Alf Hamann

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.

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.

A Helminth Immunomodulator Reduces Allergic and Inflammatory Responses by Induction of IL-10-Producing Macrophages

The coincidence between infections with parasitic worms and the reduced prevalence of allergic disease in humans and in animal models has prompted the search for helminth molecules with antiallergic and antiinflammatory potential. We report herein that filarial cystatin, a secreted protease inhibitor of filarial nematodes, suppresses Th2-related inflammation and the ensuing asthmatic disease in a murine model of OVA-induced allergic airway responsiveness. Treatment with recombinant filarial cystatin inhibited eosinophil recruitment, reduced levels of OVA-specific and total IgE, down-regulated IL-4 production, and suppressed allergic airway hyperreactivity when applied during or after sensitization and before challenge with the allergen. Depletion of macrophages by clodronate-containing liposomes prevented the curative effects and restored the levels of infiltrating cells, IgE, and allergic airway reactivity. Blocking of IL-10 by application of anti-IL-10 receptor Abs restored the reduced number of infiltrating cells and the levels of OVA-specific IgE. In contrast, depletion of regulatory T cells by anti-CD25 Abs had only limited effects. Cystatin also modulated macrophage-mediated inflammation in a murine model of dextran sulfate sodium-induced colitis, leading to reduction of inflammatory infiltrations and epithelial damage. Our data demonstrate that treatment with a single helminth protein can exert the antiallergic effects of helminth infections.

Mechanisms and regulation of lymphocyte migration

Lymphocyte traffic seems to be an essential requirement for an adequate immune response both in lymphoid tissues and local inflammatory sites. In this review, Adrian Duijvestijn and Alf Hamann discuss how selective migration of lymphocytes is directed by lymphocyte-endothelial interactions and what mechanisms may control this.

Selective depletion of Foxp3+ regulatory T cells improves effective therapeutic vaccination against established melanoma.

Tumor-bearing individuals have been reported to harbor increased numbers of Foxp3(+) regulatory T cells (Treg), which prevent the development of efficient antitumor immune responses. Thus, Treg depletion has already been tested as a promising therapeutic approach in various animal models and entered clinical trials. However, the use of nonspecific Treg targeting agents such as CD25 depleting antibodies, which in addition to CD25(+) Tregs also deplete recently activated CD25(+) effector T cells, potentially masked the tremendous potential of this therapeutic strategy. To avoid such nonspecific effects, we used transgenic DEREG (depletion of regulatory T cells) mice, which express a diphtheria toxin receptor under control of the Foxp3 locus, allowing selective depletion of Foxp3(+) Tregs even during ongoing immune responses. We showed that Foxp3(+) Treg depletion induced partial regression of established ovalbumin (OVA)-expressing B16 melanoma, which was associated with an increased intratumoral accumulation of activated CD8(+) cytotoxic T cells. The antitumor effect could be significantly enhanced when Treg depletion was combined with vaccination against OVA. To further assess whether this therapeutic approach would break self-tolerance, we crossed DEREG mice with RipOVA(low) mice, expressing OVA as neo-self-antigen under control of the rat insulin promoter. In these mice, combined Treg depletion and vaccination also induced tumor regression without the onset of diabetes. Together, our data suggest that selective Treg targeting strategies combined with vaccinations against tumor-associated (self) antigens have the potential to evoke efficient antitumor responses without inducing overt autoimmunity. These findings might have implications for future therapeutic interventions in cancer patients.