Epigenetic and transcriptional control of Foxp3+ regulatory T cells.

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Review

Epigenetic and transcriptional control of Foxp3+ regulatory T cells Jochen Huehn a,∗ , Marc Beyer b,∗∗ a b

Experimental Immunology, Helmholtz Centre for Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany LIMES-Institute, Laboratory for Genomics and Immunoregulation, University of Bonn, Carl-Troll-Str. 31, 53115 Bonn, Germany

a r t i c l e

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Article history: Received 5 January 2015 Accepted 8 February 2015 Keywords: Regulatory T cells Foxp3 Epigenetic regulation Immunoregulation

a b s t r a c t Regulatory T cells (Treg cells) present a unique T-cell lineage that plays a key role for the initiation and maintenance of immunological tolerance. Treg cells are characterized by the expression of the forkhead box transcription factor Foxp3, which acts as a lineage-specifying factor and determines the unique properties of these immunosuppressive cells. Work over the past few years has shown that well-defined and precisely controlled events on transcriptional and epigenetic level are required to ensure stable expression of Foxp3 in Treg cells. More recent work suggested that in addition to stable Foxp3 expression, epigenetic modifications of Treg -cell specific genes contribute to the unique phenotype of Treg cells by imprinting their transcriptional program and stabilizing the expression of molecules being essential for the suppressive properties of Treg cells. In this review, we will highlight how Foxp3 expression itself is epigenetically and transcriptionally controlled, how the Treg -cell specific epigenetic signature is achieved, how Foxp3 as transcription factor influences the gene expression programs in Treg cells and how unique properties of Treg -cell subsets are defined by other transcription factors. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction: development and functional properties of Foxp3+ regulatory T cells Peripheral tolerance is required to protect our body from unwanted and harmful immune responses toward self-antigens or innocuous environmental antigens such as food or commensal microbiota. Work over the past two decades has suggested that regulatory T cells (Treg cells) play a key role for the initiation and maintenance of peripheral tolerance [1,2]. Initially, the surface marker CD45RB was used to categorize CD4+ T cells into immunosuppressive (CD45RBlow ) and inflammatory (CD45RBhigh ) subsets [3]. In 1995, Sakaguchi et al. identified a subset of CD4+ T cells constitutively expressing the ␣-chain of the IL-2 receptor (CD25) as primary mediators of self-tolerance [4], and named this cell type regulatory T cells. They showed that Treg cells can prevent the development of organ-specific and systemic autoimmune disease caused by postnatal thymectomy or by transfer of CD25-depleted CD4+ T cells into immunodeficient recipient mice [4,5]. In the following years, broad evidence has been accumulated that Treg cells not only play a key role for self-tolerance, but also can modulate

∗ Corresponding author. Tel.: +49 531 6181 3310. ∗∗ Corresponding author. Tel.: +49 228 73 62787 E-mail addresses: [email protected] (J. Huehn), [email protected] (M. Beyer).

the strength of pathogen-specific immune responses, inhibit transplant rejection, promote tumor immune escape and contribute to feto-maternal tolerance [1,6]. Since CD25 is also expressed on conventional CD4+ T cells upon their activation, a search for more specific Treg -cell markers was initiated. In 2003, the forkhead box transcription factor Foxp3 was reported to be specifically expressed in Treg cells and to act as a lineage specification factor [7–9]. Nowadays, Foxp3 is the most widely used marker for Treg cells and is known to play a critical role for the unique properties of these immunosuppressive cells (see Section 2). The vast majority of Foxp3+ Treg cells are generated already during thymic development (thymus-derived, tTreg cells) and it is assumed that tTreg cells preferentially recognize self-antigens (Fig. 1). Development of tTreg cells relies on factors such as a high-affinity T-cell receptor (TCR) binding to antigens presented on thymic antigen-presenting cells (APCs), low clonal frequency of antigen-specific Treg -cell precursors, and a specific cytokine environment in combination with co-stimulation by thymic APCs [10]. In addition to their thymic generation, Treg cells can also be converted from Foxp3− conventional CD4+ T cells in the periphery [11–17]. These peripherally-induced Treg cells (pTreg cells) are required to complement the TCR repertoire of tTreg cells with specificities directed against non-pathogenic foreign antigens, including commensal microbiota, food, and fetal antigens. Indeed, pTreg cells have been shown to be important for the acquisition of oral, mucosal, and feto-maternal tolerance [18–20]. In addition to tTreg

http://dx.doi.org/10.1016/j.smim.2015.02.002 1044-5323/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Huehn J, Beyer M. Epigenetic and transcriptional control of Foxp3+ regulatory T cells. Semin Immunol (2015), http://dx.doi.org/10.1016/j.smim.2015.02.002

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Fig. 1. Development of Foxp3-expressing Treg cells. Foxp3-expressing Treg cells can either differentiate in the thymus from Treg -cell precursors giving rise to thymus-derived Treg cells or in the periphery from naive conventional Foxp3− T cells giving rise to peripherally-induced Treg cells.

and pTreg cells, in vitro induced Treg cells (iTreg cells) encompass another frequently studied group of Treg cells [21]. For the generation of iTreg cells, naïve conventional CD4+ T cells require triggering of their TCR together with cytokine stimulation through IL-2 and transforming growth factor-␤ (TGF-␤) [22]. All three Treg -cell types, tTreg , pTreg , and iTreg cells have in common that they rely on proper Foxp3 expression for the acquisition of the immunosuppressive phenotype and continuous expression of Foxp3 for maintenance of their phenotype and function, particularly under inflammatory conditions. How the stability of the transcriptional Treg -cell program is controlled and maintained has been under intense scrutiny and only very recently the molecular mechanism governing this could be elucidated. In this review, we will discuss how epigenetic and transcriptional events contribute to the induction and maintenance of Treg cells through regulating the expression of the lineage-defining transcription factor Foxp3 and highlight some of the ensuing downstream events. 2. Foxp3 as a master regulator of Treg cells The development and function of Treg cells is governed by Foxp3, a winged-helix family transcription factor, which acts as a master controller of gene expression in Treg cells. Mutations within the Foxp3 gene or deletion of Foxp3 in transgenic mouse models results in the development of fatal autoimmunity [7–9]. Already very early after its initial discovery as lineage-specification factor for Treg cells it could be shown that the transcriptional program of Treg cells is critically dependent on its expression and its ability to bind to DNA [23,24]. Foxp3 expression is required throughout the life-span of animals as deletion of Treg cells or disruption of Foxp3 expression in adult animals induces auto-reactivity comparable to the complete loss of Foxp3 expression [25,26]. Foxp3 is a multidomain protein with an N-terminal repressor domain, a zinc-finger, a leucine-zipper, and a C-terminal forkhead DNA-binding domain [27]. It has been suggested that Foxp3 can act in concert with other transcription factors including Ikaros family zinc finger 4 (Eos),

activator protein (AP)-1, GATA-binding protein 3 (Gata3), etc. to regulate gene expression in Treg cells through binding to promoter regions thereby influencing gene transcription. Using transgenic animals and elegant biochemical studies it could be demonstrated that the N-terminal repressor domain not only interacts with Eos, but also with a chromatin-remodeling complex which contains histone deacetylase 7 (HDAC7), HDAC9 and the histone acetyltransferase TIP60, indicating that Foxp3 can alter the histone landscape in Treg cells [28–30]. Global analysis of Foxp3 interaction partners has revealed that Foxp3 also binds several RNA binding proteins, suggesting that in addition to its activity as a transcription factor Foxp3 can also regulate gene expression by post-transcriptional mechanisms influencing RNA stability [31]. From genome-wide binding studies of Foxp3 by chromatin immunoprecipitation and subsequent global analysis it is known that Foxp3 binds to a number of distinct genomic loci in Treg cells, where depending on its interaction partners it can either induce or repress expression of genes [32–35]. One open question concerning its function is whether Foxp3 also can act as a factor bridging distant DNA regions and thereby facilitating regulation of genomic loci. Taken together, it is evident that Foxp3 is crucial for the induction and maintenance of the unique immunosuppressive properties of Treg cells by exerting its regulatory activity on transcriptional, epigenetic, and post-transcriptional level as well as probably also influencing the localization and distribution of DNA within the nucleus. 3. Epigenetic control of Foxp3 expression Epigenetic processes are known to play a key role in gene regulation during development as they can consolidate pre-established genetic signatures and provide an inheritable memory of transcriptional activities without changing the DNA sequence [36,37]. Particularly within the immune system, where disturbances within this delicate balance of fine-tuned developmental processes can result in overt pathological consequences, epigenetic control of gene regulation is of utmost importance. DNA methylation and



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histone modifications are two major epigenetic mechanisms that participate in establishing and maintaining chromatin structures [37,38]. While post-translational modifications of histones can be rapidly altered in response to environmental signals, active DNA demethylation of genomic loci is a much slower process [37,38]. Methylation patterns within a given cell lineage are stable under homeostatic conditions, and relatively rarely active demethylation is used to control expression of a gene being necessary for cell activation or differentiation [37,38]. Such processes are mainly controlled via regulation of histone modifications or through the activity of response-specific transcription factors inducing or repressing gene expression [37,38]. To allow for this stimulus-specific control of gene expression it has been shown that pioneering transcription factors are often present at these loci, thereby allowing for rapid activation of gene expression [39]. Recent evidence suggests that Foxp3 expression in Treg cells is under epigenetic control [40,41]. A distinct DNA methylation pattern combined with the formation of characteristic histone modifications establishes an open chromatin structure, thereby imprinting Foxp3 expression in Treg cells [42]. Within the Foxp3 locus, three conserved non-coding sequences (CNS) besides the promoter are the primary targets of epigenetic regulation and are necessary to modulate its expression depending on the environmental cues T cells receive (Fig. 2). The Foxp3 promoter lies 6.5 kb upstream of exon 1 and is defined by a TATA box, a GC box and a CAAT box as well as multiple transcription factor binding sites within approximately 500 bp upstream of the transcriptional start site [43,44]. The CpG motifs within the Foxp3 promoter are almost completely demethylated in ex vivo isolated Treg cells, whilst conventional CD4+ T cells have a much more pronounced methylation at the promoter which is even increased upon activation [45–47]. In iTreg cells, Foxp3 promoter methylation declines to the level of ex vivo isolated Treg cells [45,46]. Mechanistically, the SUMO E3 ligase protein inhibitor of activated STAT-1 (PIAS1) acts as a negative regulator of Treg -cell development and recruits DNA methyltransferases and heterochromatin protein 1 to the Foxp3 promoter, thereby preventing its demethylation and maintaining an epigenetically repressive state [48]. Upon TCR signaling this state is resolved and the promoter becomes accessible for the binding of additional transcription factors to initiate Foxp3 transcription [48]. In accordance with an enhanced demethylation of the Foxp3 promoter in ex vivo isolated Treg cells [45,48], permissive histone modifications such as histone 3 (H3) and H4 acetylation as well as di- and trimethylated H3-lysine4 (H3K4) were found at the Foxp3 promoter in Treg cells but not in CD4+ conventional T cells [44,46,49,50]. On the contrary, trimethylation of H3K27 was observed in conventional CD4+ T cells, but not in Treg cells [51]. It was reported that a Polycomb response element/Krueppel-like factor (KLF) binding site within the Foxp3 promoter is associated with Polycomb repressor complex 2 (PRC2) containing the histone methyltransferase enhancer of Zeste 2 (Ezh2), which keeps the Foxp3 promoter silenced in conventional CD4+ T cells by trimethylation of H3K27 [51]. In Treg cells, the Polycomb repressor complex is replaced by p300/CREB-binding protein-associated factor (PCAF), a histone acetyltransferase recruited via KLF10 to the Foxp3 promoter, a process that finally results in the opening of the Foxp3 promoter by permissive histone modifications [51]. CNS1 is located within the first intron, approximately 2 kb downstream of the promoter [44]. CNS1 is a TGF-␤-sensitive enhancer element, which is critical for the generation of both iTreg cells [44,46,52] and pTreg cells [19,53], but at the same time being dispensable for the generation of tTreg cells [19,46]. CNS1 does not contain any CpG motifs and thus is solely regulated via histone modifications. Indeed, permissive histone modifications such as H3/H4 acetylation and H3K4 di- and trimethylation were found to

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be enriched in both tTreg cells and iTreg cells compared to conventional CD4+ T cells [44,46,54,55]. CNS2, also named Treg -cell specific demethylated region (TSDR), is a CpG-rich element that lies in the first intron, roughly 4 kb downstream of the promoter. The selective demethylation of its CpG motifs is critical for the stabilization of Foxp3 expression in Treg cells. While transient Foxp3 expression is possible even if this region is fully methylated, demethylation of this CpG motif-rich region is mandatory for stable Foxp3 expression [46,56–58]. Along with its demethylated DNA, CNS2 contains increased levels of H3K4 methylation as well as H3/H4 acetylation in Treg cells [46,54,56], suggesting that DNA demethylation and permissive histone modification generate an open chromatin status at CNS2 that promotes stabilization of Foxp3 expression. In the thymus, double-positive thymocytes show high methylation of CNS2 which is retained in CD4 single-positive Foxp3− thymocytes while already partial DNA demethylation can be observed within developing Foxp3+ thymocytes, which results in complete demethylation once the cell is fully matured [56,59]. Importantly, also pTreg cells display a fully demethylated CNS2 and can stably express Foxp3 [42,58]. Thus, demethylation of CNS2 represents an epigenetic marker for stable Foxp3 expression and true Treg -cell lineage identity, but does not allow discrimination between tTreg and pTreg cells [57,60]. Mechanistic data point toward an integrative model where methyl-binding domain 2 (Mbd2) binds to CNS2 and recruits tet methylcytosine dioxygenase 2 (Tet2), which subsequently results in demethylation of CNS2 in Treg cells [59,61,62]. On the other hand, methylation of CNS2 can also prevent Foxp3 expression in non-Treg cells, as demonstrated by induction of Foxp3 expression in CD8+ T cells and NK cells upon Dnmt1 deletion or 5-aza-2 -deoxycytidine treatment, respectively [63,64]. These results suggest that the presence of enzymes maintaining DNA methylation in T cells is also important for cell lineage identity. Together, these data support a model, in which demethylation of the genomic locus of a lineagedefining transcription factor results in the engraving of a definitive cell identity and lineage definition into the DNA of the cell which cannot easily be altered. Very recent data now further support the functional importance of CNS2 as a ‘memory module’ being particularly important for the maintenance of Foxp3 expression in Treg cells under inflammatory conditions [65,66]. Although Foxp3 expression could be maintained upon deletion of CNS2 under homeostatic conditions, the stability of a subset of Treg cells showing a high degree of activation was dependent on the presence of CNS2 [66]. Furthermore, Treg cells exposed to inflammatory cytokines such as IL-4 and IL6 under conditions of limited IL-2 supply or strong TCR signaling also showed reduced stability [65,66]. Whether this was a direct consequence of the complete loss of CNS2 or whether the demethylation of CNS2 itself can stabilize Foxp3 expression and the Treg -cell phenotype via some indirect feedback mechanisms remains elusive. For stable expression of Foxp3 the action of both Runx1 (runt-related transcription factor 1) and Cbf-␤ (core-binding factor subunit ␤) is mandatory. They form a trimeric complex with Foxp3 and bind to CNS2 thereby establishing a feed forward loop fostering Foxp3 expression [49,67]. Furthermore, the hypomethylation of CNS2 is shielded from methylating enzymes by the Foxp3-Runx1Cbf-␤ complex [49,67]. In line with the observation that Foxp3 expression is stabilized by binding of additional transcription factors, it was reported that E26 avian leukemia oncogene 1 (Ets-1) is an additional transcription factor that specifically binds to the demethylated CpG motifs within CNS2 with disruption of Ets-1 binding sites reducing its transcriptional enhancer activity [68]. The critical role of CNS2 as an element containing multiple transcription factor binding sites that can potentially respond to the activation of different signaling pathways with stable Foxp3 expression has been further documented by a recent study, demonstrating that



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Fig. 2. Transcription factors regulating Foxp3 expression. T cell receptor (TCR) and CD28 costimulatory signals as well as Interleukin-2 and an autocrine Foxp3-dependent 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 (orange): AP-1, activator protein 1; c-Rel, member of NF-␬B transcription factor family; CREB, cyclic-AMP-responsive-element-binding protein; NFAT, nuclear factor of activated T cells; p65 (Rel-A), member of NF-␬B transcription factor family. IL-2 signaling (blue): STAT5, signal transducer and activator of transcription 5. TGF-␤ signaling (green): SMAD3, mothers against decapentaplegic homologue 3. Foxp3 signaling (orange): Cbf-␤, core-binding factor, beta subunit; Foxp3, forkhead box P3; RUNX, runt-related transcription factor 1. Constitutive factors (gray): PCAF, p300/CREB-binding protein-associated factor; Nr4a, nuclear receptor 4a; Ets-1, E26 avian leukemia oncogene 1. Inhibitory factors (dark green): Foxo, Forkhead box O; PRC2, Polycomb repressor complex 2; PIAS1, protein inhibitor of activated STAT-1.

upon TCR activation, nuclear factor of activated T cells (NFAT) activity is critical for CNS2 to enhance and stabilize Foxp3 expression in proliferating Treg cells [66]. Binding of NFAT to CNS2 results in a conformational change in the 3D-DNA structure of the Foxp3 locus by bringing CNS2 in close proximity to the Foxp3 promoter as demonstrated by chromosome conformation capture assays [66]. Interference with this DNA interaction resulted in destabilized Foxp3 expression suggesting a functional role for this interaction in protecting Treg -cell identity. CNS3 is located immediately downstream of the first coding exon, approximately 7 kb downstream of the promoter. CNS3 is also called ‘pioneer element’ since it plays a critical role for the initiation of Foxp3 expression in both tTreg and pTreg cells, but is dispensable once Foxp3 is expressed [46]. In Treg cells, it is enriched in H3K4 mono- and dimethylation (but not trimethylation), and these permissive histone modifications are already significantly increased in Foxp3− thymocyte subsets [46], suggesting that CNS3 facilitates the opening of the Foxp3 locus already in Treg -cell precursors [46]. Viewed as a whole, the current data support a model where gradual demethylation of the Foxp3 promoter and CNS2 along with the acquisition of permissive histone modifications in all conserved genetic element of the Foxp3 locus occurs during Treg -cell development and supports stabilization of Foxp3 expression by allowing transcription factors to better access these genomic regions. 4. Epigenetic control of the global gene expression program of Treg cells Recent evidence suggests that not only the Foxp3 gene, but also other Treg -cell specific genes are under epigenetic control [40,41]. Using whole-genome methylated DNA immunoprecipitation sequencing and bisulfite sequencing of Treg cells and their thymic precursors Ohkura et al. demonstrated that Treg cells have a closely related, but still distinct DNA methylation pattern in comparison to conventional CD4+ T cells [42]. The establishment of this Treg -cell specific DNA hypomethylation pattern was induced by TCR signaling, independently of Foxp3 expression [42]. Both, Foxp3 expression and hypomethylation have to occur concurrently for T cells to develop into stable Treg cells. This global regulation of Treg cell associated transcription through DNA hypomethylation was further supported by the combined analysis of transcriptional start sites with genome-wide patterns of DNA methylation [69]. DNA hypomethylated regions in non-activated Treg cells were highly enriched for actively transcribed genes, while in activated Treg cells

down-regulated genes showed increased Foxp3 binding in combination with DNA hypomethylation. In line with this observation, in human Treg cells hypomethylated Treg -cell specific genomic regions were mainly located at promoter-distal regions of Treg -cell associated genes [70], supporting a model where Treg -cell specific DNA hypomethylation is critical for the expression of genes necessary to generate and maintain Treg cells with Foxp3 further modulating the transcriptional state of Treg cells upon their activation. Besides DNA methylation the epigenetic regulation of gene transcription also can occur via positioning of histone proteins regulating accessibility for transcription factors and the posttranslational modification of histone proteins. Based on the general concept of lineage definition and commitment where closely related cells should be similar in their epigenetic profile, chromatin accessibility of Treg cells and conventional CD4+ T cells was highly correlated with only a small group of Treg -cell specific DNase hypersensitive sites located near or within many Treg -cell characteristic genes [32]. The majority of these enhancer regions are either occupied by Foxp3 cofactors before Foxp3 is expressed or are bound by Foxo1, a related Forkhead transcription family member, which acts as a pioneer factor and can be replaced by Foxp3 upon its expression. For the Treg -cell specific enhancers it was demonstrated that similar to the Treg -cell specific demethylation of genomic regions the accessibility of these Treg -cell specific DNase hyper-sensitive sites was dependent on TCR signaling prior to the acquisition of Foxp3 expression through NFAT-AP1-assisted chromatin remodeling [32]. This rather limited acquisition of Treg -cell specific epigenetic modifications is also reflected by the analysis of genome-wide maps of permissive and repressive histone modifications [71]. While effector genes partially follow the concept of terminal differentiation with Treg cells and conventional CD4+ T cells showing distinct permissive and repressive histone modification patterns, particularly upstream regulators like transcription factors exhibited a more neutral spectrum of epigenetic states. This suggests that these genes are mainly regulated through transcriptional events rather than epigenetic remodeling of their genomic loci. Foxp3 itself seems to be actively involved in this epigenetic modification of Treg cell specific loci through its interaction with Tip60, HDAC7, and Eos as initially suggested using a hypomorphic Foxp3 reporter mouse [30]. This was particularly apparent under inflammatory conditions, an observation that could be recently confirmed in a study by Arvey et al. demonstrating that Foxp3-bound enhancer elements in the DNA were poised for repression only in activated Treg cells




[72]. This was mediated by decreased chromatin accessibility and selective trimethylation of H3K27 as a result of the activation of the PRC2 complex containing Ezh2, leading to downregulation of the transcriptional activity of nearby genes [72]. Taken together, these observations foster the idea that DNA methylation, histone modifications, and nucleosome positioning as major epigenetic modifications contribute to lineage determination and commitment of Treg cells by altering the accessibility of gene loci for transcription factors and maintain this epigenetic status allowing for transcriptional accessibility over a long period of time independently of Foxp3. 5. Transcriptional control of Foxp3 expression Transcription of Foxp3 in Treg cells is controlled not only on epigenetic level but also through a highly variable interplay of transcriptional events depending on the signals T cells receive either in the thymus or in the periphery and the concomitant environmental factors converging in the induction of Foxp3 (Fig. 2). Highly important for the induction of Foxp3 expression are the aforementioned CNS enhancer elements within the Foxp3 locus with two of them showing high activity in these distinct developmental processes. 5.1. Transcriptional control of Treg -cell development in the thymus The development of Treg cells in the thymus can be sub-divided into two steps. Upon receipt of TCR- and APC-dependent signals CD4SP thymocytes can either upregulate CD25 expression and develop into a Treg -cell precursor state (CD25+ Foxp3− ) or induce Foxp3 expression and develop into apoptosis-prone, immature tTreg cells (Foxp3+ CD25− ) [73–75]. This first step is then followed by a second ‘maturation’ step that is independent of TCR stimulation, but dependent on common ␥-chain cytokines (especially IL-2, but also IL-7 or IL-15) to either induce stable Foxp3 expression in CD25+ Foxp3− Treg precursor cells or to prevent apoptosis induction in Foxp3+ CD25− cells [73–75]. Both CNS2 and CNS3 have been shown to play an important role for the induction of Foxp3 expression during tTreg -cell development in addition to the Foxp3 promoter. While transcription factors binding to CNS3 are responsible for the initiation of Foxp3 expression, factors binding to hypomethylated CNS2 are required for the heritable maintenance of Foxp3 expression [46]. In this process, TCR signaling and CD28-mediated costimulation are absolutely necessary for thymocytes to acquire the signal to develop into Treg cells [2,40,41]. Downstream of these two events, nuclear factor-␬B (NF␬B) signaling through c-Rel, which directly binds to the promoter, CNS2 and CNS3 regions, is required to reorganize the corresponding regions to allow for active transcription of Foxp3 [46,76–78]. In addition, formation of a c-Rel enhanceosome at the Foxp3 promoter can further support its expression [78]. This is followed by the recruitment of additional transcription factors resulting in Foxp3 transcription. AP-1 binding to the Foxp3 promoter [43] and cyclic AMP response element-binding protein (CREB) binding to CNS2 [45] have been implicated as important events downstream of TCR signaling and CD28-mediated costimulation promoting Foxp3 expression in the thymus. The role of NFAT and its family members is still controversial. Although it has been shown that Ca2+ signaling results in NFAT recruitment to the Foxp3 promoter in vitro [43,44] and that combined genetic deletion of NFAT family members can result in decreased Treg -cell generation in the thymus [79], there seems to be a high redundancy in the action of single NFAT family members. TCR signaling further induces the expression of members of the nuclear receptor 4a (Nr4a) family of orphan nuclear receptors [50,54,80]. These cofactors bind to the Foxp3 promoter

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and induce Foxp3 expression [80]. Their genetic ablation results in a deficiency in tTreg -cell generation [50], suggesting a role for Nr4a family members in translating the strength of TCR signaling into the transcriptional control of Foxp3 expression. In addition to TCR signaling and proper costimulation, cytokine signaling is directly involved in the induction of Foxp3 expression during tTreg cell development. Whereas the contribution of common ␥-chain cytokines and their downstream targets to the induction and stabilization of Foxp3 expression is unquestionable, resulting in the binding of signal transducer and activator of transcription (Stat)5 to the Foxp3 promoter and CNS2 [81,82], the role TGF-␤ and SMAD proteins as direct targets of TGF-␤ binding to its receptor still remains controversial. Initially, TGF-␤ signaling has been suggested to solely play a role in the maintenance of Foxp3 expression and homeostasis of Treg cells in the periphery [83]. However, more recent work demonstrated that ablation of TGF-␤ signaling resulted in deficiency in tTreg -cell development in neonatal mice [84,85]. To add another layer of complexity, TCR activation and costimulation result in activation of the phosphoinositide 3kinase (PI3K)-Akt pathway, thereby leading to phosphorylation of Foxo1/Foxo3 and their nuclear exclusion, which precludes Foxo1/Foxo3 from binding to CNS2 and the Foxp3 promoter, finally resulting in increased tTreg -cell generation [15,86,87]. Once tTreg cell induction is initiated the termination of antigen contact enables Foxo1/Foxo3 to translocate back to the nucleus to consolidate Foxp3 expression by binding to the Foxp3 promoter [86]. Taken together, in the thymus antigen contact via the TCR, costimulation, and cytokine signals all are necessary to induce and reinforce Foxp3 expression mainly via binding of transcription factors to the Foxp3 promoter and the CNS2 and CNS3 elements with CNS3 mainly acting as pioneer enhancer element being critical for the initiation of Foxp3 expression. 5.2. Development of peripherally induced Treg cells Peripheral conversion of Foxp3− naïve conventional CD4+ T cells into Foxp3+ pTreg cells can be mediated by exposure to low-dose antigen in the presence of cytokines promoting pTreg -cell induction and is mainly taking place at barrier surfaces where constant antigen exposure to neo-antigens is present [11–13,16,17]. This generation of pTreg cells is highly dependent on CNS1 as deletion of CNS1 resulted in defective generation of pTreg cells [19,20,46]. Similar to the generation of tTreg cells, NF-␬B and Nr4a family members as downstream effector molecules of TCR activation have been linked to the induction of Foxp3 expression in pTreg cells [50,77,78]. NFAT and probably also AP-1 contribute to Foxp3 induction by binding to the CNS1 element as mediators of direct TCR activation [55,88]. In contrast to tTreg cells, however, costimulation does not promote Foxp3 induction but rather prevents its expression. Strong costimulation via CD28 blocks pTreg -cell induction [89] while coinhibitory molecules like CTLA-4 facilitate their generation [90]. In this context, the PI3K-Akt axis through Foxo1/Foxo3 also influences pTreg -cell induction as blockade of PI3K-Akt signaling can foster pTreg -cell differentiation via nuclear translocation of Foxo1/Foxo3 [86,87,91–93]. As cytokines, TGF-␤ and IL-2 are the main factors supporting Foxp3 expression and pTreg -cell induction. While data on the role of TGF-␤ in tTreg -cell induction are still contradictory, it is widely accepted that binding of SMAD3 downstream of TGF-␤ receptor signaling to CNS1 is a prerequisite for the induction of Foxp3 expression in naïve conventional CD4+ T cells [44,46,52]. TGF-␤ driven pTreg -cell induction can be further supported by the action of the vitamin A derivate retinoic acid generated by intestinal dendritic cells [89,94,95]. Retinoic acid can act directly on the Foxp3 locus via induction of histone acetylation at the promoter [96] and via recruitment of retinoic acid receptors to the CNS1 element [55],



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or indirectly by interfering with production of effector cytokines by memory T cells [94]. IL-2 signaling via Stat5 further fosters and solidifies Foxp3 expression in pTreg cells through its binding to the Foxp3 locus [97], while simultaneously preventing TH 17-cell differentiation [98,99].

6. Induction of Treg -cell associated transcriptional programs Following the epigenetic reorganization of the genome downstream of Treg -cell lineage commitment and the induction of Foxp3 as the main lineage transcription factor necessary for Treg -cell identity the transcriptional program of Treg cells is initiated and sustained through additional transcription factors acting in concert with Foxp3. Among these is Runx1, which can physically interact with Foxp3. This complex can not only suppress the expression of effector cytokines (e.g. IL-2, IL-4, and IFN-␥), but can also induce the production of Treg -cell associated molecules such as CD25, CTLA-4 and GITR [100]. Another transcription factor interacting with Foxp3 is Eos, which binds to the N-terminal region of Foxp3. This binding was shown to be crucial for the ability of Foxp3 to repress the transcription of its target genes by inducing chromatin modifications that result in gene silencing in Treg cells [101]. In addition, the N-terminal domain is also involved in the interaction with ROR␥t and ROR␣, thereby antagonizing the action of these transcription factors and inhibiting TH 17-cell differentiation [102,103]. Furthermore, there have been numerous transcription factors downstream of TCR signaling and costimulation which have been associated with Foxp3 and its transcriptional activity, including NFAT, NF-␬B, and AP-1. Indeed, Foxp3 cooperates with NFAT through the forkhead domain to form a DNA-binding complex that is important for Treg -cell function by competing with AP-1 [104]. In addition, by interacting with c-Jun/AP-1 Foxp3 can sequester activated AP-1 in the nucleus in Treg cells, altering the subnuclear distribution of activated AP-1 and disrupting chromatin-binding of activated AP-1 [105]. While Foxp3 can physically interact with both the Rel-A and c-Rel subunits of the NF-␬B complex, the functional relevance of this binding is still elusive [106,107]. Global analysis of the interactome of Foxp3 further supported a model, in which Foxp3 and its binding partners form a multiprotein complex that can bind to DNA and regulate transcription depending on the recruited interaction partners [31]. As outlined above, this binding exploits a pre-existing enhancer landscape within the CD4+ T-cell lineage, showing particular enrichment in regions, which show pre-bound Foxp3 interaction partners [32]. This is further supported by the analysis of genomic regions bound by Foxo1 as Foxp3 can replace Foxo1 at a subset of loci, thereby fine-tuning gene expression in Treg cells [32]. In addition, analysis of Foxo1 target genes identified by combined assessment of genome-wide binding regions with transcriptome analysis of Foxo1-deficient Treg cells showed a separate subset of >300 putative direct target genes for Foxo1 which are not bound by Foxp3, proposing that Foxo1 binds an additional set of genes with a largely non-overlapping genetic program to Foxp3 [87]. This has been implicated in the inhibition of Teffector -cell function in Treg cells as lack of Foxo1/Foxo3 in Treg cells is sufficient to induce TH 1 and TH 17 effector cytokines but not TH 2 cytokines [86,87]. Although in the recent past several important mechanisms how Foxp3 influences the Treg -cell transcriptional program have been elucidated, several questions remain how precisely the network of transcription factors co-expressed with Foxp3 actively controls, modulates and governs Foxp3-dependent transcriptional modules in Treg cells.

7. Transcriptional control of effector properties in Treg cells Despite the fact that Foxp3 is the most important transcriptional regulator of Treg -cell function, accumulating evidence suggests that additional transcription factors are required for the development of functionally unique subsets of Treg cells. The Treg -cell population is not homogeneous and does not utilize a universal suppressor mechanism to suppress all target cells, but rather constitutes a very diverse and dynamic repertoire that changes depending on the inflammatory context as well as the location of the response. Thus, on a population level, Treg cells exhibit a high degree of diversity that corresponds to the expression of transcription factors in subsets of Treg cells, which were originally found to be expressed and of functional importance for inflammatory TH -cell subsets. This was first reported for the acquisition of TH 2-like properties in Treg cells induced by expression of the transcription factor IRF4 inducing TH 2-like transcriptional modules and enabling Treg cells to control TH 2-driven autoimmunity [108]. A similar observation could be made for the TH 1-specification factor Tbx21 (T-bet), which can also be expressed by Treg cells under type 1 inflammatory conditions, resulting in the acquisition of TH 1-like transcriptional programs in this Treg -cell population [109,110]. T-bet induces the expression of the chemokine receptor CXCR3 in this subpopulation of Treg cells, which allows them to migrate to sites of TH 1-type inflammation and to suppress tissular TH 1 immune responses, further supporting the idea that the expression of this TH 1-cell specific transcription factor controls migration and function of these Treg cells [109,110]. This concept has since then been extended to the expression of Gata3 as a TH 2-cell specific transcription factor inducing transcriptional TH 2-like programs in Treg cells [111,112]. Concordantly, TH 17-cell properties are induced in Treg cells trough the expression of Stat3, resulting in expression of the TH 17-cell specific homing receptor CCR6 and the induction of TH 17-cell transcriptional programs [113,114]. Together, these findings suggest that subsetspecific transcription factors impart specificity based on chemokine receptor expression and functional properties, which allows Treg cells to better co-localize with and more specifically suppress their diverse target cells.

8. Transcriptional control of tissue-specific programs in Treg cells Treg cells can also be found in non-lymphoid tissues such as skin, intestinal mucosa, lung, liver, adipose tissue, autoimmune target tissues, infected tissues, grafts, placenta, tumors, atherosclerotic plaques, and injured skeletal muscle. These cells play a direct role in controlling local inflammation and in the maintenance of tissue homeostasis. Accumulating evidence suggests that the phenotype and function of such tissue-resident Treg cells are not identical to ‘classical’ Treg cells that can be found within lymphoid-organs [115]. For visceral adipose tissue (VAT), the population of tissueresident Treg cells has been analyzed in detail [116,117]. While the majority of the Treg -cell signature genes are also expressed by VAT Treg cells, they differentially express several chemokine receptors and immunomodulatory cytokines [116,117]. Responsible for these unique transcriptional and functional properties of VAT Treg cells is the transcription factor PPAR-␥, which is a member of the nuclear receptor superfamily required for adipocyte differentiation and function [118] and can interact with Foxp3 to induce the VAT Treg -cell associated transcriptional programs [116,117]. The importance of PPAR-␥ for VAT Treg cells is reflected by reduction in Treg -cell numbers and the VAT-associated transcriptional programs in PPAR-␥ deficient Treg cells, while no changes in gene expression or frequencies could be observed in lymphoid organs [116,117]. These data for the first time could link a tissue-specific




transcription factor with the acquisition of tissue-specific transcriptional programs in Treg cells. Treg cells were also identified in injured and regenerating skeletal muscle, where they exert highly specialized functions and contribute to muscle regeneration [119]. Transcriptome analysis of these muscle Treg cells revealed unique transcriptional programs, which would suggest a closer relationship of muscle Treg cells to VAT Treg cells when compared to lymphoid Treg cells with high expression of genes encoding for effector molecules like IL10, the growth factor amphiregulin, and TIM-3 [119]. In summary, these pioneer data support a model where tissue-resident Treg cells acquire tissue-specific transcriptional properties being imprinted by environmental factors. 9. Conclusions Recent data suggest that the development of Treg cells is dependent on epigenetic changes preceding induction of the Treg -cell lineage specific transcription factor Foxp3. This involves signaling events downstream of the TCR and costimulation with environmental signals further contributing to the induction and stabilization of Foxp3 expression through the concerted binding of transcription factors to the promoter and several enhancer elements within the genomic locus of Foxp3. Once Foxp3 transcription is induced, Foxp3 itself with the help of additional transcription factors can further reinforce its expression. In inflammatory environments or after tissue homing, Treg -cell subsets coopt transcriptional programs by expression of transcription factors also required for the specific immune reaction or tissue condition to fulfill their highly specialized function as regulators of immune reactions. For long-term lineage commitment and stability of Treg cells, the expression of Foxp3 and other Treg -cell signature genes is ensured by epigenetic modifications including hypomethylation of respective DNA regions. How the differentiation of Treg cells and their effector function is controlled in peripheral tissues during active immune responses is an area, which is far from being understood. Further investigation in this direction will elucidate the mechanisms how Treg cells can modulate immune responses during chronic inflammatory conditions as in age- or western-diet-associated diseases, enabling us to devise new approaches for clinical manipulation of these powerful and multi-talented cells. Acknowledgements M.B. has been funded by the Wilhelm-Sander-Foundation and the German Research Foundation (SFB 832, BE 4427/3-1). References [1] Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell 2008;133:775–87. [2] Josefowicz SZ, Lu LF, Rudensky AY. Regulatory T cells: mechanisms of differentiation and function. Annu Rev Immunol 2012;30:531–64. [3] Powrie F, Leach MW, Mauze S, Caddle LB, Coffman RL. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C. B-17 scid mice. Int Immunol 1993;5:1461–71. [4] Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alphachains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995;155:1151–64. [5] Asano M, Toda M, Sakaguchi N, Sakaguchi S. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J Exp Med 1996;184:387–96. [6] Shevach EM. Regulatory T cells in autoimmmunity. Annu Rev Immunol 2000;18:423–49. [7] Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+ CD25+ regulatory T cells. Nat Immunol 2003;4:330–6. [8] Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003;299:1057–61.

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Transcriptional control of regulatory T cells.

Differential roles of epigenetic changes and Foxp3 expression in regulatory T cell-specific transcriptional regulation.

Transcriptional control of regulatory T-cell differentiation.

high)Foxp3(+) regulatory T cells.

Lineage stability and phenotypic plasticity of Foxp3⁺ regulatory T cells.

FOXP3+ regulatory T cells and their functional regulation.

Enhanced suppressor function of TIM-3+ FoxP3+ regulatory T cells.

Transcriptome profiling of human FoxP3+ regulatory T cells.

Development of thymic Foxp3(+) regulatory T cells: TGF-β matters.

IL-17+Foxp3+ T cells: an intermediate differentiation stage between Th17 cells and regulatory T cells.

Adipose tissue macrophages induce PPARγ-high FOXP3(+) regulatory T cells.

Hepatocytes induce Foxp3⁺ regulatory T cells by Notch signaling.

Foxp3+ regulatory T cells promote lung epithelial proliferation.

Unstable FoxP3+ T regulatory cells in NZW mice.

Harnessing FOXP3+ regulatory T cells for transplantation tolerance.

γδ T cells restrain extrathymic development of Foxp3+-inducible regulatory T cells via IFN-γ.

Regulatory T cells, especially ICOS+ FOXP3+ regulatory T cells, are increased in the hepatocellular carcinoma microenvironment and predict reduced survival.

The balance of intestinal Foxp3+ regulatory T cells and Th17 cells and its biological significance.

RelB+ Steady-State Migratory Dendritic Cells Control the Peripheral Pool of the Natural Foxp3+ Regulatory T Cells.

Androgen receptor modulates Foxp3 expression in CD4+CD25+Foxp3+ regulatory T-cells.

Induction of antigen specific CD4(+)CD25(+)Foxp3(+)T regulatory cells from naïve natural thymic derived T regulatory cells.

UXT is a novel regulatory factor of regulatory T cells associated with Foxp3.

Differential control of immune cell homeostasis by Foxp3(+) regulatory T cells in murine peripheral lymph nodes and spleen.

Regulatory T Cells in Melanoma Revisited by a Computational Clustering of FOXP3+ T Cell Subpopulations.