Plasticity of Treg cells: Is reprogramming of Treg cells possible in the presence of FOXP3?

International Immunopharmacology 11 (2011) 555–560

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Plasticity of Treg cells: Is reprogramming of Treg cells possible in the presence of FOXP3? Marc Beyer ⁎, Joachim L. Schultze LIMES-Institute, Laboratory for Genomics and Immunoregulation, University of Bonn, Carl-Troll-Str. 31, D-53115 Bonn, Germany

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Article history: Received 31 August 2010 Received in revised form 11 November 2010 Accepted 15 November 2010 Available online 27 November 2010 Keywords: Regulatory T cells FOXP3 Plasticity

a b s t r a c t Regulatory T cells (Treg cells) are involved in self tolerance, immune homeostasis, prevention of autoimmunity, and suppression of immunity to pathogens or tumors. The forkhead transcription factor FOXP3 is essential for Treg-cell development and function as mutations in FOXP3 cause severe autoimmune diseases in mice and humans. Over the last years it has been postulated that FOXP3 expression in Treg prevents effector T-cell (Teffector-cell) lineage commitment, yet several recent studies suggest that the co-existence of effector and regulatory T-cell programs can occur and might help to enable Treg cells with properties necessary to exert their function in peripheral tissues. Furthermore, downregulation of FOXP3 in the periphery might help Treg cells to lose suppressive functions and gain memory properties with specificity for self-antigens and an effector phenotype including the ability to produce IFN-γ and IL-17. This plasticity might have an impact on their reactivity towards autoimmunity as well as tumors or infections. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Under homeostatic conditions the immune system has the role to recognize and destroy pathogens respectively foreign proteins or RNA/DNA in the context of danger signals while at the same time prohibiting reactivity against proteins presented without activating signals but also overt autoreactivity against self. Among the mechanisms to prevent this reactivity are negative selection in the thymus as a mechanism of central tolerance, but also peripheral tolerance including negative regulatory circuits such as clonal deletion, anergy, suppression, neglection, ignorance and active suppression mediated by specific cells including Treg cells, natural killer (NK) T cells or CD8+CD28− T cells. All of these mechanisms are central to suppress autoreactivity induced by T cells expressing a selfreactive TCR that have escaped negative selection in the thymus because of recognition of tissue-specific proteins not expressed in the thymus or by expressing low-avidity TCRs. The major effector arm of active suppression are T cells with regulatory or suppressive properties. Over the last years several types of regulatory T cells have been identified and characterized, chief among them are cells defined as natural regulatory cells (Treg) characterized by the expression of the forkhead transcription factor FOXP3 [1]. Loss of FOXP3 expression either by genetic targeting in mice or by spontaneously occurring point mutations resulting in a lack of FOXP3

⁎ Corresponding author. Tel.: +49 228 73 62792; fax: +49 228 73 62646. E-mail address: [email protected] (M. Beyer). 1567-5769/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2010.11.024

functions in humans and in scurfy mice leads to a fatal mutiorgan autoimmune disease named in humans IPEX syndrome (Immunodysregulation, Polyendocrinopathy and Enteropathy X-linked syndrome) [2,3] or XLAAD syndrome (X-linked Autoimmunity–Allergic Disregulation syndrome) [4] and scurfy disease in mice [5–8]. The suppressive nature of FOXP3 expressing T cells could be introduced into effector T cells by gene transfer of FOXP3 suggesting that the expression of FOXP3 is sufficient to induce a translational program in FOXP3-expressing effector T cells similar to natural Treg cells while other transcriptional effector programs might not necessarily be altered [5–7]. Functionally, Treg cells can actively suppress other T cells, dendritic cells, NK or NKT cells, as well as antibody production and affinity maturation by B cells in order to prevent unwanted immune responses or inflammatory reactions and subsequent tissue destruction. A diverse set of molecular mechanisms, how Treg cells exert suppression has been described, yet their overall contribution to the Treg-cell mediated suppressive function is still under intense debate. Unifying observations are the need for cell–cell contact between Treg cells and responder cell and that suppressive function is dose-dependent. 2. Induction and maintenance of FOXP3 expression in Treg cells Expression of the forkhead transcription factor FOXP3 is required for Treg-cell differentiation and its high expression is a characteristic feature of Treg cells [5–7]. FOXP3 expression in a subset of single positive thymocytes is induced by a multi-step process. T-cell receptor (TCR) signaling of increased strength has been reported as prerequisite of thymic FOXP3 induction [9,10] which results from high-affinity interactions between their TCR and self-peptide–MHC complexes

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presented by thymic stromal cells [11]. This leads to CD25 upregulation. Next, IL-2 signaling via CD25 drives activation of STAT5 which is also required for thymic generation of Treg cells [12–15]. Integration of signals via CD28 upon binding of its ligands from the B7 family is critical for the induction of FOXP3 expression, as CD28-deficient and CD80– CD86-deficient mice show a marked decrease in frequencies of Treg cells [16,17]. An important downstream event is the activation of lck as the lck-binding domain of the CD28 cytoplasmic tail has been shown to be critical for the induction of FOXP3 [16]. As a result of the integration of these signaling pathways a number of transcription factors are recruited to the genomic FOXP3 locus and regulate its expression (Fig. 1). Besides STAT5 which is induced by common γ-chain cytokine signaling, NFAT and AP1 have been reported to bind to the FOXP3 promoter [18]. CREB–ATF-1 on the other hand can bind to an intronic regulatory region at the genomic FOXP3 locus and have been suggested to enhance FOXP3 expression [19]. Several mouse models targeting molecules of the NFκB signaling cascade (e.g. PKC-θ, Bcl10, CARMA1, and IκB kinase 2) have supported an important role of NFκB in the induction of FOXP3 [20–24], yet only recently the NFκB transcription factor c-Rel could be demonstrated to bind to the genomic FOXP3 locus in an enhanceosome containing c-Rel, p65, NFAT, Smad, and CREB and inducing its transcription [25–28]. In addition, RUNX/Cbfβ also regulates the initiation and maintenance of FOXP3 gene expression by binding to regulatory elements within the FOXP3 locus [29–31]. The importance of TGF-β for the expression of FOXP3 in thymocytes is more controversial. While it is necessary for FOXP3 induction in naïve T cells in the periphery, it was proposed that in thymocytes TGF-β-induced binding of Smad to a conserved SmadNFAT response element in the genomic FOXP3 locus results in an activation of the FOXP3 locus essential for Treg-cell generation [32]. Using TGF-βRI knock-out animals, Liu et al. reported an impairment in FOXP3 induction and Treg-cell generation during the first week of life yet this decrease could be fully rescued at later stages of life [33]. This suggested that TGF-β is necessary during early Treg-cell development but that additional factors can substitute for TGF-β requirements in later stages of life. The finding that TGF-β is not necessary at later stages of life is in line with earlier reports analyzing Treg-cell

generation in 1–2 week-old mice lacking TGF-β1 or subjected to TGF-βRII ablation in double-positive thymocytes [34–36]. An elegant study by Zheng et al. could furthermore delineate the complex nature of binding of enhancers and repressors to the FOXP3 promoter and several conserved non-coding regulatory elements within the genomic FOXP3 locus [37]. One important counterplayer to the induction of FOXP3 is the PI3K–Akt pathway as its activation opposes FOXP3 induction in the thymus [38–40]. This could be shown by blockade of PI3K–Akt signaling using chemical inhibitors which imparted FOXP3 expression in CD4+ T cells while sustained Akt activation inhibits stable FOXP3 induction [38,39]. Very recently the downstream events of PI3K–Akt signaling in Treg cells have been further deciphered. Akt-mediated phosphorylation is known to inactivate transcription factors from the FOXO family. In induced Treg cells FOXO1 and FOXO3a act as transcription factors promoting FOXP3 gene expression while FOXO3a deletion did not alter the numbers of FOXP3-expressing thymic Treg cells [41]. Ouyang et al. demonstrated that T-cell specific deletion of FOXO1 and FOXO3a resulted in reduced numbers of thymic Treg cells in 3 week-old mice [42]. While Treg-cell numbers were restored after 6 weeks of age, the Treg cells that were generated were functionally impaired. A further layer of decision making in the process of Treg-cell induction has been proposed recently. The differentiation of TH1 cells and Treg cells is reciprocally regulated by S1P(1), a receptor for the bioactive lipid sphingosine 1-phosphate (S1P) signaling through the mTOR pathway thereby antagonizing the function of TGF-β by modulating activity of Smad3 [43]. This results in inhibited generation of adaptive and natural Treg cells while simultaneously inducing TH1 differentiation and disrupting immune homeostasis. Once FOXP3 expression is induced in Treg cells, its sustained expression is necessary to maintain Treg-cell phenotype and function. This has been demonstrated in mouse models where loss of FOXP3 or its diminished expression in Treg cells resulted in the acquisition of Teffector-cell properties including production of effector cytokines such as IL-2, IL-4, IL-17, and IFN-γ [44,45]. Critical for the stable maintenance of FOXP3 expression in Treg cells is the demethylation

Fig. 1. Transcription factors regulating FOXP3 expression. T-cell receptor (TCR) and CD28 costimulatory signals as well as interleukin-2 and an autocrine feed-back loop are important for induction of FOXP3. FOXP3 expression is regulated by several transcription factors binding to the promoter region of FOXP3 as well as 3 highly conserved non-coding sequences (CNS1-3) in the genomic FOXP3 region. Upstream pathways inducing expression of the downstream transcription factors are depicted in the same color. TCR/CD28 signaling (violet): AP1, activator protein 1; c-Rel, member of NF-kappaB transcription factor family; CREB, cyclic-AMP—responsive-element-binding protein; NFAT, nuclear factor of activated T cells; p65 (RelA), member of NF-kappaB transcription factor family—IL-2 signaling (blue): STAT5, signal transducer and activator of transcription 5—TGF-β signaling (green): SMAD3, mothers against decapentaplegic homologue 3—FOXP3 signaling (orange): Cbf- β, core-binding factor, beta subunit; FOXP3, forkhead box P3; RUNX1, runt-related transcription factor 1.

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of CpG motifs at the genomic FOXP3 locus, while these motifs remain methylated in induced Treg cells not stably expressing FOXP3 [46–48]. The transcription factor Ets-1 is contributing to the stable expression of FOXP3 as an additional molecular player that specifically binds to the demethylated CpG motifs at the genomic FOXP3 locus in a demethylation-dependent manner. Disruption of the Ets-1 binding sites reduced its transcriptional enhancer activity, thereby restricting stable FOXP3 expression to the Treg-cell lineage [49]. Furthermore, permissive chromatin modifications, e.g. histone methylation and acetylation as well as miRNA status are major determinants for stable FOXP3 gene expression [50,51]. 3. FOXP3 suppresses Teffector-cell differentiation On a molecular level FOXP3 expression is necessary to suppress the induction of several key Teffector-cell molecules either by binding directly to the genomic locus or indirectly acting in concert with other proteins. The direct actions of FOXP3 have been elegantly demonstrated in heterozygous female mice that express either a functional or non-functional FOXP3 allele [52–54]. The cells that lack a functional FOXP3 allele in these animals, although displaying a certain degree of resemblance towards Treg cells expressing a functional FOXP3 protein, have no suppressive activity, altered proliferative potential and metabolic fitness while expressing Teffector-cytokines and producing high amounts of Teffector-cytokines, e.g. IL17 [52,54]. Expression of an attenuated FOXP3 allele in Treg cells resulted in the impairment of suppressor function as well and induced the production of IL-4 [45,55]. Targets of FOXP3 in Treg cells have been identified over the last years using genome-wide analyses of FOXP3-binding genes [56–58]. These studies suggest that FOXP3 binds to several cytokine loci, e.g. the IFN-γ or TH2 locus [58], and can act as a transcriptional repressor or activator depending on the co-factors bound by FOXP3 and inhibitory or permissive histone modifications at the target loci. Global mapping of histone modifications in Treg cells have been performed by Wei et al. and have further deepened our understanding of the epigenetic modifications underlying the transcriptional programs in Treg cells [51]. Still only a handful of important FOXP3 targets genes have been studied in greater detail and central mechanisms downstream of FOXP3 regulating local cytokine expression including contextdependent epigenetic regulation are yet to be discovered. Several studies over the last years have focused on the identification of factors binding to FOXP3 and acting in concert to regulate gene expression. One of the best studied interactions of FOXP3 consists of its cooperation with NFAT through the forkhead domain to form a DNAbinding complex that is important for Treg-cell function competing with AP-1 [59]. In addition, FOXP3 can also directly interact with c-JUN/AP-1 in Treg cells sequestering activated AP-1 in the nucleus, altering the subnuclear distribution of activated AP-1 and disrupting chromatinbinding of activated AP-1 [60]. FOXP3 can also form a transcriptional repressor complex with several post-translational modification enzymes such as histone deacetylase HDAC1, HDAC7, and HDAC9 or histone acetyltransferase TIP60 (Tat-interactive protein, 60 kDa) [61]. RUNX1, which is crucially required for normal hematopoiesis, binds to the promoters of IL-2 and IFN-γ in conventional CD4+ T cells and induces their expression. In Treg cells FOXP3 physically interacts with RUNX1 which in turn suppresses IL-2 and IFN- γ production and induces Treg-cell associated molecules such as CD25, CTLA-4 or GITR [62]. Tregcell specific deficiency of Cbfβ, a cofactor for all RUNX proteins, induced altered gene expression profiles including attenuated expression of FOXP3 and resulted in high expression of IL-4. The FOXP3–RUNX1–Cbfβ complex binds to a large number of gene loci in Treg cells, including the FOXP3 and the IL-4 silencer indicating that the RUNX1–Cbfβ heterodimer is indispensable for optimal expression of FOXP3, maintenance of suppressive function and suppression of Teffector-cell properties [30]. Selective dysregulation of TH2 responses and enhanced germinal center formation have been reported in Treg cells lacking IRF4 [63]. IRF4

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expression endows Treg cells with the ability to suppress TH2 responses. Furthermore, direct interactions of IRF4 and FOXP3 are necessary to exert this suppressive effect. Besides FOXP3 itself, downstream molecules induced by FOXP3 also help to establish the transcriptional Treg-cell program. Eos, a zinc-finger transcription factor of the Ikaros family, is an important mediator of FOXP3-dependent gene silencing in Treg cells as Eos directly interacts with FOXP3 and induces chromatin modifications that result in gene silencing in Treg cells [64]. Loss of Eos results in a loss of suppressive function and partial induction of Teffector-cell functions supporting an important role for Eos in maintaining transcriptional Treg-cell programs while at the same time suppressing Teffector-cell differentiation. The FOXO transcription factors FOXO1 and FOXO3a have also been implicated in the inhibition of Teffector-cell function in Treg cells as lack of FOXO1 and FOXO3a in Treg cells is sufficient to induce TH1 and TH17 effector cytokines but not TH2 cytokines [42]. HOPX is an additional transcription factor that has been recently identified as an important mediator of suppressive function of Treg cells [65]. Primarily identified in induced Treg cells after low-dose antigen administration, HOPX is essential for the downregulation of the expression of the transcription factor AP-1 complex and suppression of other T cells. Loss of HOPX resulted in high expression of the AP-1 complex and subsequent loss of suppressive function. In vivo FOXP3 also suppresses Teffector-cell differentiation as demonstrated in mouse models where FOXP3 expression was ablated or reduced in the periphery. These CD4+ T cells differentiate into Teffector cells and can cause pronounced tissue inflammation upon transfer into lymphopenic recipients [44]. This autoreactivity is probably due to the TCR repertoire of these cells as an enrichment of self-reactive TCR has been reported in these T-cell populations. Taken together, FOXP3 acts mostly as a transcriptional repressor inhibiting Teffector-cell differentiation of Treg cells by acting in concert with a number of transcription factors as well as post-transcriptional regulators. Still, these studies keep open the option that FOXP3 expression in Treg cells in vivo is not permanently stable and raise the question how fixed the transcriptional Treg-cell program is and whether conversion of Treg cells can occur in vivo. 4. Conversion of Treg cells into Teffector cells in vivo Possibilities that might favor conversion of Treg cells into Teffector cells could be tissue-specific factors or an inflammatory environment. As an example of the latter, in the presence of high amounts of IL-6, the FOXP3+ Treg-cell population can differentiate into TH17 cells [66,67]. From mice lacking TGF-β an intimate link between the differentiation programs of FOXP3+ Treg cells and TH17 cells could be established [68,69]. In both mice and humans, T cells that express both RORγt and FOXP3 have been identified in vivo [66,69,70]. These cells however produce less IL-17 than TH17 cells thereby suggesting that FOXP3 antagonizes RORγt-induced IL-17 expression by direct interaction [69]. By genetic fate mapping using mice expressing Cre recombinase under the regulation of the FOXP3 locus and crossing these mice with Rosa26-stop–YFP reporter mice to mark cells that had expressed FOXP3 at some stage of development it was shown that at least 25% of IL-17-producing cells previously expressed FOXP3 during their ontogeny [69]. In humans, Treg cells can secrete IL-17 ex vivo and constitutively express RORγt [71] yet retain their suppressive activity in vitro [70,72]. These IL-17-secreting Treg cells share some phenotypic and functional properties with TH17 cells like high expression of CCR4 and CCR6 or low expression of CXCR3 while other properties like low levels of CD161 expression and secretion of IL-22 and TNF-α are confined to the TH17 cells. This differentiation of Treg cells into IL17-producing cells depends on histone/protein deacetylase activity as treatment with an histone deacetylase inhibitor negatively influenced TH17 differentiation suggesting epigenetic modifications are essential for the conversion of Treg cells towards TH17 cells [73]. As conversion

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of Treg cells into IL17-producing cells has also been reported upon coculture with dendritic cells which had been activated by a C-type lectin receptor (dectin-1) involved in fungal recognition [74], one might speculate that TH17 conversion of Treg cells might be important in the response against invading pathogens, e.g. in the gut. Differentiation of Treg cells into Teffector cells other than TH17 cells has also been reported as several studies over the last years have assessed whether Treg cells maintain a stable phenotype upon adoptive transfer in vivo: adoptive transfer of Treg cells to immunocompetent mice could demonstrate stability of Treg cells and FOXP3 expression [46,75], while adoptive transfer of Treg cells into lymphopenic hosts results in the conversion of a fraction of FOXP3+ Treg cells that lose FOXP3 expression and differentiate into effector T-cell lineages as a consequence of cell division which might be associated with modifications in DNA methylation and other epigenetic regulations associated with cell division [75–77]. Furthermore, the adoptive transfer of Treg cells into lymphopenic hosts can also result in the differentiation of Treg cells into follicular helper T cells (TFH) in Peyer's patches which then participate in germinal center formation and IgA synthesis [77]. The differentiation into TFH cells required interaction with B cells through CD40 and the concomitant loss of FOXP3 expression. A recent study in a more physiological setting could demonstrate that differentiation of Treg cells into effector-memory T cells is taking place in vivo [76]. In this model utilizing a ROSA26–YFP recombination reporter allele combined with a Foxp3 BAC transgene expressing Cre recombinase a population of CD4+ T cells could be observed that had lost FOXP3 expression. These “exFOXP3” Treg cells differentiated into effector-memory T cells expressing proinflammatory cytokines, most prominently IFN-γ, induced rapid onset of diabetes upon adoptive transfer and the TCR repertoire of these cells suggested that both natural and adaptive Treg cells can convert into “exFOXP3” Treg cells [76]. A very recent report by Rubtsov et al. has questioned these observations using an inducible FOXP3–eGFP–Cre–ERT2 mouse model crossed to ROSA26– YFP recombination reporter mice [78]. Although the authors could observe a small but detectable population of YFP+GFP− cells which might correspond to the aforementioned “exFOXP3” Treg cells the authors conclude from their studies that the Treg-cell lineage is stable in vivo under physiologic and inflammatory conditions as an accumulation of YFP+GFP− cells was not detectable over time [78]. In vitro a subpopulation of Treg cells has been reported that can express both IFN-γ and TH1-specifying transcription factor T-bet while maintaining FOXP3 expression under TH1 polarizing culture conditions [51], but it is unclear if the IFN-γ-expressing Treg cells retain their suppressive capacity or whether these cells can undergo this change of phenotype under homeostatic conditions [66]. In inflamed tissues in response to IFN-γ, Treg cells can upregulate T-bet which promotes expression of CXCR3 on Treg cells resulting in the accumulation of T-bet-expressing Treg cells at sites of inflammation [79]. A further striking example of acquisition of characteristic TH1 effector cell features by Treg cells has been recently reported in a study of lethal Toxoplasma gondii infection in mice where environmental cues provided by both local dendritic cells and Teffector cells can induce the expression of T-bet and IFN-γ by Treg cells [80]. Two elegant studies performed by the Rudensky lab have further highlighted the plasticity of Treg cells in the periphery. Analyzing Treg cells which were deficient in either the TH2-driving transcription factor IRF-4 or the TH-17 inducing transcription factor STAT3, they could demonstrate that Treg cells use transcriptional programs necessary for TH2 resp. TH17 differentiation to migrate to and suppress the respective immune response in the corresponding tissue [63,81]. In contrast to the mouse where FOXP3 is solely expressed in Treg cells [82], FOXP3 is expressed transiently by most activated human T cells without inducing suppressive function [83]. Moreover, in humans a subset of activated Treg cells with low levels of FOXP3 expression has been described. These cells produce IL17 and have lost their inhibitory properties [84]. One aspect highlighting the importance of the

conversion of Treg cells into Teffector cells is that non-functional Treg cells have been implied in the pathogenesis of autoimmune disorders, e.g. multiple sclerosis [85] and that numbers of FOXP3low Treg cells producing IL17 are augmented in peripheral blood of patients with active systemic lupus erythematosus [84]. Taken together, in both humans and mice Treg cells have been described that have not only lost their suppressive function but also gained expression of Teffector-cell programs. These cells might have pathogenic potential and could be involved in the induction or maintenance of autoimmunity. Whether central mechanisms exist that determine the functional fate of Treg cells however remains to be seen. 5. Further events influencing Treg-cell stability A further layer of complexity has been introduced by epigenetic and post-translational modifications which can also impact stability of FOXP3 expression in Treg cells and influence Teffector-cell differentiation. In Treg cells CpG islands at the FOXP3 promoter, at an intronic enhancer (CNS1), and at a highly conserved element upstream of exon 1 referred to as the Treg-cell-specific demethylated region (TSDR, CNS2) have been described that are fully demethylated in natural Treg cells but not in induced Treg cells or activated Tconv cells transiently expressing FOXP3 [19,46,48,86]. In the abovementioned in vivo model, where Treg cells could be observed that had lost FOXP3 and gained effector-memory functions, the majority of “exFOXP3”-Treg cells have methylated CpG islands most closely resembling Tconv cells or methylation patterns that are at a transitional stage from Treg cells to Tconv cells [76]. Histone modifications at the FOXP3 locus might be additional factors modulating FOXP3 expression and stability of the Treg cells phenotype. Our understanding of the regulation of FOXP3 could be further enhanced by the characterization of conserved sites at the FOXP3 locus that are a docking position for several transcription factors of which accessibility is highly regulated by histone modifications [37]. Wei et al. could demonstrate that Treg cells are a distinct subset of T cells when looking at the global transcriptome mapping of permissive and repressive histone modifications (H3K4me4 and H3K27me27) in Treg cells and naïve T cells, yet the lineage defining factors T-bet, GATA3, and RORC are all bivalently modified in Treg cells demonstrating both repressive and permissive histone modifications. This results in a non-fixed phenotype with a subpopulation of Treg cells being able to express T-bet and IFN-γ under TH1-polarizing conditions [51]. This suggests that global epigenetic changes might allow for lineage differentiation and hold T cells in such a functional differentiation state but that certain conditions might be able to reverse this differentiation and that T cells thereby retain a certain degree of plasticity [87]. In addition to epigenetic changes, miRNAs act as post-transcriptional regulators of the Treg-cell phenotype as in mice with a disruption of the maturation of miRNAs by specific deletion of either DICER or DROSHA, both RNaseIII enzymes necessary for miRNA maturation and conversion of FOXP3expressing Treg cells into TH1 or TH2 cell has been reported [50]. Using conditional DICER–FOXP3–cre mice crossed to ROSA26–YFP mice these experiments clearly could demonstrate that Treg cells that had once expressed FOXP3 had differentiated into IL-4 or IFN-γ producing Teffector cells. In line with this finding in a second in vivo model, where DROSHA had been deleted in all T cells, higher levels of TH1 and TH17 cells could be observed, although it was not determined whether this was a result of Treg-cell conversion [88]. Observations in a third model where DICER had been specifically deleted in Treg cells confirmed that miRNA are critical for the homeostatic potential of Treg cells as well as Treg-cell function under inflammatory conditions while the authors did not address stability of the Treg-cell lineage itself [89]. Assessing function of single miRNAs in Treg cells, Rudensky and colleagues could identify a critical role for miR-155 and miR-146a in Treg cells. While miR-155 is essential for the maintenance of competitive fitness of Treg cells by modulating suppressor of cytokine signaling 1 (SOCS1) expression to tightly regulate the expression of the transcription factor signal

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transducer and activator of transcription 5 (STAT5) in response to limiting amounts of interleukin-2 [90], miR-146a is important to suppress pathogenic TH1-responses by downregulating STAT1 expression in Treg cells [91]. Together these findings point to a hierarchy of repressive mechanisms ensuring suppression of Teffector-cell function in Treg cells. Nevertheless, the first level within such hierarchy downstream of FOPX3 regulation is still elusive. 6. Concluding remarks A common feature for all Teffector cells and Treg cells is the expression or lack of unique cytokines and transcription factors essential for the transcriptional programs regulating their differentiation. This differentiation was mainly believed to be mutually exclusive, yet over the last years it has become increasingly clear that Treg cells and also other T-cell subsets are not as stable as previously assumed and that under certain conditions during their development, maintenance, and differentiation conversion of Treg cells into Teffector cells can occur. Even an aiding rather than suppressing role of Treg cells during inflammation has been reported [92], clearly demonstrating that the extra- and intracellular signals and molecular events that regulate FOXP3 expression and differentiation of Treg cells need to be further elucidated to complete our understanding of these complex processes. This will have impact on therapeutic settings such as Treg-cell therapies or drugs expanding Treg cells as they are being considered for treatment of autoimmune diseases and for preventing transplant rejection while depletion or conversion of Treg cells is thought to be a viable therapeutic strategy in cancer or infectious diseases. Acknowledgements This work was supported by the German Jose-Carreras-Leukemia Foundation and the German Research Foundation (SFB 832 and 704). References [1] Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell 2008;133:775–87. [2] Bennett CL, Christie J, Ramsdell F, Brunkow ME, Ferguson PJ, Whitesell L, et al. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet 2001;27:20–1. [3] Wildin RS, Ramsdell F, Peake J, Faravelli F, Casanova JL, Buist N, et al. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet 2001;27:18–20. [4] Chatila TA, Blaeser F, Ho N, Lederman HM, Voulgaropoulos C, Helms C, et al. JM2, encoding a fork head-related protein, is mutated in X-linked autoimmunityallergic disregulation syndrome. J Clin Invest 2000;106:R75–81. [5] Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003;299:1057–61. [6] Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 2003;4:330–6. [7] Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat Immunol 2003;4:337–42. [8] Brunkow ME, Jeffery EW, Hjerrild KA, Paeper B, Clark LB, Yasayko SA, et al. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet 2001;27:68–73. [9] Hsieh CS, Liang Y, Tyznik AJ, Self SG, Liggitt D, Rudensky AY. Recognition of the peripheral self by naturally arising CD25+ CD4+ T cell receptors. Immunity 2004;21:267–77. [10] Lio CW, Hsieh CS. A two-step process for thymic regulatory T cell development. Immunity 2008;28:100–11. [11] Liston A, Nutsch KM, Farr AG, Lund JM, Rasmussen JP, Koni PA, et al. Differentiation of regulatory Foxp3+ T cells in the thymic cortex. Proc Natl Acad Sci USA 2008;105: 11903–8. [12] Burchill MA, Yang J, Vogtenhuber C, Blazar BR, Farrar MA. IL-2 receptor betadependent STAT5 activation is required for the development of Foxp3+ regulatory T cells. J Immunol 2007;178:280–90. [13] Burchill MA, Yang J, Vang KB, Moon JJ, Chu HH, Lio CW, et al. Linked T cell receptor and cytokine signaling govern the development of the regulatory T cell repertoire. Immunity 2008;28:112–21. [14] Vang KB, Yang J, Mahmud SA, Burchill MA, Vegoe AL, Farrar MA. IL-2, -7, and -15, but not thymic stromal lymphopoeitin, redundantly govern CD4+Foxp3+ regulatory T cell development. J Immunol 2008;181:3285–90.

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