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Seminars in Immunology 23 (2011) 401– 409

Contents lists available at ScienceDirect

Seminars in Immunology

jo u rn al hom epage: www.elsev ier .com/ locate /ysmim

eview

egulatory T cell lineage commitment in the thymus

udger Klein ∗, Ksenija Jovanovicniversity of Munich, Institute for Immunology, Goethestr. 31, 80336 Munich, Germany

r t i c l e i n f o

eywords:hymusegulatory T celloxp3

a b s t r a c t

A substantial fraction of the Foxp3+ CD4+ regulatory T (Treg) cell repertoire is generated through instructiveand/or selective processes in the thymus, and there is some consensus that clonal deviation into theTreg lineage is a result of self-antigen recognition. Paradoxically, the same holds true for a diametrically

egative selectionineage commitment

different cell fate decision of developing thymocytes, namely their removal from the repertoire throughapoptotic cell death (clonal deletion). Here, we will review our current understanding of how T cellreceptor stimulation, cytokine signaling, co-stimulation, epigenetic modifications and T cell intrinsicdevelopmental tuning synergize during Treg cell differentiation, and how instructive signals converge atthe Foxp3 gene-locus during entry into the Treg cell lineage. We will also discuss how these parametersrelate to known determinants of negative selection.

. Introduction: clonal deletion and clonal deviation aslternative fates of autoreactive thymocytes

Seminal experiments in the late 1980s showed that expres-ion of an autoreactive antigen receptor can result in the physicallimination or functional inactivation of immature T cells in thehymus [1–4]. Because clonal deletion and clonal inactivationanergy) both represent cell intrinsic mechanisms, they are alsoermed recessive tolerance. Around the same time, it was found thatransplantation of allo- and even xenogeneic thymic epitheliumefore colonization by hematopoietic precursors resulted in life-

ong tolerance to grafted tissues of the donor-type [5,6]. This worknd a subsequent series of experiments established that thymicpithelium-mediated tolerance could not be explained by reces-ive tolerance mechanisms, but instead must somehow rely onhe dominant action of autoreactive “suppressor” cells [7]. Inter-stingly, whereas recessive modalities of central tolerance rapidlyecame an accepted paradigm, this dominant mode of tolerancenly slowly gained a wider acceptance, perhaps owing to the facthat the precise nature of these thymus-dependent “suppressor”r “regulatory” cells remained elusive (apart from residing within

he CD4+ T cell compartment [8]), but certainly also because theerm “suppressor” had fallen into disrepute due to earlier con-roversies surrounding largely unrelated phenomena (reviewed in

Abbreviations: APC, antigen presenting cell; Foxp3, forkhead box transcrip-ion factor 3; TCR, T cell receptor; tTreg cell, thymus-derived regulatory T cell;Treg cell, induced regulatory T cell; TSDR, Treg-specific-demethylated-region; mTEC,

edullary thymic epithelial cell; MHC II, major histocompatibility complex class II.∗ Corresponding author. Tel.: +49 89 218075696; fax: +49 89 51602236.

E-mail address: ludger.klein@med.uni-muenchen.de (L. Klein).

044-5323/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.smim.2011.06.003

© 2011 Elsevier Ltd. All rights reserved.

[9]). Nevertheless, in the mid 1990s several other labs more or lesssimultaneously showed that elimination of particular subsets ofCD4+ T cells (by selective depletion prior to adoptive transfer intolymphopenic hosts or as a consequence of neonatal thymectomy)would unleash an otherwise suppressed auto-destructive potentialwithin the T cell repertoire [10–12]. Although these investigatorsall used distinct surface phenotypes – CD45RBlow or CD25high inmice and CD45RClow in rats, respectively – to characterize the par-ticular regulatory population of CD4+ T cells, it is in hindsight clearthat all these reports in essence described the immunoregulatoryfunction of what is now known as CD25+Foxp3+ regulatory T (Treg)cells [13]. A plausible “missing link” between Foxp3+ Treg cells andthymic epithelium-induced dominant tolerance was then estab-lished when a critical function of the thymus for their generationwas discovered [14] and by evidence that T cell receptor (TCR)transgenic thymocytes can be deviated into the Treg cell lineagewhen cognate antigen is expressed by thymic epithelium [15–17].

To date, there is some consensus that a substantial fraction ofthe Treg cell repertoire originates from the thymus, as supportedby a repertoire comparison of thymic and peripheral Treg cells [18].However, there is little doubt that phenotypically similar and func-tionally related (although not necessarily identical) Treg cells canunder particular circumstances also arise through conversion ofperipheral naïve T cells into so-called adaptive or induced (i)Treg

cells [19]. In the following, we will refer to thymus-derived Treg cellsas tTreg cells (instead of the commonly used but perhaps somewhatmisleading term “natural (n)Treg”).

Entry into the Treg lineage during thymocyte development is

believed to depend upon instructive processes ensuing from self-antigen recognition. Evidence for this has not only been obtainedin TCR transgenic systems, but also stems from observations thatpolyclonal thymocytes bearing superantigen-reactive TCRs are

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ubstantially enriched in Foxp3+ cells [20,21]. Also consistent withnhanced autoreactivity being a key driving force of Treg lineageommitment, T cells transduced with Treg derived TCRs are acti-ated upon transfer into non-lymphopenic hosts [18] (althoughther investigators failed to observe such overt autoreactivity ofreg TCRs [22]), and the Treg repertoire harbors a significant fre-uency of cells that show reactivity to syngeneic antigen presentingells (APCs) in vitro [23].

Together, a substantial body of evidence supports the idea thathe expression of an autoreactive TCR is a decisive determinantpecifying the entry of MHC II-restricted thymocytes into the tTreg

ineage. Hence, there is the paradox that autoreactivity appearso be a common attribute of clonally deleted TCR specificities asell as of thymocytes that are deviated into the Treg lineage. In this

eview, we will discuss thymocyte-intrinsic and -extrinsic parame-ers that have been implicated in tTreg differentiation and, whereverossible, try to relate these factors to known determinants of neg-tive selection.

. The avidity model of Treg differentiation

Current models of thymocyte selection posit that MHC/self-eptide interactions of intermediate “strength” are required forositive selection, whereas very strong interactions lead to negativeelection. Of note, this commonly used concept in fact amalga-ates two models with quite distinct assumptions: whereas a

trictly affinity-based model centers on properties of the individ-al TCR/MHC–peptide interaction [24,25], the avidity model insteadssumes that the product of the TCR/MHC–peptide affinity multi-lied by the number of interactions is critical [26,27]. How doeshe presumed agonist-driven entry into the Treg lineage fit into thiscenario? Several lines of evidence are consistent with the idea thatreg differentiation ensues from interactions that lie in between theignaling strength required for positive selection on the one sidend clonal deletion on the other side [28]. First, the combination of

particular MHC II-restricted TCR transgenic system with multipleines of cognate antigen-transgenic mice revealed that the num-er of emerging Treg cells inversely correlated with the “antigenose” (as measured by the abundance of cognate antigen mRNA

n the thymus), whereas the extent of concomitant negative selec-ion increased with the overall “antigen dose” [29]. Second, similaresults were obtained in vitro upon titration of agonist peptidesnto TCR transgenic fetal thymic organ cultures (FTOCs) [30]. Third,ntravenous administration of graded amounts of cognate antigenn vivo preferentially lead to Treg induction at very low levels ofntigen, whereas increasing amounts of injected antigen favoredegative selection [31].

Most of these experiments bear the inherent caveat that, besidesuantitative parameters (i.e. the level of antigen-expressionr–presentation), also qualitative variables (e.g. the type of APChich ultimately presents the respective antigen and/or the devel-

pmental stage at which antigen is “seen”) can only insufficientlye controlled. In an attempt to circumvent these confound-

ng issues, we used an alternative strategy to test the avidityodel: in AIRE-HA × TCR-HA double transgenic mice, medullary

hymic epithelial cell (mTEC)-specific antigen-expression and -resentation results in negative selection of about two thirds ofemagglutinin (HA)-specific thymocytes, whereas a substantial

raction of the remaining cells differentiate into Treg cells [32].hen antigen presentation by mTECs in this system was attenu-

ted through RNAi mediated knock-down of MHC class II (throughilencing of its transcriptional master regulator CIITA), a diminishedxtent of negative selection and an increased emergence of Treg cellsas observed, which is fully consistent with the notion that inter-

munology 23 (2011) 401– 409

mediate avidities favor Treg differentiation over negative selection[33].

In sum, most of the available data are compatible with an aviditymodel of Treg differentiation. Accordingly, quantitative variationsin the level of cognate antigen-presentation, i.e. in the ensuingavidity of antigen recognition, can drive cells that express thevery same TCR into either apoptotic cell death or Treg differenti-ation. Therefore, the quality, that is, the “pure” affinity of a givenTCR/agonist interaction, is highly unlikely to be the central, binarydeterminant of Treg differentiation versus negative selection (atleast as far as the available TCR transgenic specificities that havebeen used to test this idea are concerned). It remains to be seenhow these findings can be reconciled with the perplexingly nar-row affinity threshold that was suggested to separate positiveselection from negative selection [34]. As these latter observa-tions were made with CD8T cells, it remains possible that onlyCD4T cell-differentiation accommodates a lineage-specific “third”avidity-window between positive and negative selection.

3. Interleukin-2 (IL-2) in Treg differentiation

3.1. The essential function of IL-2

Interleukin-2 (IL-2) was cloned in 1983 on the basis of its capac-ity to enhance T cell proliferation in vitro [35]. The view that IL-2smain function in vivo is to support immunity by acting as a T cellgrowth factor was challenged by the at first paradoxical finding thatmice deficient for IL-2, IL-2R� (CD25) or IL-2R� (CD122), ratherthan being immune deficient, succumbed to systemic autoimmu-nity [36–39]. Subsequently, two seminal papers demonstrated thatIL-2 or IL-2 receptor deficiency led to reduced frequencies of CD25+

Treg, and that adoptive transfer of CD25+ Treg rescued the autoim-munity of IL-2R� and IL-2R� deficient mice [40,41]. To date, asubstantial body of evidence supports the corresponding view thatthe essential, non-redundant function of IL-2 is the maintenance ofimmune homeostasis through its role in Treg biology [42].

Adoptive transfer of Treg cells into IL-2 deficient hosts results intheir rapid loss [43], and neutralization of IL-2 promotes autoim-mune manifestations through depletion of Treg cells [44], consistentwith a role of IL-2 as an essential survival factor for Treg cells. Asecond, mutually not exclusive function of IL-2 as differentiationfactor in thymic Treg development remained more controversialfor some time owing to the fact that it is to some degree redun-dant. Thus, the thymic population of Foxp3+ Treg cells is only mildlyaffected in IL-2 or IL-2R� (CD25) deficient mice [43]. However, a farmore dramatic decrease is seen in mice with targeted mutations ofthe IL-2R� chain (common �-chain) [45], JAK3 [46] or STAT5 [47],i.e. in the absence of signaling molecules that are shared by theIL-7- and IL-15-signaling pathways. Because neither IL-7- nor IL-15-deficiency by itself affects the thymic production of Treg cells,it is the prevailing view that IL-2 is the principal common �-chaincytokine required for intrathymic Treg cell development, but thatin its absence IL-7 and IL-15 can at least in part compensate for itsloss [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 signalingthrough 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 pre-cursors of Foxp3+CD25+ Treg cells [50,51]. Consistent with this,work in a TCR transgenic model of Treg differentiation indicatedthat within 12 h after the instructive TCR stimulus, developing

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reg cells acquire a Foxp3−CD25+ phenotype, whereas expres-ion of measurable amounts of Foxp3 protein is not seen beforet least another 48 h [52]. Importantly, these Foxp3−CD25+ pre-ursor cells require common �-chain cytokines but no furtherCR stimulation for acquisition of the “mature” Foxp3+ Treg

henotype. Thus, Treg development seems to segregate into aCR-driven “instructive” phase and a cytokine-driven “consolida-ion” phase, and this scenario was termed two-step model of tTreg

ifferentiation [50,51].The mechanistic details of how molecular events downstream

f cytokine- and common �-chain signaling impinge on Treg differ-ntiation remain to be fully established. On the one hand, activatedTAT5 is likely to directly bind to and regulate the Foxp3 genet the promoter and at an intronic enhancer region [47] (Fig. 1).owever, several mutually not exclusive other modes-of-action of

ess “instructive” nature remain possible: first, cytokine-signalingight establish permissive chromatin modifications at critical

lineage-defining” gene loci (including, but not restricted to,oxp3). Second, common �-chain signaling might also promote thexpansion and/or the survival of committed Treg precursors.

Little is known as to how this function of cytokines in Treg

ifferentiation relates to clonal deletion of autoreactive MHCI-restricted thymocytes. For instance, whereas competition forL-2 is very likely to shape the Treg repertoire at the “TCR-rimed” Foxp3−CD25+ precursor stage, the fate of those cellshat fail to receive appropriate IL-2 stimulation remains to bestablished. Whereas it is possible that these cells revert pheno-ypically and escape from central tolerance as naïve, conventional

cells, it appears more likely that they undergo apoptotic celleath (which would represent a form of “negative selectiony neglect”). Considering the latter scenario, it cannot even bexcluded that all thymocytes (or all CD4 SP cells) that haveeceived a TCR stimulus exceeding a certain threshold-value enter

corresponding state in which common �-chain signaling mightrotect from clonal deletion. To our knowledge, this idea hasot been rigorously tested. However, it may deserve mentioninghat IL-7 was suggested to have the capacity to negate negativeelection [53].

. CD28 co-stimulation in Treg differentiation

.1. CD28 and the “two-step” model of Treg differentiation

Ablation of the genes encoding for CD28 or its ligands CD80 andD86 (B7.1 and B7.2, respectively) results in a dramatic decrease

n the number of thymic and peripheral Treg cells [54–56]. How-ver, overt autoimmunity is not seen under these circumstances,resumably because of a compensatory effect of diminished acti-ation of potentially dangerous “conventional” effector T cells [54].lthough co-stimulation has been implicated in IL-2 production,

he inefficient entry of CD28−/− thymocytes into the Treg lineage isot corrected by the presence of bystander CD28+/+ cells, suggest-

ng that the paucity of thymic Treg cells in co-stimulation deficientice primarily reflects a T cell-intrinsic function of the CD28/B7

xis, largely independent of IL-2 [57].At what stage of Treg differentiation does CD28 operate? Recent

ata indicate that CD25+Foxp3−Treg precursors are strongly dimin-shed in the thymus of CD28−/− mice, suggesting an “early”equirement of CD28 co-stimulation simultaneous to or in rela-ively close temporal proximity to the presumed initiating TCRignal [58,59]. Because abrogation of Treg differentiation at this pre-

ursor stage precludes an analysis of subsequent stages, it remainsossible that CD28 signals are continually required throughoutoth the TCR driven first and the cytokine dependent second phasef Treg differentiation.

munology 23 (2011) 401– 409 403

4.2. CD28 and NF-�B

Generally, CD28 co-signals in T cells can stabilize messengerRNAs and amplify the activation of nuclear factor of activated Tcells (NFAT) and nuclear factor-�B (NF-�B), thereby supportingcytokine production, T cell survival and proliferation [60]. CD28addresses several downstream signaling cascades through distinctSH2- or SH3-domain-binding motifs in its cytoplasmic tail thatmediate among others interactions with Lck and the PI3K path-way, respectively. The ability of CD28 to support efficient tTreg

cell generation was found not to require an intact PI3K-bindingmotif [57–59]. Consistent with this, PI3K-signaling through Akt andmTOR might in fact even antagonize tTreg differentiation by seques-tering Foxo1/Foxo3a from the nucleus [61–65] (Fig. 1). By contrast,the Lck-interacting P187YAPP motif in the cytoplasmic tail of CD28seems to be crucial for tTreg differentiation, as point mutations to alarge 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. Thispossibility cannot be excluded at present; however, in this case,one would expect the residual Treg cell repertoire generated inthe absence of CD28 to be altered at the level of TCR specifici-ties. Surprisingly, this does not seem to be the case, as the relativeabundance of individual TCR specificities within the contractedTreg pool of CD28−/− mice resembles that of WT mice (at leastas far as abundant specificities are concerned) [58]. Second, CD28signaling might operate through promotion of cytokine produc-tion. However, although the P187YAPP motif has been implicatedin CD28-driven IL-2 production, it is hard to see how this shouldaccount for the (partial) block of thymic Treg development at “stepone”, which is believed to be TCR-driven but cytokine independent.Third, CD28, in conjunction with TCR stimulation, may gener-ate signals distinct from those elicited by TCR triggering alone.For instance, in Jurkat cells, cross-linking of the TCR togetherwith CD28 stimulation was found to activate NF-�B, whereas TCRtriggering alone was largely unable to do so [66]. Likewise, a muta-tion of the CD28 P187YAPP motif strongly diminishes TCR/CD28mediated NF-�B activation [67]. Consistent with a role of NF-�Bactivation downstream of an integrated TCR/CD28 signal in Treg

differentiation, conditional targeting of genes involved in NF-�Bactivation (PKC-�, CARMA-1, Bcl-10, IKK-2) impairs thymic Treg

differentiation [68–70]. Therefore, it is conceivable that in Treg pre-cursors, concurrent CD28/TCR stimulation signals via PKC-� and theCARMA-1/Bcl-10/Malt-1 (CBM) complex to activate IKK-2, whichultimately allows for NF-�B family transcription factor-mediatedinduction of genes involved in Treg differentiation. The identityof these NF-�B-induced genes for the most part remains to beestablished. However, the recent identification of c-Rel as essentialNF-�B family transcription factor during tTreg differentiation sup-ports that NF-�B-activation serves a bona fide “lineage-instructing”function [71–75]. Thus, c-Rel (most likely as a homodimer) directlycontrols the Foxp3 gene through binding to a conserved noncod-ing sequence (CNS3) which is located 3′ of exon 1 and contains asequence element resembling the CD28-response element in theIL-2 gene [76] (Fig. 1). Presumably, c-Rel coordinates the initialphase of Treg development at least in part through opening andremodeling of the Foxp3 locus. Consistent with such a role of CNS3as “pioneer element”, Treg differentiation of thymocytes carrying atargeted mutation of the CNS3 is significantly impaired [76].

Taken together, the available data provide a plausibleframework of how CD28 co-stimulation supports thymic Treg dif-ferentiation (at least in part) through c-Rel-mediated control of the

Foxp3 gene. Exactly how c-Rel brings about the presumed chro-matin modifications that poise the Foxp3 gene for transcriptionremains to be established. Given that c-Rel is believed to exert atruly “instructive” function, it is surprising that, despite an over-

404 L. Klein, K. Jovanovic / Seminars in Immunology 23 (2011) 401– 409

Fig. 1. Known and presumed determinants that control the expression of Foxp3 in immature thymocytes and committed Treg cells. (Above) In immature thymocytes aswell 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 Foxp3promoter is thought to be occupied by PIAS1, which might recruit histone deacetylases (HDACs) and heterochromatin protein 1 (HP1), thereby maintaining a repressivechromatin 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 promoteraccessibility 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, transcriptionfactors 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 atthe 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 andhow “inappropriate” activation of PI3K and Akt is actively suppressed during tTreg differentiation. Subsequently, STAT5 downstream of the IL-2 receptor (and other commongamma chain cytokine receptors) may synergize with these transcription factors to consolidate the activation of Foxp3 transcription (“step two”). At a relatively late stage oftTreg development, the CNS2/TSDR becomes progressively demethylated through as yet unknown mechanisms (active demethylation or lack of maintenance methylationduring DNA replication). The CNS2/TSDR thereby acts as a “stabilizer” of Treg lineage commitment through conferring robustness to Foxp3 expression, for instance via a feed-forward-loop that involves binding of Foxp3 itself to the CNS2/TSDR. The CNS1 (“TGF-� sensor”), which acts downstream of TGF-� receptor signaling during TGF-�-driveni ifferen ressio

amTndtrotvetth

n vitro conversion of mature T cells and presumably also during extrathymic iTreg dot exclude that functions of TGF-� signaling other than direct effects on Foxp3 exp

ll contraction of the Treg pool, the TCR repertoire of CD28−/−

ice shows a substantial overlap with that of CD28+/+ mice [58].herefore, it remains possible that NF-�B-signaling or other sig-aling pathways downstream of CD28, most likely in parallel, alsoeliver crucial and as yet unknown “permissive” signals and “sethe stage” for the second step of early Treg differentiation by up-egulating components of the IL-2 receptor [58,59]. It also remainspen how the apparently paradoxical situation can be reconciledhat TCR/CD28 signaling on the one hand supports Foxp3 inductionia NF-�B-activation, but on the other hand may exert a negative

ffect through PI3K- and Akt-signaling, which sequesters Foxo pro-eins from the nucleus [77] (Fig. 1). The latter might be relatedo the key issue that we are still lacking an integrated model ofow CD28 co-stimulation differentially affects Treg differentiation

ntiation in vivo, is not essential for intrathymic Treg development. Of note, this doesn somehow modulate thymic Treg development.

versus clonal deletion. In light of controversial findings concerningthe role of CD28 in negative selection [78–81], it remains possiblethat CD28/B7 co-stimulation supports Treg development not onlythrough participating in the molecular orchestration of the earlyphases of Treg differentiation, but also though protecting devel-oping Treg cells from negative selection. Consistent with this, wefound that “presumptive” Treg cells in a TCR × neo-antigen double-transgenic model are in fact lost from the T cell repertoire in CD28deficient mice (our unpublished observation).

5. Transforming growth factor-� (TGF-�)

TGF-� regulates multiple facets of T cell development, home-ostasis, tolerance and immune responses [82]. For instance, TCR

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timulation in the presence of TGF-� promotes Foxp3 expressionn mature T cells in vitro [83] and is believed to mimic certainspects of the in vivo conversion of peripheral naïve T cells intoo-called adaptive or induced (i)Treg cells. For TGF-�-driven con-ersion of naïve cells, Smad3 downstream of the TGF-�-receptor ishought to co-operate with NFAT downstream of the TCR throughirectly binding to and activating a conserved Smad3-NFAT respon-ive enhancer element in the Foxp3 gene (also known as conservedoncoding sequence 1; CNS1) which is located approximately 2 kbownstream of the promoter [84] (Fig. 1).

Does a general dependence of Foxp3 expression upon TGF-�echanistically link peripheral iTreg conversion and the dif-

erentiation of tTreg cells? Initially, T cell specific ablation ofhe TGF-�-receptor failed to reveal a detrimental effect on thentrathymic development of tTreg cells; by contrast, the periph-ral Treg cell pool was severely diminished [85,86], giving rise tohe notion that TGF-� was crucial for peripheral Treg cell home-stasis and maintenance of Foxp3 expression, whereas it wouldot have a non-redundant function for intrathymic Treg differenti-tion [87]. Upon closer inspection, it turned out that T cell-specificblation of the TGF-�-receptor resulted in a significant diminu-ion of the first wave of neonatal thymic Treg production arounday four after birth [88], suggesting that Treg differentiation in theeonatal and adult thymus might differ in their requirement forGF-�. However, further evidence that intrathymic Treg differen-iation and TGF-�-driven iTreg conversion of mature CD4T cellsollow different molecular rules (at least as far as the role of TGF-

is concerned) was obtained more recently in mice engineered toack the TGF-�-responsive 5′ enhancer element (CNS1) of the Foxp3ene. Thus, CNS1 was dispensable for thymic Treg cell differentia-ion, whereas both the TGF-�-driven iTreg differentiation in vitro asell as the size of the Treg population in gut-associated lymphoid

issues (GALT) and mesenteric lymph nodes (both believed to bereferential sites of extrathymic iTreg differentiation) were stronglyffected [76].

In sum, it seems possible that the apparently conflicting resultsoncerning the requirement for TGF-� during thymic Treg differ-ntiation might reflect effects of TGF-� on thymocytes that areot directly related to the induction of Foxp3 via the TGF-�-/Smad3/Foxp3-CNS1 axis. Indeed, a recent report showed thatGF-�-signaling can protect developing thymocytes from deathignals that emanate from “strong” TCR stimulation [89]. Of note,his anti-apoptotic function was not restricted to developing Treg

ells, but likewise applied to thymocytes exposed to stimulihat otherwise promoted clonal deletion. These findings suggesthat during thymic Treg differentiation, TGF-� signaling serves apermissive” (as opposed to “instructive”) function distinct fromnduction of Foxp3 expression.

. Epigenetic aspects of intrathymic Treg differentiation

Eventually, the integration of Treg inducing-stimuli throughhe various signal transduction pathways described above culmi-ates in the stable expression of Foxp3. Epigenetic modificationsuch as DNA methylation at CpG motifs and histone-methylationr -acetylation are critical in the control of gene expressionhrough altering the higher order chromatin structure and thusetermining the accessibility of DNA to transcription factors.ot surprisingly, accumulating evidence suggests that epigenetic

egulation of Foxp3 is critically involved in the establish-ent and maintenance of the Treg phenotype [90]. This has

ost extensively been described for the Foxp3 promoter region

s well as for a conserved noncoding sequence (CNS) thats known as CNS2 or Treg-specific-demethylated-region (TSDR)Fig. 1).

munology 23 (2011) 401– 409 405

6.1. Epigenetic control of the Foxp3 promoter

Once fully active in mature peripheral Treg cells, the promoterof Foxp3 is largely demethylated at CpG motifs and shows a strongassociation with acetylated histones [91], indicative of an “open”chromatin configuration permissive for access by key transcriptionfactors such as AP1, NFAT, NF-�B, STAT5 and Runx. How is pre-mature binding of these factors and thus aberrant expression ofFoxp3 at early stages of thymocyte development prevented, espe-cially given that some of these transcription factors (e.g. NF-�B andNFAT [92]) are activated and fulfill functions in thymocyte devel-opment prior to the presumed TCR-driven entry point into the Treg

lineage? A recent study has shed light on this issue by suggestingthat the SUMO E3 ligase PIAS1 is involved in the epigenetic controlof intrathymic Treg differentiation [93]. Specifically, in immatureCD4+CD8+ and “mainstream” CD4 SP thymocytes, PIAS1 occupiesthe Foxp3 promoter and recruits DNA methyltransferases and het-erochromatin protein 1 (HP1), which in turn maintain a repressivechromatin state at the Foxp3 promoter (Fig. 1). Conceivably, a TCRsignal of appropriate strength or a concurrent co-signal of unknownnature then mediates the dissociation of PIAS1 form the promoter,which would allow for promoter demethylation, reduced histonemethylation and enhanced promoter accessibility. Consistent withthis idea, PIAS1−/− mice displayed an increased thymic Treg cellpopulation [93].

6.2. The Treg-specific-demethylated-region (TSDR)

The TSDR is located in an intronic region approximately 4.5 kbdownstream of the transcriptional start-site. It is fully demethy-lated at several CpG motifs in “mature” peripheral Treg cells,whereas these residues are highly methylated in conventional, non-Treg cells and also in immature CD4+CD8+ thymocytes [91,94]. Themethylation status of the TSDR is controlled by as yet unknownmechanisms that apparently do not involve PIAS1 [93,95]. Impor-tantly, whereas demethylation of the TSDR is critically involved insustained Foxp3 expression by mature Treg cells and thus stabilityof the Treg phenotype [94] through recruitment of Ets-1 [96,97],CREB [91], NF-�B/c-Rel [73] and Foxp3 itself (which in complexwith Runx1 establishes a feed-forward loop [98,99]), it is unlikelythat the TSDR methylation-status impinges on the entry of thymo-cytes into the Treg lineage. For instance, in thymic CD4 SP Foxp3+

cells (in contrast to their peripheral progeny) the TSDR is only par-tially demethylated [91,94], and targeted disruption of the TSDRaffects 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 ofthe 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 DNAmethylation during cell cycling – remains an interesting question.

7. Thymic antigen presenting cells (APCs) in Tregdifferentiation

Reminiscent of the classical work by Le Douarin and colleagueson thymic epithelium transplantation and dominant tolerance,early findings in TCR × model-antigen double transgenic systemshinted at a connection between self-antigen expression in thymic

epithelium and tTreg generation [16,17]. However, none of theseexperimental systems formally established the nature of the APCthat ultimately presented the respective antigen to developingthymoctes. Several recent studies on the surprisingly dynamic

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ransfer of self-determinants within the thymic microenvironmentighlight the relevance of this issue [100,101]. Specifically, self-ntigens and even functionally intact MHC molecules seem toomehow be “shuttled” unidirectionally from TECs to DCs. Thenderlying mechanistic details remain to be established; however,his phenomenon certainly adds another layer of complexity tour understanding of how individual subsets of thymic stromalells might differentially contribute to deletional or non-deletionalodes of central tolerance. For instance, in one particular model

ystem (RIP-mOVA × OTII), it was reported that deletion of spe-ific CD4T cells as a consequence of cognate antigen-expressionn mTECs was dependent on antigen transfer to and presentationy DCs [102]. By contrast, in another model (Aire-HA × TCR-HA),here a substantial fraction of specific CD4T cells differentiated

nto the Treg lineage, presentation of mTEC-derived antigen by DCsas found to be dispensable, indicating an autonomous function ofTECs as both expressers and presenters of endogenously expressed

elf-antigens for Treg induction [32].Can one generalize these observations and postulate a quali-

ative model whereby self-antigen presentation by mTECs or DCsould promote either Treg differentiation or clonal deletion, respec-

ively? We deem this rather unlikely. On the one hand, there isccruing evidence that mTECs also substantially contribute to neg-tive selection. For instance, reducing the surface expression ofHC II on mTECs to about 10% of the physiological levels throughTEC-specific silencing of C2TA indicates that at least 30% of CD4

P T cells are normally negatively selected by mTECs [33]. Viceersa, there is little reason to believe that antigen recognition onCs invariably results in negative selection. For instance, thymic

tromal lymphopoietin (TSLP) conditioned DCs [103] or plasmacy-oid DCs [104,105] were suggested to promote Treg differentiationn the human thymus, and studies in mouse models have like-

ise been interpreted to indicate that thymic DCs can supportreg differentiation [106,107]. Furthermore, a reductionist cell cul-ure system indicated that different thymic DC-subtypes similar tohymic epithelial cells efficiently induced Treg development in vitroiven that optimal doses of cognate antigen were provided [52].

In sum, an APC-centered binary model postulating that qual-tatively distinct co-signals delivered by the stromal interactionartner govern the decision-making by autoreactive CD4T cellsppears highly unlikely. In line with the idea that intrathymic Treg

ifferentiation does not require a dedicated APC but rather entails high degree of flexibility in the stromal cell types involved, sev-ral genetically manipulated mouse models that lack either DCs orubsets of mTECs, or with absent or aberrant MHC II expression onubclasses of thymic APCs, display for the most part normal num-ers of polyclonal Treg cells [33,108–113]. Nevertheless, it remainso be seen in how far this apparent principle redundancy betweenhymic APCs translates into a true “functional” redundancy at theevel of distinct TCR specificities that are guided into the Treg reper-oire (or deleted).

. Thymocyte-intrinsic developmental tuning and Tregifferentiation

Early concepts of immune tolerance predicted that immatureymphocytes bear an inherent predisposition towards a toler-nce response, whereas only after further maturation they wouldespond to antigenic stimulation by activation and acquisition offfector function [114]. This scenario has become a well acceptedaradigm in immunology. In its most simplistic experimental man-

festation, this becomes evident from the fact that a vast majorityf immature CD4+CD8+ (DP) thymocytes will undergo apoptosishen confronted with an anti-CD3 anti-CD28 coated plastic sur-

ace [115], whereas mature naïve T cells from spleen or lymph node

munology 23 (2011) 401– 409

will respond to the very same stimulus with robust proliferativeexpansion and IL-2 secretion.

Where and when in the life of a T cell does this transition froma tolerance-sensitive stage to a subsequent fully responsive stateoccur? Most of the available information indicates that this hap-pens during continual maturation at the single-positive (SP) stageof thymocyte differentiation. Thus, although there are also someindications that recent thymic emigrants within the first few daysafter exiting the thymus undergo further phenotypic and functionalmaturation [116], it appears that the most dramatic alteration inthe “intrinsic wiring” of T cells occurs while cells are still retainedin the thymic medulla. Specifically, medullary SP thymocytes withan “immature” (HSAhi) phenotype were found to be susceptible tonegative selection, whereas more mature (HSAlo) cells are largelyresistant to tolerance induction and instead respond to antigenicstimulation with proliferation, very much alike their progeny insecondary lymphoid organs [117]. Considering that APCs in thethymic medulla have acquired several characteristics that obvi-ously have evolved to optimize the efficacy of central toleranceinduction [118] (most prominently the “promiscuous” expressionof peripheral antigens by mTECs [119]), it is intuitively obviouswhy subsequent to positive selection and relocating to the medulla,T cells remain sensitive to tolerance induction for a certain timebefore entering a tolerance-resistant state.

How does agonist-driven Treg cell differentiation relate to thisT cell-intrinsic developmental control of tolerance-susceptibility?Whereas it is well established that DP thymocytes are susceptibleto antigen-driven clonal deletion as soon as they express a func-tionally rearranged TCR on their surface [120], it is much less clearat which developmental stage the capacity to enter the Treg lineageis established. The latter issue is intimately related to the ques-tion of when during thymocyte differentiation the Treg cell lineagebranches of from “mainstream” thymocyte development. Someinvestigators have proposed that Treg differentiation is the con-sequence of “altered positive selection” of cortical DP thymocytes[30,109,121–123]. By contrast, others have presented evidence thatTregs arise at the CD4 SP stage through what may be called “alterednegative selection” in the thymic medulla [32,33,124]. Part of thiscontroversy perhaps stems from the fact that most of these studiesdeduced the developmental stage (i.e. the time point) at which Treg

differentiation was initiated from particular modalities (i.e. the spa-tial 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 cellsmay, at first glance, suggest that Treg induction must have occurredat the DP stage [109]. However, “illegitimate” contacts of SP cellswith cTECs cannot be formally excluded because the chemokinegradients that orchestrate thymocyte positioning are unlikely toimpose a strict demarcation between cortex and medulla. Viceversa, and on the basis of the same considerations, the fact thatconfined expression and presentation of an agonist self-antigen inmedullary epithelial cells can result in Treg differentiation of specificTCR transgenic CD4T cells does not provide conclusive informa-tion as to the exact developmental stage at which the presumedinstructive stimulus occurred [32,33].

In an attempt to eliminate some of the inherent complexitiesof studying Treg differentiation in the steady state thymus, weused the intrathymic (i.t) transfer of post-positive selection “naïve”CD4 SP thymocytes of known antigen-specificity into a cognateantigen-expressing host thymus. This approach formally estab-lished 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 developmen-tal stage the principle responsiveness towards Treg inducing stimuliis established, they clearly showed that the permissive “window

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f opportunity” extends well into the CD4 SP stage. Importantly,hen CD4 SP cells of consecutive maturation stages or periph-

ral CD4T cells were i.t. injected, the efficiency of Treg inductionnversely correlated with T cell “age”. This inclination of immature

cells towards Treg differentiation was likewise seen in an APC-freen vitro system providing only TCR stimulation and IL-2, indicatinghat it is indeed a thymocyte intrinsic property.

Taken together, the declining inclination towards Treg differ-ntiation that goes along with progressive intrathmyic T cellaturation bears striking resemblance to a similar developmental

witch from susceptibility to resistance for clonal deletion. Possibly,oncomitant to gradually losing the susceptibility to being deleted,hymocytes may enter a transient phase of exquisite predispositionor Treg development. However, it appears that the developmen-al “windows of opportunity” for Treg differentiation or negativeelection are largely overlapping, so that a clear developmentalemarcation between these conditions is unlikely to exist. Impor-antly, the underlying molecular control-mechanisms remain as yetargely unexplored.

. Concluding remarks

There is a remarkably precise understanding of how genetic andpigenetic events orchestrate the expression of Foxp3, including alausible explanation of how extrinsic information such as anti-en recognition, co-stimulation and cytokines control the Foxp3ene locus (Fig. 1). The avidity model provides a reasonable concep-ual framework for the cell-fate-choice between Treg differentiationnd negative selection. However, this model fails to incorporatehenomena such as thymocyte intrinsic developmental tuning52,117], the dynamic nature of thymocyte/APC interactions [125]nd the (in all probability) non-homogeneous distribution of self-ntigens within the thymic microenvironment [126,127]. Theseatter two parameters may together lead to a dynamic interplayf the likelihood and the duration of self-antigen-encounters, anday explain the as yet enigmatic intraclonal competition between

reg precursors of identical specificity [128,129]. In the same vein,nderstanding how discontinuous or intermittent TCR signaling

mpinges on thymocyte fate may hold the key to the fundamentaluestion how quantitatively different input signals are integratednd translated into qualitatively different cell fate decisions.

cknowledgements

We like all members of the lab for critical reading of thisanuscript. We would like to acknowledge funding through theeutsche Forschungsgemeinschaft (grants KL 1228/2-1, KL 1228/3-

and Sonderforschungsbereich 571) and the Hertie FoundationGrant 1.01.1/09/11).

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