Regulatory T cell lineage commitment in the thymus

Seminars in Immunology 23 (2011) 401–409

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Review

Regulatory T cell lineage commitment in the thymus Ludger Klein ∗ , Ksenija Jovanovic University of Munich, Institute for Immunology, Goethestr. 31, 80336 Munich, Germany

a r t i c l e

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Keywords: Thymus Regulatory T cell Foxp3 Negative selection Lineage commitment

a b s t r a c t A substantial fraction of the Foxp3+ CD4+ regulatory T (Treg ) cell repertoire is generated through instructive and/or selective processes in the thymus, and there is some consensus that clonal deviation into the Treg lineage is a result of self-antigen recognition. Paradoxically, the same holds true for a diametrically different cell fate decision of developing thymocytes, namely their removal from the repertoire through apoptotic cell death (clonal deletion). Here, we will review our current understanding of how T cell receptor stimulation, cytokine signaling, co-stimulation, epigenetic modifications and T cell intrinsic developmental tuning synergize during Treg cell differentiation, and how instructive signals converge at the Foxp3 gene-locus during entry into the Treg cell lineage. We will also discuss how these parameters relate to known determinants of negative selection. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction: clonal deletion and clonal deviation as alternative fates of autoreactive thymocytes Seminal experiments in the late 1980s showed that expression of an autoreactive antigen receptor can result in the physical elimination or functional inactivation of immature T cells in the thymus [1–4]. Because clonal deletion and clonal inactivation (anergy) both represent cell intrinsic mechanisms, they are also termed recessive tolerance. Around the same time, it was found that transplantation of allo- and even xenogeneic thymic epithelium before colonization by hematopoietic precursors resulted in lifelong tolerance to grafted tissues of the donor-type [5,6]. This work and a subsequent series of experiments established that thymic epithelium-mediated tolerance could not be explained by recessive tolerance mechanisms, but instead must somehow rely on the dominant action of autoreactive “suppressor” cells [7]. Interestingly, whereas recessive modalities of central tolerance rapidly became an accepted paradigm, this dominant mode of tolerance only slowly gained a wider acceptance, perhaps owing to the fact that the precise nature of these thymus-dependent “suppressor” or “regulatory” cells remained elusive (apart from residing within the CD4+ T cell compartment [8]), but certainly also because the term “suppressor” had fallen into disrepute due to earlier controversies surrounding largely unrelated phenomena (reviewed in

Abbreviations: APC, antigen presenting cell; Foxp3, forkhead box transcription factor 3; TCR, T cell receptor; tTreg cell, thymus-derived regulatory T cell; iTreg cell, induced regulatory T cell; TSDR, Treg -specific-demethylated-region; mTEC, medullary thymic epithelial cell; MHC II, major histocompatibility complex class II. ∗ Corresponding author. Tel.: +49 89 218075696; fax: +49 89 51602236. E-mail address: [email protected] (L. Klein). 1044-5323/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.smim.2011.06.003

[9]). Nevertheless, in the mid 1990s several other labs more or less simultaneously showed that elimination of particular subsets of CD4+ T cells (by selective depletion prior to adoptive transfer into lymphopenic hosts or as a consequence of neonatal thymectomy) would unleash an otherwise suppressed auto-destructive potential within the T cell repertoire [10–12]. Although these investigators all used distinct surface phenotypes – CD45RBlow or CD25high in mice and CD45RClow in rats, respectively – to characterize the particular regulatory population of CD4+ T cells, it is in hindsight clear that all these reports in essence described the immunoregulatory function of what is now known as CD25+ Foxp3+ regulatory T (Treg ) cells [13]. A plausible “missing link” between Foxp3+ Treg cells and thymic epithelium-induced dominant tolerance was then established when a critical function of the thymus for their generation was discovered [14] and by evidence that T cell receptor (TCR) transgenic thymocytes can be deviated into the Treg cell lineage when cognate antigen is expressed by thymic epithelium [15–17]. To date, there is some consensus that a substantial fraction of the Treg cell repertoire originates from the thymus, as supported by a repertoire comparison of thymic and peripheral Treg cells [18]. However, there is little doubt that phenotypically similar and functionally related (although not necessarily identical) Treg cells can under particular circumstances also arise through conversion of peripheral naïve T cells into so-called adaptive or induced (i)Treg cells [19]. In the following, we will refer to thymus-derived Treg cells as tTreg cells (instead of the commonly used but perhaps somewhat misleading term “natural (n)Treg ”). Entry into the Treg lineage during thymocyte development is believed to depend upon instructive processes ensuing from selfantigen recognition. Evidence for this has not only been obtained in TCR transgenic systems, but also stems from observations that polyclonal thymocytes bearing superantigen-reactive TCRs are

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substantially enriched in Foxp3+ cells [20,21]. Also consistent with enhanced autoreactivity being a key driving force of Treg lineage commitment, T cells transduced with Treg derived TCRs are activated upon transfer into non-lymphopenic hosts [18] (although other investigators failed to observe such overt autoreactivity of Treg TCRs [22]), and the Treg repertoire harbors a significant frequency of cells that show reactivity to syngeneic antigen presenting cells (APCs) in vitro [23]. Together, a substantial body of evidence supports the idea that the expression of an autoreactive TCR is a decisive determinant specifying the entry of MHC II-restricted thymocytes into the tTreg lineage. Hence, there is the paradox that autoreactivity appears to be a common attribute of clonally deleted TCR specificities as well as of thymocytes that are deviated into the Treg lineage. In this review, we will discuss thymocyte-intrinsic and -extrinsic parameters that have been implicated in tTreg differentiation and, wherever possible, try to relate these factors to known determinants of negative selection.

mediate avidities favor Treg differentiation over negative selection [33]. In sum, most of the available data are compatible with an avidity model of Treg differentiation. Accordingly, quantitative variations in the level of cognate antigen-presentation, i.e. in the ensuing avidity of antigen recognition, can drive cells that express the very same TCR into either apoptotic cell death or Treg differentiation. Therefore, the quality, that is, the “pure” affinity of a given TCR/agonist interaction, is highly unlikely to be the central, binary determinant of Treg differentiation versus negative selection (at least as far as the available TCR transgenic specificities that have been used to test this idea are concerned). It remains to be seen how these findings can be reconciled with the perplexingly narrow affinity threshold that was suggested to separate positive selection from negative selection [34]. As these latter observations were made with CD8T cells, it remains possible that only CD4T cell-differentiation accommodates a lineage-specific “third” avidity-window between positive and negative selection. 3. Interleukin-2 (IL-2) in Treg differentiation

2. The avidity model of Treg differentiation Current models of thymocyte selection posit that MHC/selfpeptide interactions of intermediate “strength” are required for positive selection, whereas very strong interactions lead to negative selection. Of note, this commonly used concept in fact amalgamates two models with quite distinct assumptions: whereas a strictly affinity-based model centers on properties of the individual TCR/MHC–peptide interaction [24,25], the avidity model instead assumes that the product of the TCR/MHC–peptide affinity multiplied by the number of interactions is critical [26,27]. How does the presumed agonist-driven entry into the Treg lineage fit into this scenario? Several lines of evidence are consistent with the idea that Treg differentiation ensues from interactions that lie in between the signaling strength required for positive selection on the one side and clonal deletion on the other side [28]. First, the combination of a particular MHC II-restricted TCR transgenic system with multiple lines of cognate antigen-transgenic mice revealed that the number of emerging Treg cells inversely correlated with the “antigen dose” (as measured by the abundance of cognate antigen mRNA in the thymus), whereas the extent of concomitant negative selection increased with the overall “antigen dose” [29]. Second, similar results were obtained in vitro upon titration of agonist peptides into TCR transgenic fetal thymic organ cultures (FTOCs) [30]. Third, intravenous administration of graded amounts of cognate antigen in vivo preferentially lead to Treg induction at very low levels of antigen, whereas increasing amounts of injected antigen favored negative selection [31]. Most of these experiments bear the inherent caveat that, besides quantitative parameters (i.e. the level of antigen-expression or–presentation), also qualitative variables (e.g. the type of APC which ultimately presents the respective antigen and/or the developmental stage at which antigen is “seen”) can only insufficiently be controlled. In an attempt to circumvent these confounding issues, we used an alternative strategy to test the avidity model: in AIRE-HA × TCR-HA double transgenic mice, medullary thymic epithelial cell (mTEC)-specific antigen-expression and presentation results in negative selection of about two thirds of hemagglutinin (HA)-specific thymocytes, whereas a substantial fraction of the remaining cells differentiate into Treg cells [32]. When antigen presentation by mTECs in this system was attenuated through RNAi mediated knock-down of MHC class II (through silencing of its transcriptional master regulator CIITA), a diminished extent of negative selection and an increased emergence of Treg cells was observed, which is fully consistent with the notion that inter-

3.1. The essential function of IL-2 Interleukin-2 (IL-2) was cloned in 1983 on the basis of its capacity to enhance T cell proliferation in vitro [35]. The view that IL-2s main function in vivo is to support immunity by acting as a T cell growth factor was challenged by the at first paradoxical finding that mice deficient for IL-2, IL-2R␣ (CD25) or IL-2R␤ (CD122), rather than being immune deficient, succumbed to systemic autoimmunity [36–39]. Subsequently, two seminal papers demonstrated that IL-2 or IL-2 receptor deficiency led to reduced frequencies of CD25+ Treg , and that adoptive transfer of CD25+ Treg rescued the autoimmunity of IL-2R␣ and IL-2R␤ deficient mice [40,41]. To date, a substantial body of evidence supports the corresponding view that the essential, non-redundant function of IL-2 is the maintenance of immune homeostasis through its role in Treg biology [42]. Adoptive transfer of Treg cells into IL-2 deficient hosts results in their rapid loss [43], and neutralization of IL-2 promotes autoimmune manifestations through depletion of Treg cells [44], consistent with a role of IL-2 as an essential survival factor for Treg cells. A second, mutually not exclusive function of IL-2 as differentiation factor in thymic Treg development remained more controversial for some time owing to the fact that it is to some degree redundant. Thus, the thymic population of Foxp3+ Treg cells is only mildly affected in IL-2 or IL-2R␣ (CD25) deficient mice [43]. However, a far more dramatic decrease is seen in mice with targeted mutations of the IL-2R␥ chain (common ␥-chain) [45], JAK3 [46] or STAT5 [47], i.e. in the absence of signaling molecules that are shared by the IL-7- and IL-15-signaling pathways. Because neither IL-7- nor IL15-deficiency by itself affects the thymic production of Treg cells, it is the prevailing view that IL-2 is the principal common ␥-chain cytokine required for intrathymic Treg cell development, but that in its absence IL-7 and IL-15 can at least in part compensate for its loss [48,49]. 3.2. Common--chain cytokines and the two-step model of Treg differentiation What is the function of IL-2, or of common ␥-chain signaling through STAT5 in general, during thymic Treg cell differentiation? Analyses of polyclonal thymocytes showed that a relatively small Foxp3− CD25+ subset of CD4 SP thymocytes is enriched in precursors of Foxp3+ CD25+ Treg cells [50,51]. Consistent with this, work in a TCR transgenic model of Treg differentiation indicated that within 12 h after the instructive TCR stimulus, developing

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Treg cells acquire a Foxp3− CD25+ phenotype, whereas expression of measurable amounts of Foxp3 protein is not seen before at least another 48 h [52]. Importantly, these Foxp3− CD25+ precursor cells require common ␥-chain cytokines but no further TCR stimulation for acquisition of the “mature” Foxp3+ Treg phenotype. Thus, Treg development seems to segregate into a TCR-driven “instructive” phase and a cytokine-driven “consolidation” phase, and this scenario was termed two-step model of tTreg differentiation [50,51]. The mechanistic details of how molecular events downstream of cytokine- and common ␥-chain signaling impinge on Treg differentiation remain to be fully established. On the one hand, activated STAT5 is likely to directly bind to and regulate the Foxp3 gene at the promoter and at an intronic enhancer region [47] (Fig. 1). However, several mutually not exclusive other modes-of-action of less “instructive” nature remain possible: first, cytokine-signaling might establish permissive chromatin modifications at critical “lineage-defining” gene loci (including, but not restricted to, Foxp3). Second, common ␥-chain signaling might also promote the expansion and/or the survival of committed Treg precursors. Little is known as to how this function of cytokines in Treg differentiation relates to clonal deletion of autoreactive MHC II-restricted thymocytes. For instance, whereas competition for IL-2 is very likely to shape the Treg repertoire at the “TCRprimed” Foxp3− CD25+ precursor stage, the fate of those cells that fail to receive appropriate IL-2 stimulation remains to be established. Whereas it is possible that these cells revert phenotypically and escape from central tolerance as naïve, conventional T cells, it appears more likely that they undergo apoptotic cell death (which would represent a form of “negative selection by neglect”). Considering the latter scenario, it cannot even be excluded that all thymocytes (or all CD4 SP cells) that have received a TCR stimulus exceeding a certain threshold-value enter a corresponding state in which common ␥-chain signaling might protect from clonal deletion. To our knowledge, this idea has not been rigorously tested. However, it may deserve mentioning that IL-7 was suggested to have the capacity to negate negative selection [53].

4. CD28 co-stimulation in Treg differentiation 4.1. CD28 and the “two-step” model of Treg differentiation Ablation of the genes encoding for CD28 or its ligands CD80 and CD86 (B7.1 and B7.2, respectively) results in a dramatic decrease in the number of thymic and peripheral Treg cells [54–56]. However, overt autoimmunity is not seen under these circumstances, presumably because of a compensatory effect of diminished activation of potentially dangerous “conventional” effector T cells [54]. Although co-stimulation has been implicated in IL-2 production, the inefficient entry of CD28−/− thymocytes into the Treg lineage is not corrected by the presence of bystander CD28+/+ cells, suggesting that the paucity of thymic Treg cells in co-stimulation deficient mice primarily reflects a T cell-intrinsic function of the CD28/B7 axis, largely independent of IL-2 [57]. At what stage of Treg differentiation does CD28 operate? Recent data indicate that CD25+ Foxp3− Treg precursors are strongly diminished in the thymus of CD28−/− mice, suggesting an “early” requirement of CD28 co-stimulation simultaneous to or in relatively close temporal proximity to the presumed initiating TCR signal [58,59]. Because abrogation of Treg differentiation at this precursor stage precludes an analysis of subsequent stages, it remains possible that CD28 signals are continually required throughout both the TCR driven first and the cytokine dependent second phase of Treg differentiation.

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4.2. CD28 and NF-B Generally, CD28 co-signals in T cells can stabilize messenger RNAs and amplify the activation of nuclear factor of activated T cells (NFAT) and nuclear factor-␬B (NF-␬B), thereby supporting cytokine production, T cell survival and proliferation [60]. CD28 addresses several downstream signaling cascades through distinct SH2- or SH3-domain-binding motifs in its cytoplasmic tail that mediate among others interactions with Lck and the PI3K pathway, respectively. The ability of CD28 to support efficient tTreg cell generation was found not to require an intact PI3K-binding motif [57–59]. Consistent with this, PI3K-signaling through Akt and mTOR might in fact even antagonize tTreg differentiation by sequestering Foxo1/Foxo3a from the nucleus [61–65] (Fig. 1). By contrast, the Lck-interacting P187 YAPP motif in the cytoplasmic tail of CD28 seems to be crucial for tTreg differentiation, as point mutations to a large degree recapitulate the full CD28 knockout [57–59]. So, what exactly does CD28 co-stimulation do to support Treg differentiation? First, it might merely amplify TCR signaling. This possibility cannot be excluded at present; however, in this case, one would expect the residual Treg cell repertoire generated in the absence of CD28 to be altered at the level of TCR specificities. Surprisingly, this does not seem to be the case, as the relative abundance of individual TCR specificities within the contracted Treg pool of CD28−/− mice resembles that of WT mice (at least as far as abundant specificities are concerned) [58]. Second, CD28 signaling might operate through promotion of cytokine production. However, although the P187 YAPP motif has been implicated in CD28-driven IL-2 production, it is hard to see how this should account for the (partial) block of thymic Treg development at “step one”, which is believed to be TCR-driven but cytokine independent. Third, CD28, in conjunction with TCR stimulation, may generate signals distinct from those elicited by TCR triggering alone. For instance, in Jurkat cells, cross-linking of the TCR together with CD28 stimulation was found to activate NF-␬B, whereas TCR triggering alone was largely unable to do so [66]. Likewise, a mutation of the CD28 P187 YAPP motif strongly diminishes TCR/CD28 mediated NF-␬B activation [67]. Consistent with a role of NF-␬B activation downstream of an integrated TCR/CD28 signal in Treg differentiation, conditional targeting of genes involved in NF-␬B activation (PKC-␪, CARMA-1, Bcl-10, IKK-2) impairs thymic Treg differentiation [68–70]. Therefore, it is conceivable that in Treg precursors, concurrent CD28/TCR stimulation signals via PKC-␪ and the CARMA-1/Bcl-10/Malt-1 (CBM) complex to activate IKK-2, which ultimately allows for NF-␬B family transcription factor-mediated induction of genes involved in Treg differentiation. The identity of these NF-␬B-induced genes for the most part remains to be established. However, the recent identification of c-Rel as essential NF-␬B family transcription factor during tTreg differentiation supports that NF-␬B-activation serves a bona fide “lineage-instructing” function [71–75]. Thus, c-Rel (most likely as a homodimer) directly controls the Foxp3 gene through binding to a conserved noncoding sequence (CNS3) which is located 3 of exon 1 and contains a sequence element resembling the CD28-response element in the IL-2 gene [76] (Fig. 1). Presumably, c-Rel coordinates the initial phase of Treg development at least in part through opening and remodeling of the Foxp3 locus. Consistent with such a role of CNS3 as “pioneer element”, Treg differentiation of thymocytes carrying a targeted mutation of the CNS3 is significantly impaired [76]. Taken together, the available data provide a plausible framework of how CD28 co-stimulation supports thymic Treg differentiation (at least in part) through c-Rel-mediated control of the Foxp3 gene. Exactly how c-Rel brings about the presumed chromatin modifications that poise the Foxp3 gene for transcription remains to be established. Given that c-Rel is believed to exert a truly “instructive” function, it is surprising that, despite an over-

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Fig. 1. Known and presumed determinants that control the expression of Foxp3 in immature thymocytes and committed Treg cells. (Above) In immature thymocytes as well as in peripheral non-Treg cells, the Foxp3 promoter and the CNS2 (also known as TSDR; Treg -specific-demethylated-region) are fully methylated. The methylated Foxp3 promoter is thought to be occupied by PIAS1, which might recruit histone deacetylases (HDACs) and heterochromatin protein 1 (HP1), thereby maintaining a repressive chromatin state. (Below) At the onset of tTreg development (“step one”), a TCR/CD28 signal of appropriate strength might on the one hand mediate enhanced Foxp3 promoter accessibility through dissociation of PIAS1, and on the other hand (via PKC␪, the Carma1–Bcl10–Malt1 complex and IKK) might result in binding of c-Rel to the CNS3 (“Pioneer element”), which presumably coordinates the initial phase of Treg development through opening and remodeling of the Foxp3 locus. Concurrently, transcription factors downstream of TCR/CD28 signaling (NFAT, AP-1, CREB, c-Rel) occupy distinct elements in the Foxp3 gene locus and also enhance the cytokine responsiveness of Treg precursors through up-regulation of IL-2 receptor subunits. Paradoxically, Foxo factors are believed to be positive regulators of Foxp3 expression through direct binding at the Foxp3 locus, although TCR/CD28 signals should activate PI3K/Akt, which in turn would lead to sequestration of Foxo from the nucleus. It remains open whether and how “inappropriate” activation of PI3K and Akt is actively suppressed during tTreg differentiation. Subsequently, STAT5 downstream of the IL-2 receptor (and other common gamma chain cytokine receptors) may synergize with these transcription factors to consolidate the activation of Foxp3 transcription (“step two”). At a relatively late stage of tTreg development, the CNS2/TSDR becomes progressively demethylated through as yet unknown mechanisms (active demethylation or lack of maintenance methylation during DNA replication). The CNS2/TSDR thereby acts as a “stabilizer” of Treg lineage commitment through conferring robustness to Foxp3 expression, for instance via a feedforward-loop that involves binding of Foxp3 itself to the CNS2/TSDR. The CNS1 (“TGF-␤ sensor”), which acts downstream of TGF-␤ receptor signaling during TGF-␤-driven in vitro conversion of mature T cells and presumably also during extrathymic iTreg differentiation in vivo, is not essential for intrathymic Treg development. Of note, this does not exclude that functions of TGF-␤ signaling other than direct effects on Foxp3 expression somehow modulate thymic Treg development.

all contraction of the Treg pool, the TCR repertoire of CD28−/− mice shows a substantial overlap with that of CD28+/+ mice [58]. Therefore, it remains possible that NF-␬B-signaling or other signaling pathways downstream of CD28, most likely in parallel, also deliver crucial and as yet unknown “permissive” signals and “set the stage” for the second step of early Treg differentiation by upregulating components of the IL-2 receptor [58,59]. It also remains open how the apparently paradoxical situation can be reconciled that TCR/CD28 signaling on the one hand supports Foxp3 induction via NF-␬B-activation, but on the other hand may exert a negative effect through PI3K- and Akt-signaling, which sequesters Foxo proteins from the nucleus [77] (Fig. 1). The latter might be related to the key issue that we are still lacking an integrated model of how CD28 co-stimulation differentially affects Treg differentiation

versus clonal deletion. In light of controversial findings concerning the role of CD28 in negative selection [78–81], it remains possible that CD28/B7 co-stimulation supports Treg development not only through participating in the molecular orchestration of the early phases of Treg differentiation, but also though protecting developing Treg cells from negative selection. Consistent with this, we found that “presumptive” Treg cells in a TCR × neo-antigen doubletransgenic model are in fact lost from the T cell repertoire in CD28 deficient mice (our unpublished observation). 5. Transforming growth factor-␤ (TGF-␤) TGF-␤ regulates multiple facets of T cell development, homeostasis, tolerance and immune responses [82]. For instance, TCR

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stimulation in the presence of TGF-␤ promotes Foxp3 expression in mature T cells in vitro [83] and is believed to mimic certain aspects of the in vivo conversion of peripheral naïve T cells into so-called adaptive or induced (i)Treg cells. For TGF-␤-driven conversion of naïve cells, Smad3 downstream of the TGF-␤-receptor is thought to co-operate with NFAT downstream of the TCR through directly binding to and activating a conserved Smad3-NFAT responsive enhancer element in the Foxp3 gene (also known as conserved noncoding sequence 1; CNS1) which is located approximately 2 kb downstream of the promoter [84] (Fig. 1). Does a general dependence of Foxp3 expression upon TGF-␤ mechanistically link peripheral iTreg conversion and the differentiation of tTreg cells? Initially, T cell specific ablation of the TGF-␤-receptor failed to reveal a detrimental effect on the intrathymic development of tTreg cells; by contrast, the peripheral Treg cell pool was severely diminished [85,86], giving rise to the notion that TGF-␤ was crucial for peripheral Treg cell homeostasis and maintenance of Foxp3 expression, whereas it would not have a non-redundant function for intrathymic Treg differentiation [87]. Upon closer inspection, it turned out that T cell-specific ablation of the TGF-␤-receptor resulted in a significant diminution of the first wave of neonatal thymic Treg production around day four after birth [88], suggesting that Treg differentiation in the neonatal and adult thymus might differ in their requirement for TGF-␤. However, further evidence that intrathymic Treg differentiation and TGF-␤-driven iTreg conversion of mature CD4T cells follow different molecular rules (at least as far as the role of TGF␤ is concerned) was obtained more recently in mice engineered to lack the TGF-␤-responsive 5 enhancer element (CNS1) of the Foxp3 gene. Thus, CNS1 was dispensable for thymic Treg cell differentiation, whereas both the TGF-␤-driven iTreg differentiation in vitro as well as the size of the Treg population in gut-associated lymphoid tissues (GALT) and mesenteric lymph nodes (both believed to be preferential sites of extrathymic iTreg differentiation) were strongly affected [76]. In sum, it seems possible that the apparently conflicting results concerning the requirement for TGF-␤ during thymic Treg differentiation might reflect effects of TGF-␤ on thymocytes that are not directly related to the induction of Foxp3 via the TGF-␤R/Smad3/Foxp3-CNS1 axis. Indeed, a recent report showed that TGF-␤-signaling can protect developing thymocytes from death signals that emanate from “strong” TCR stimulation [89]. Of note, this anti-apoptotic function was not restricted to developing Treg cells, but likewise applied to thymocytes exposed to stimuli that otherwise promoted clonal deletion. These findings suggest that during thymic Treg differentiation, TGF-␤ signaling serves a “permissive” (as opposed to “instructive”) function distinct from induction of Foxp3 expression.

6. Epigenetic aspects of intrathymic Treg differentiation Eventually, the integration of Treg inducing-stimuli through the various signal transduction pathways described above culminates in the stable expression of Foxp3. Epigenetic modifications such as DNA methylation at CpG motifs and histone-methylation or -acetylation are critical in the control of gene expression through altering the higher order chromatin structure and thus determining the accessibility of DNA to transcription factors. Not surprisingly, accumulating evidence suggests that epigenetic regulation of Foxp3 is critically involved in the establishment and maintenance of the Treg phenotype [90]. This has most extensively been described for the Foxp3 promoter region as well as for a conserved noncoding sequence (CNS) that is known as CNS2 or Treg -specific-demethylated-region (TSDR) (Fig. 1).

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6.1. Epigenetic control of the Foxp3 promoter Once fully active in mature peripheral Treg cells, the promoter of Foxp3 is largely demethylated at CpG motifs and shows a strong association with acetylated histones [91], indicative of an “open” chromatin configuration permissive for access by key transcription factors such as AP1, NFAT, NF-␬B, STAT5 and Runx. How is premature binding of these factors and thus aberrant expression of Foxp3 at early stages of thymocyte development prevented, especially given that some of these transcription factors (e.g. NF-␬B and NFAT [92]) are activated and fulfill functions in thymocyte development prior to the presumed TCR-driven entry point into the Treg lineage? A recent study has shed light on this issue by suggesting that the SUMO E3 ligase PIAS1 is involved in the epigenetic control of intrathymic Treg differentiation [93]. Specifically, in immature CD4+ CD8+ and “mainstream” CD4 SP thymocytes, PIAS1 occupies the Foxp3 promoter and recruits DNA methyltransferases and heterochromatin protein 1 (HP1), which in turn maintain a repressive chromatin state at the Foxp3 promoter (Fig. 1). Conceivably, a TCR signal of appropriate strength or a concurrent co-signal of unknown nature then mediates the dissociation of PIAS1 form the promoter, which would allow for promoter demethylation, reduced histone methylation and enhanced promoter accessibility. Consistent with this idea, PIAS1−/− mice displayed an increased thymic Treg cell population [93]. 6.2. The Treg -specific-demethylated-region (TSDR) The TSDR is located in an intronic region approximately 4.5 kb downstream of the transcriptional start-site. It is fully demethylated at several CpG motifs in “mature” peripheral Treg cells, whereas these residues are highly methylated in conventional, nonTreg cells and also in immature CD4+ CD8+ thymocytes [91,94]. The methylation status of the TSDR is controlled by as yet unknown mechanisms that apparently do not involve PIAS1 [93,95]. Importantly, whereas demethylation of the TSDR is critically involved in sustained Foxp3 expression by mature Treg cells and thus stability of the Treg phenotype [94] through recruitment of Ets-1 [96,97], CREB [91], NF-␬B/c-Rel [73] and Foxp3 itself (which in complex with Runx1 establishes a feed-forward loop [98,99]), it is unlikely that the TSDR methylation-status impinges on the entry of thymocytes into the Treg lineage. For instance, in thymic CD4 SP Foxp3+ cells (in contrast to their peripheral progeny) the TSDR is only partially demethylated [91,94], and targeted disruption of the TSDR affects Treg stability, but not the intrathymic differentiation of Treg cells [76]. In sum, the available data argue that early epigenetic events at the Foxp3 promoter are critically involved in intrathymic Treg differentiation, whereas it appears that epigenetic modifications of the TSDR are a consequence of, rather than a prerequisite for, Treg differentiation. How TSDR demethylation is ultimately achieved – through the action of demethylases or lack of maintenance DNA methylation during cell cycling – remains an interesting question. 7. Thymic antigen presenting cells (APCs) in Treg differentiation Reminiscent of the classical work by Le Douarin and colleagues on thymic epithelium transplantation and dominant tolerance, early findings in TCR × model-antigen double transgenic systems hinted at a connection between self-antigen expression in thymic epithelium and tTreg generation [16,17]. However, none of these experimental systems formally established the nature of the APC that ultimately presented the respective antigen to developing thymoctes. Several recent studies on the surprisingly dynamic

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transfer of self-determinants within the thymic microenvironment highlight the relevance of this issue [100,101]. Specifically, selfantigens and even functionally intact MHC molecules seem to somehow be “shuttled” unidirectionally from TECs to DCs. The underlying mechanistic details remain to be established; however, this phenomenon certainly adds another layer of complexity to our understanding of how individual subsets of thymic stromal cells might differentially contribute to deletional or non-deletional modes of central tolerance. For instance, in one particular model system (RIP-mOVA × OTII), it was reported that deletion of specific CD4T cells as a consequence of cognate antigen-expression in mTECs was dependent on antigen transfer to and presentation by DCs [102]. By contrast, in another model (Aire-HA × TCR-HA), where a substantial fraction of specific CD4T cells differentiated into the Treg lineage, presentation of mTEC-derived antigen by DCs was found to be dispensable, indicating an autonomous function of mTECs as both expressers and presenters of endogenously expressed self-antigens for Treg induction [32]. Can one generalize these observations and postulate a qualitative model whereby self-antigen presentation by mTECs or DCs would promote either Treg differentiation or clonal deletion, respectively? We deem this rather unlikely. On the one hand, there is accruing evidence that mTECs also substantially contribute to negative selection. For instance, reducing the surface expression of MHC II on mTECs to about 10% of the physiological levels through mTEC-specific silencing of C2TA indicates that at least 30% of CD4 SP T cells are normally negatively selected by mTECs [33]. Vice versa, there is little reason to believe that antigen recognition on DCs invariably results in negative selection. For instance, thymic stromal lymphopoietin (TSLP) conditioned DCs [103] or plasmacytoid DCs [104,105] were suggested to promote Treg differentiation in the human thymus, and studies in mouse models have likewise been interpreted to indicate that thymic DCs can support Treg differentiation [106,107]. Furthermore, a reductionist cell culture system indicated that different thymic DC-subtypes similar to thymic epithelial cells efficiently induced Treg development in vitro given that optimal doses of cognate antigen were provided [52]. In sum, an APC-centered binary model postulating that qualitatively distinct co-signals delivered by the stromal interaction partner govern the decision-making by autoreactive CD4T cells appears highly unlikely. In line with the idea that intrathymic Treg differentiation does not require a dedicated APC but rather entails a high degree of flexibility in the stromal cell types involved, several genetically manipulated mouse models that lack either DCs or subsets of mTECs, or with absent or aberrant MHC II expression on subclasses of thymic APCs, display for the most part normal numbers of polyclonal Treg cells [33,108–113]. Nevertheless, it remains to be seen in how far this apparent principle redundancy between thymic APCs translates into a true “functional” redundancy at the level of distinct TCR specificities that are guided into the Treg repertoire (or deleted).

8. Thymocyte-intrinsic developmental tuning and Treg differentiation Early concepts of immune tolerance predicted that immature lymphocytes bear an inherent predisposition towards a tolerance response, whereas only after further maturation they would respond to antigenic stimulation by activation and acquisition of effector function [114]. This scenario has become a well accepted paradigm in immunology. In its most simplistic experimental manifestation, this becomes evident from the fact that a vast majority of immature CD4+ CD8+ (DP) thymocytes will undergo apoptosis when confronted with an anti-CD3 anti-CD28 coated plastic surface [115], whereas mature naïve T cells from spleen or lymph node

will respond to the very same stimulus with robust proliferative expansion and IL-2 secretion. Where and when in the life of a T cell does this transition from a tolerance-sensitive stage to a subsequent fully responsive state occur? Most of the available information indicates that this happens during continual maturation at the single-positive (SP) stage of thymocyte differentiation. Thus, although there are also some indications that recent thymic emigrants within the first few days after exiting the thymus undergo further phenotypic and functional maturation [116], it appears that the most dramatic alteration in the “intrinsic wiring” of T cells occurs while cells are still retained in the thymic medulla. Specifically, medullary SP thymocytes with an “immature” (HSAhi ) phenotype were found to be susceptible to negative selection, whereas more mature (HSAlo ) cells are largely resistant to tolerance induction and instead respond to antigenic stimulation with proliferation, very much alike their progeny in secondary lymphoid organs [117]. Considering that APCs in the thymic medulla have acquired several characteristics that obviously have evolved to optimize the efficacy of central tolerance induction [118] (most prominently the “promiscuous” expression of peripheral antigens by mTECs [119]), it is intuitively obvious why subsequent to positive selection and relocating to the medulla, T cells remain sensitive to tolerance induction for a certain time before entering a tolerance-resistant state. How does agonist-driven Treg cell differentiation relate to this T cell-intrinsic developmental control of tolerance-susceptibility? Whereas it is well established that DP thymocytes are susceptible to antigen-driven clonal deletion as soon as they express a functionally rearranged TCR on their surface [120], it is much less clear at which developmental stage the capacity to enter the Treg lineage is established. The latter issue is intimately related to the question of when during thymocyte differentiation the Treg cell lineage branches of from “mainstream” thymocyte development. Some investigators have proposed that Treg differentiation is the consequence of “altered positive selection” of cortical DP thymocytes [30,109,121–123]. By contrast, others have presented evidence that Tregs arise at the CD4 SP stage through what may be called “altered negative selection” in the thymic medulla [32,33,124]. Part of this controversy perhaps stems from the fact that most of these studies deduced the developmental stage (i.e. the time point) at which Treg differentiation was initiated from particular modalities (i.e. the spatial distribution) of agonist antigen – or MHC class II – expression. For instance, the existence of an apparently normal compartment (as far as absolute numbers are concerned) of Treg cells in mice (K14–Ab␤) that express MHC class II only on cortical epithelial cells may, at first glance, suggest that Treg induction must have occurred at the DP stage [109]. However, “illegitimate” contacts of SP cells with cTECs cannot be formally excluded because the chemokine gradients that orchestrate thymocyte positioning are unlikely to impose a strict demarcation between cortex and medulla. Vice versa, and on the basis of the same considerations, the fact that confined expression and presentation of an agonist self-antigen in medullary epithelial cells can result in Treg differentiation of specific TCR transgenic CD4T cells does not provide conclusive information as to the exact developmental stage at which the presumed instructive stimulus occurred [32,33]. In an attempt to eliminate some of the inherent complexities of studying Treg differentiation in the steady state thymus, we used the intrathymic (i.t) transfer of post-positive selection “naïve” CD4 SP thymocytes of known antigen-specificity into a cognate antigen-expressing host thymus. This approach formally established that Treg induction by agonist encounter in vivo does not obligatorily require cognate interactions at the CD4+ CD8+ DP stage [52]. Although these experiments left open at which developmental stage the principle responsiveness towards Treg inducing stimuli is established, they clearly showed that the permissive “window

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of opportunity” extends well into the CD4 SP stage. Importantly, when CD4 SP cells of consecutive maturation stages or peripheral CD4T cells were i.t. injected, the efficiency of Treg induction inversely correlated with T cell “age”. This inclination of immature T cells towards Treg differentiation was likewise seen in an APC-free in vitro system providing only TCR stimulation and IL-2, indicating that it is indeed a thymocyte intrinsic property. Taken together, the declining inclination towards Treg differentiation that goes along with progressive intrathmyic T cell maturation bears striking resemblance to a similar developmental switch from susceptibility to resistance for clonal deletion. Possibly, concomitant to gradually losing the susceptibility to being deleted, thymocytes may enter a transient phase of exquisite predisposition for Treg development. However, it appears that the developmental “windows of opportunity” for Treg differentiation or negative selection are largely overlapping, so that a clear developmental demarcation between these conditions is unlikely to exist. Importantly, the underlying molecular control-mechanisms remain as yet largely unexplored. 9. Concluding remarks There is a remarkably precise understanding of how genetic and epigenetic events orchestrate the expression of Foxp3, including a plausible explanation of how extrinsic information such as antigen recognition, co-stimulation and cytokines control the Foxp3 gene locus (Fig. 1). The avidity model provides a reasonable conceptual framework for the cell-fate-choice between Treg differentiation and negative selection. However, this model fails to incorporate phenomena such as thymocyte intrinsic developmental tuning [52,117], the dynamic nature of thymocyte/APC interactions [125] and the (in all probability) non-homogeneous distribution of selfantigens within the thymic microenvironment [126,127]. These latter two parameters may together lead to a dynamic interplay of the likelihood and the duration of self-antigen-encounters, and may explain the as yet enigmatic intraclonal competition between Treg precursors of identical specificity [128,129]. In the same vein, understanding how discontinuous or intermittent TCR signaling impinges on thymocyte fate may hold the key to the fundamental question how quantitatively different input signals are integrated and translated into qualitatively different cell fate decisions. Acknowledgements We like all members of the lab for critical reading of this manuscript. We would like to acknowledge funding through the Deutsche Forschungsgemeinschaft (grants KL 1228/2-1, KL 1228/31 and Sonderforschungsbereich 571) and the Hertie Foundation (Grant 1.01.1/09/11).

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