factor Foxp3 has been identified as the molecular cause of this multiorgan autoimmune disease. Foxp3 is a central control element in the development and function of regulatory T cells (T reg cells), which are necessary for the maintenance of self-tolerance. However, it is unclear whether dysfunction or a lack of T reg cells is etiologically involved in scurfy pathogenesis and its human correlate, the IPEX syndrome. We describe the generation of bacterial artificial chromosome–transgenic mice termed “depletion of regulatory T cell” (DEREG) mice expressing a diphtheria toxin (DT) receptor–enhanced green fluorescent protein fusion protein under the control of the foxp3 gene locus, allowing selective and efficient depletion of Foxp3+ T reg cells by DT injection. Ablation of Foxp3+ T reg cells in newborn DEREG mice led to the development of scurfy-like symptoms with splenomegaly, lymphadenopathy, insulitis, and severe skin inflammation. Thus, these data provide experimental evidence that the absence of Foxp3+ T reg cells is indeed sufficient to induce a scurfy-like phenotype. Furthermore, DEREG mice will allow a more precise definition of the function of Foxp3+ T reg cells in immune reactions in vivo.

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

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‐β‐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 T cells, but none beside FOXP3 turned out to be entirely specific to Treg 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.

DNA methylation controls Foxp3 gene expression.

Compelling evidence suggests that Foxp3‐expressing CD25+CD4+ regulatory T cells (Treg) are generated within the thymus as a separate lineage. However, Foxp3+CD4+ Treg can also be generated de novo in a TGF‐β‐dependent process from naive T cells by TCR triggering. Recently, we have shown that naturally occurring, but not in vitro TGF‐β‐induced Foxp3+ Treg display stable Foxp3 expression that was associated with selective demethylation of an evolutionarily conserved element within the Foxp3 locus named TSDR (Treg‐specific demethylated region). Here, we report that inhibition of DNA methylation by azacytidine, even in absence of exogenous TGF‐β, not only promoted de novo induction of Foxp3 expression during priming, but also conferred stability of Foxp3 expression upon restimulation. Most notably, such stable Foxp3 expression was found only for cells displaying enhanced TSDR demethylation. In contrast, in vitro TSDR methylation diminished its transcriptional activity. Foxp3+ Treg generated in vivo by DEC‐205‐mediated targeting of agonist ligands to dendritic cells showed long‐term survival in the absence of the inducing antigen and exhibited efficient TSDR demethylation. Together, our data suggest that TSDR is an important methylation‐sensitive element regulating Foxp3 expression and demonstrate that epigenetic imprinting in this region is critical for establishment of a stable Treg lineage.

Expression of the integrin αEβ7 identifies unique subsets of CD25+ as well as CD25− regulatory T cells

Regulatory CD25+CD4+ T cells are considered as important players in T cell homeostasis and self-tolerance. Here we report that the integrin αEβ7, which recognizes epithelial cadherin, identifies the most potent subpopulation of regulatory CD25+ T cells. Strikingly, CD25-negative αE+CD4+ T cells displayed regulatory activity. Both αE+ subsets, CD25+ and CD25−, express CTLA-4, suppress T cell proliferation in vitro, and protect mice from colitis in the severe combined immunodeficient model (SCID) in vivo. Whereas αE+CD25+ T cells produce almost no cytokines, αE+CD25− T cells represent a unique subset in which high IL-2, IFN-γ and T helper 2-cytokine production is linked with suppressive function. Thus, the integrin αEβ7 can be regarded as a novel marker for subsets of highly potent, functionally distinct regulatory T cells specialized for crosstalk with epithelial environments.

Epigenetic control of FOXP3 expression: the key to a stable regulatory T-cell lineage?

Regulatory T (TReg) 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 TReg 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.

T Cell Receptor Stimulation-Induced Epigenetic Changes and Foxp3 Expression Are Independent and Complementary Events Required for Treg Cell Development

The transcription factor Foxp3 is essential for the development of regulatory T (Treg) cells, yet its expression is insufficient for establishing the Treg cell lineage. Here we showed that Treg cell development was achieved by the combination of two independent processes, i.e., the expression of Foxp3 and the establishment of Treg cell-specific CpG hypomethylation pattern. Both events were induced by T cell receptor stimulation. The Treg cell-type CpG hypomethylation began in the thymus and continued to proceed in the periphery and could be fully established without Foxp3. The hypomethylation was required for Foxp3(+) T cells to acquire Treg cell-type gene expression, lineage stability, and full suppressive activity. Thus, those T cells in which the two events have concurrently occurred are developmentally set into the Treg cell lineage. This model explains how Treg cell fate and plasticity is controlled and can be exploited to generate functionally stable Treg cells.

Maternal farm exposure modulates neonatal immune mechanisms through regulatory T cells

BACKGROUND Cross-sectional studies suggest that maternal exposure to farming decreases the risk of allergic diseases in offspring. The potential underlying immunologic mechanisms are not understood. OBJECTIVE We sought to assess whether maternal farm exposure activates regulatory T (Treg) cells in cord blood, exerting T(H)2-suppressive effects after microbial stimulation. METHODS Eighty-four pregnant mothers were recruited before delivery. Detailed questionnaires (60 nonfarming and 22 farming mothers with 2 exclusions) assessed the farming exposures. Cord blood was stimulated with the microbial stimulus peptidoglycan (Ppg), the mitogen PHA, house dust mite extracts (Der p 1), and combinations. Treg cells (CD4+CD25(high) cells; intracellular forkhead/winged-helix family transcriptional repressor p3 [FOXP3] expression, FOXP3 levels, lymphocyte activation gene 3 mRNA expression, functional studies, and DNA methylation of the FOXP3 locus), proliferation, and T(H)2/T(H)1/T(H)17 cytokines were examined. RESULTS Cord blood Treg cell counts (both unstimulated and PHA stimulated) were increased with maternal farming exposures and associated with higher FOXP3 (Der p 1 + Ppg stimulation) and trendwise higher lymphocyte activation gene 3 (Ppg) expression. Furthermore, Treg cell function was more efficient with farming exposure (effector cell suppression, P = .004). In parallel, T(H)2 cytokine (IL-5) levels were decreased and associated with decreased lymphoproliferation and increased IL-6 levels (Ppg stimulation, Der p 1 + Ppg stimulation, or both; P < .05). Maternal exposure to increasing numbers of farm animals and stables was discovered to exert distinct effects on Treg cells, T(H)1/T(H)2 cells, or both. Additionally, FOXP3 demethylation in offspring of mothers with farm milk exposure was increased (P = .02). CONCLUSIONS Farm exposures during pregnancy increase the number and function of cord blood Treg cells associated with lower T(H)2 cytokine secretion and lymphocyte proliferation on innate exposure. One fascinating speculation is that maternal farm exposure might reflect a natural model of immunotherapy, potentially including a selection of innate stimuli in addition to allergen, shaping a childs immune system at an early stage.

Quantitative DNA Methylation Analysis of FOXP3 as a New Method for Counting Regulatory T Cells in Peripheral Blood and Solid Tissue

Regulatory T-cells (Treg) have been the focus of immunologic research due to their role in establishing tolerance for harmless antigens versus allowing immune responses against foes. Increased Treg frequencies measured by mRNA expression or protein synthesis of the Treg marker FOXP3 were found in various cancers, indicating that dysregulation of Treg levels contributes to tumor establishment. Furthermore, they constitute a key target of immunomodulatory therapies in cancer as well as transplantation settings. One core obstacle for understanding the role of Treg, thus far, is the inability of FOXP3 mRNA or protein detection methods to differentiate between Treg and activated T cells. These difficulties are aggravated by the technical demands of sample logistics and processing. Based on Treg-specific DNA demethylation within the FOXP3 locus, we present a novel method for monitoring Treg in human peripheral blood and solid tissues. We found that Treg numbers are significantly increased in the peripheral blood of patients with interleukin 2-treated melanoma and in formalin-fixed tissue from patients with lung and colon carcinomas. Conversely, we show that immunosuppressive therapy including therapeutic antibodies leads to a significant reduction of Treg from the peripheral blood of transplantation patients. In addition, Treg numbers are predictively elevated in the peripheral blood of patients with various solid tumors. Although our data generally correspond to data obtained with gene expression and protein-based methods, the results are less fluctuating and more specific to Treg. The assay presented here measures Treg robustly in blood and solid tissues regardless of conservation levels, promising fast screening of Treg in various clinical settings.

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

The adoptive transfer of CD4+CD25+ natural regulatory T cells (Treg) is a promising strategy for the treatment of autoimmune diseases and the prevention of alloresponses after transplantation. Clinical trials exploring this strategy require efficient in vitro expansion of this rare cell population. Protocols developed thus far rely on high‐grade purification of Treg prior to culture initiation, a process still hampered by the lack of Treg cell‐specific surface markers. Depletion of CD127+ cells was shown to separate activated conventional T cells from natural Treg cell populations allowing the isolation of highly enriched FOXP3+ cells with all functional and molecular characteristics of natural Treg. Here, we demonstrate that upon in vitro expansion, CpG methylation in a conserved region within the FOXP3 gene locus increased in CD4+CD25+CD127low Treg, correlating with loss of FOXP3 expression and emergence of pro‐inflammatory cytokines. Further analysis identified CD45RA−FOXP3+ memory‐type Treg as the main source of converting cells, whereas CD45RA+FOXP3+ Treg from the same donors showed no conversion within 3 wk of in vitro expansion. Thus, Treg cell lineage differentiation does not seem to represent a final fate decision, as natural Treg can lose their cell‐type‐specific characteristics after repetitive TCR stimulation.