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.

Thymus-derived CD4 ؉ CD25 ؉ regulatory T cells suppress autoreactive CD4 ؉ and CD8 ؉ T cells and thereby protect from autoimmunity. In animal models, adoptive transfer of CD4 ؉ CD25 ؉ regulatory T cells has been shown to prevent and even cure autoimmune diseases as well as pathogenic alloresponses after solid organ and stem-cell transplantations. We recently described methods for the efficient in vitro expansion of human regulatory T cells for clinical applications. We now demonstrate that only CCR7-and L-selectin (CD62L) - IntroductionSelf-tolerance within the T-cell compartment is primarily ensured by positive and negative selection during T-cell development in the thymus. Autoreactive T cells that escape central deletion are frequently controlled by peripheral tolerance mechanisms, including cell-mediated suppression by CD4 ϩ CD25 ϩ regulatory T (Treg) cells. [1][2][3] Thymic maturation and suppressive function of natural Treg cells depend on expression of the transcriptional repressor forkhead box P3 (FOXP3), as nonsense mutations in this gene result in loss of Treg-cell function and severe autoimmunity in mice and humans. [4][5][6][7][8] Apart from suppression of autoreactive T cells, FOXP3 ϩ CD4 ϩ CD25 ϩ Treg cells also dampen immune responses against infectious pathogens, 9 cancer, 10 and allogeneic organ 11 and stem-cell grafts. 12 Thus, depletion of Treg cells seems a promising strategy to augment immune responsiveness to tumors, chronic infections, and vaccination, while an enhancement of Treg-cell activity is envisaged for the prevention and treatment of T-cellinduced diseases. 13 In animal models, the adoptive transfer of CD4 ϩ CD25 ϩ Treg cells has been shown to protect from type 1 diabetes 14,15 or experimental autoimmune encephalomyelitis 16 and even revert ongoing disease in colitis 17,18 and arthritis. 19 Similarly, adoptively transferred Treg cells protected against rejection and graft-versus-host disease (GVHD) after allogeneic organ transplantation and bone marrow transplantation (BMT), respectively. 11,[20][21][22][23][24][25] In human peripheral blood, natural Treg cells mainly reside within the subpopulation of CD4 ϩ T cells with high CD25 expression levels (CD25 high ), 26 while cells with intermediate CD25 expression (CD25 int ) consist mainly of recently activated and memory T cells, with only 5% to 15% FOXP3 ϩ Treg cells (P.H. and M.E., unpublished results, June 2006). Due to lack of Treg cell-specific surface markers, isolation of CD4 ϩ CD25 high T cells is thus far considered the most promising strategy for the generation of pure Treg-cell products. 27 As they represent only 1% to 3% of peripheral-blood mononuclear cells (PBMCs), we and others recently described methods for the in vitro expansion of Treg cells for future clinical trials. [28][29][30] Cross-linking of stimulating CD3 and CD28 antibodies by beads or Fc receptor-bearing fibroblasts in combination with high-dose interleukin-2 (IL-2) resulted in a 3-to 4-log expansion within 3 to 4 weeks. Cultured cells maintained Treg-c...

The adoptive transfer of CD4 1 CD25 1 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 1 cells was shown to separate activated conventional T cells from natural Treg cell populations allowing the isolation of highly enriched FOXP3 1 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 1 CD25 1 CD127 low Treg, correlating with loss of FOXP3 expression and emergence of pro-inflammatory cytokines. Further analysis identified CD45RA À FOXP3 1 memory-type Treg as the main source of converting cells, whereas CD45RA 1 FOXP3 1 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.Key words: Cellular therapy . Immune regulation . Treg Supporting Information available online Introduction CD4 1 CD25 1 Treg are pivotal for the maintenance of peripheral self-tolerance and imbalances in this T-cell compartment have been shown to contribute to various autoimmune diseases [1]. In murine disease models, adoptively transferred Treg prevent, and in some cases, even cure autoimmunity [2,3]. In addition, they protect from graft rejection after allogeneic organ transplantation [4] as well as from graft-versus-host disease after MHCmismatched stem cell transplantation [5][6][7][8]. Recently, a limited number of Phase I clinical trials exploring the adoptive transfer of Treg have been initiated and several additional trials in various clinical settings are in preparation [9,10]. Prerequisites for the initiation of such trials are (i) the availability of efficient in vitro expansion protocols for this rare cell population and (ii) the ability to unequivocally identify Treg to avoid contamination of 1088Treg cultures with potentially harmful conventional effector T cells (Tconv). While efficient cell culture protocols have recently been established by us and others for the polyclonal as well as antigen-specific Treg cell expansion [11][12][13], the search for an exclusive surface marker for Treg is still ongoing.Contrary to earlier reports, human CD4 1 CD25 high Treg in adult peripheral blood have recently been shown to comprise not only (self-)antigen-experienced, CD45RA À central and effector memory cells, but also a subpopulation of CD45RA 1 naïve recent thymic emigrants [14,15]. We previously demonstrated that these CD45RA 1 CD4 1 CD25 high T cells (RA 1 Treg) homogen...

DNA methylation participates in establishing and maintaining chromatin structures and regulates gene transcription during mammalian development and cellular differentiation. With few exceptions, research thus far has focused on gene promoters, and little is known about the extent, functional relevance, and regulation of cell type-specific DNA methylation at promoter-distal sites. Here, we present a comprehensive analysis of differential DNA methylation in human conventional CD4+ T cells (Tconv) and CD4 + CD25 + regulatory T cells (Treg), cell types whose differentiation and function are known to be controlled by epigenetic mechanisms. Using a novel approach that is based on the separation of a genome into methylated and unmethylated fractions, we examined the extent of lineage-specific DNA methylation across whole gene loci. More than 100 differentially methylated regions (DMRs) were identified that are present mainly in cell type-specific genes (e.g., FOXP3, IL2RA, CTLA4, CD40LG, and IFNG) and show differential patterns of histone H3 lysine 4 methylation. Interestingly, the majority of DMRs were located at promoter-distal sites, and many of these areas harbor DNA methylation-dependent enhancer activity in reporter gene assays. Thus, our study provides a comprehensive, locuswide analysis of lineage-specific methylation patterns in Treg and Tconv cells, links cell type-specific DNA methylation with histone methylation and regulatory function, and identifies a number of cell type-specific, CpG methylationsensitive enhancers in immunologically relevant genes.

The adoptive transfer of donor CD4+CD25+ regulatory T cells has been shown to protect from lethal graft-versus-host disease after allogeneic bone marrow transplantation in murine disease models. Efficient isolation strategies that comply with good manufacturing practice (GMP) guidelines are prerequisites for the clinical application of human CD4+CD25+ regulatory T cells. Here we describe the isolation of CD4+CD25+ T cells with regulatory function from standard leukapheresis products by using a 2-step magnetic cell-separation protocol performed under GMP conditions. The generated cell products contained on average 49.5% CD4+CD25high T cells that phenotypically and functionally represented natural CD4+CD25+ regulatory T cells and showed a suppressive activity comparable to that of CD4+CD25+ regulatory T-cell preparations purified by non-GMP-approved fluorescence-activated cell sorting.

Acute graft-versus-host disease (aGvHD) is a frequent complication after allogeneic bone marrow/stem cell transplantation (BMT/SCT) induced by co-transplanted alloreactive conventional donor T cells. We previously demonstrated that the adoptive transfer of donor CD4+CD25+Foxp3+ regulatory T cells (Treg) at the time of BMT prevents aGvHD in murine models. Yet, the therapeutic potential of donor Treg for the treatment of established aGvHD has not yet been studied in detail. We now used in vitro expanded phenotypically and functionally stable murine Treg to explore their therapeutic efficacy in haploidentical aGvHD models. Upon transfer donor Treg ameliorate clinical and histologic signs of aGvHD and significantly improve survival. They migrate to lymphoid as well as aGvHD target organs, predominantly the gastrointestinal tract, where they inhibit the proliferation of conventional T cells, reduce the influx of myeloid cells, and the accumulation of inflammatory cytokines. Successfully treated animals restore aGvHD-induced tissue damage in target organs and lymphoid tissues, thereby supporting lymphocyte reconstitution. The therapeutically applied Treg population survives long term without conversion into pathogenic effector T cells. These results demonstrate that donor Treg not only prevent aGvHD, but are also efficacious for the treatment of this life-threatening BMT complication.

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