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HYPOTHESIS AND THEORY ARTICLE published: 09 May 2011 doi: 10.3389/fimmu.2011.00014 Models of Aire-dependent gene regulation for thymic negative selection

Dina Danso-Abeam1,2, Stephanie Humblet-Baron1,2, James Dooley 1,2* and Adrian Liston1,2*

1 VIB Autoimmune Genetics Laboratory, Leuven, Belgium 2 Department of Experimental Medicine, University of Leuven, Leuven, Belgium

Edited by: Mutations in the (AIRE) gene lead to autoimmune polyendocrinopa- Rachel R. Caspi, National Institutes of thy syndrome type 1 (APS1), characterized by the development of multi-organ autoimmune Health, USA damage. The mechanism by which defects in AIRE result in autoimmunity has been the Reviewed by: Mitsuru Matsumoto, University of subject of intense scrutiny. At the cellular level, the working model explains most of the Tokushima, Japan clinical and immunological characteristics of APS1, with AIRE driving the expression of Ludger Klein, tissue-restricted antigens (TRAs) in the epithelial cells of the thymic medulla. This TRA Ludwig-Maximilians-University, expression results in effective negative selection of TRA-reactive thymocytes, prevent- Germany Mark Anderson, University of ing autoimmune disease. At the molecular level, the mechanism by which AIRE initiates California at San Francisco, USA TRA expression in the thymic medulla remains unclear. Multiple different models for the *Correspondence: molecular mechanism have been proposed, ranging from classical transcriptional activity, James Dooley and Adrian Liston, VIB to random induction of , to epigenetic tag recognition effect, to altered Autoimmune Genetics Laboratory, cell biology. In this review, we evaluate each of these models and discuss their relative K.U.Leuven, Onderwijs en Navorsing strengths and weaknesses. 2, Campus Gasthuisberg, Herestraat 49, Box 1023, 3000 Leuven, Belgium. Keywords: Aire, , tissue-restricted antigens, autoimmunity, tolerance e-mail:james.dooley@ vib-kuleuven.be; [email protected]

INTRODUCTION Aire-deficient mouse models. Despite relatively mild disease on The autoimmune regulator, AIRE (OMIM #607358), has been the C57BL/6 background, each knockout strain develops lym- the focus of intense research since mutations in the gene were phocytic infiltrate and autoantibodies (Anderson et al., 2002; identified in 1997 as the cause of autoimmune polyendocrinopa- Kuroda et al., 2005; Su et al., 2008), with more severe clinical pre- thy syndrome type 1 (APS1, OMIM #2400300, also known sentation on alternative genetic backgrounds (Jiang et al., 2005; as autoimmune polyendocrinopathy-candidiasis-ectodermal dys- Kuroda et al., 2005). A role for Aire in central tolerance was trophy syndrome; Finnish-German APECED Consortium, 1997; first suggested through experiments demonstrating that expres- Nagamine et al., 1997). The syndrome is a rare Mendelian disease, sion of Aire is largely restricted to rare cells in the thymic medulla found more commonly in isolated populations, with prevalence (Bjorses et al., 1999; Heino et al., 1999; Ramsey et al., 2002). rates of 1/25000 among Finns (Ahonen, 1985), 1:5600 to 1/9000 Thymic transplant experiments demonstrated that the key non- among Iranian Jews (Zlotogora and Shapiro, 1992), and 1/14400 redundant function of Aire existed within the thymic stroma, among Sardinians (Rosatelli et al., 1998). The disease is clinically and that medullary thymic epithelial cells showed reduced tran- classified by the presence of any two out of three primary dis- scription of tissue-restricted antigens (TRAs) in Aire knockout orders: hypoparathyroidism, primary adrenocortical failure, and mice (Anderson et al., 2002). While TRA expression in the thymic chronic mucocutaneous candidiasis, with additional endocrine medulla had been previously documented (Jolicoeur et al., 1994), failure common (Betterle et al., 1998). Most of these clinical the finding of a specific molecular mediator to drive this expres- conditions result from progressive autoimmune destruction, with sion suggested an endogenous tolerogenic function. The function lymphocytic infiltrate and autoantibody production (Betterle and of Aire-dependent TRA expression in maintaining immunological Zanchetta, 2003). The clinical mechanism of enhanced sensitiv- tolerance was first demonstrated using a neo-self transgenic sys- ity toward candidiasis also appears to be autoimmune in nature, tem,where TRA expression in the thymus was linked to the efficacy with autoantibodies targeted against Th17-associated cytokines of negative selection of autoreactive thymocytes and consequently neutralizing anti-Candida immunity (Kisand et al., 2010; Puel to the development of autoimmune disease in the periphery (Lis- et al., 2010). ton et al., 2003, 2004). Additional experiments have demonstrated that thymic TRA expression is likewise able to drive regulatory THE FUNCTIONAL ROLE OF AIRE IN IMMUNOLOGICAL T cell conversion, with the alternative fates (negative selection or TOLERANCE regulatory T cell conversion) likely depending on antigen quantity Key insights into the mechanism by which mutations in and TCR affinity (Aschenbrenner et al., 2007; Hinterberger et al., AIRE affect tolerance have come through the development of 2010; Wirnsberger et al., 2011). Several alternative mechanisms

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of central tolerance were proposed for Aire (Anderson et al., focuses to evaluate the relative strengths and weaknesses of each of 2005; Kuroda et al., 2005), however the TRA-transcription func- the models. tion appears to be the most robust, and has been subsequently extended to multiple endogenous self-antigens, including insulin MOLECULAR BIOLOGY MODELS FOR AIRE ACTIVITY 2, salivary 1, desmoglein 3, seminal vesicle secretory pro- Aire as a classical transcription factor tein 2 (SVS2), and interphotoreceptor retinoid-binding protein The first model for Aire transcriptional activity describes Aire as a (IRBP; DeVoss et al., 2006; Yano et al., 2008; Hou et al., 2009; classical transcription factor, able to bind the promoter sequence Wada et al., 2011). of target genes (Figure 1A). Initial analysis of A potential role for Aire in peripheral tolerance remains contro- the human Aire protein identified several functional domains versial. Several groups have found extra-thymic Aire expression in indicative of a transcriptional regulator (Nagamine et al., 1997; multiple peripheral locations (Halonen et al., 2001; Kogawa et al., Kumar et al., 2001). Most importantly, Aire contains DNA bind- 2002; Gardner et al., 2008), while other studies have found Aire ing domains, which have been mapped to two zinc-finger-binding expression to be restricted to thymic epithelial cells (Hubert et al., plant homeodomains (PHD1 and PHD2), and a “human Sp100, 2008). Initial experiments suggested that the function of Aire was Aire1, NucP41/P75, and Drosophila DEAF1 domain” (SAND) restricted to the thymic epithelium, as loss of Aire in the thy- domain (Gibson et al., 1998; Bottomley et al., 2001; Kumar et al., mus was sufficient to induce disease (Anderson et al., 2002) and 2001). This model is experimentally supported by the ability of expression of selected TRAs in the lymph nodes were not Aire- Aire to bind DNA via these domains in a sequence-specific manner dependent (Kont et al., 2008). However, more recent experiments (Kumar et al., 2001). Gel-shift analysis revealed the SAND domain have suggested that Aire is expressed in the periphery and does to bind the motif TTATTA in the presence of guanine residues have a functional role (Gardner et al., 2008). Aire-expressing stro- while the zinc-finger-containing PHD domains were found to mal cells from the lymph nodes have been demonstrated to express bind to the ATTGGTTA sequence (Kumar et al., 2001; Purohit TRAs in a tolerogenic form, and this process has been demon- et al., 2005). Furthermore, when tethered to DNA Aire has the strated to be Aire-dependent for at least a subset of TRAs (Gardner capacity to activate transcription (Pitkanen et al., 2000). Also in et al., 2008). Independent experiments have also confirmed that favor of this model is the finding that Aire is capable of recruit- the lymph node stroma mediates TRA immune tolerance (Lee ing molecular partners involved in chromatin binding, transcrip- et al., 2007; Nichols et al., 2007; Magnusson et al., 2008; Fletcher tion, mRNA elongation and processing, such as TOP2a, DNA-PK, et al., 2010), including both Aire-dependent and Aire-independent Ku80, PARP-1, and H2A (Abramson et al., 2010). Importantly, the mechanisms (Cohen et al., 2010). Overall, it is likely that Aire has knockdown of these molecular partners reduces the expression some function in peripheral tolerance for a subset of TRAs,but that of Aire-dependent genes, indicating a functional role in Aire- failures in this function are not the primary cause of autoimmunity mediated transcription (Abramson et al., 2010). The most impor- in APS1. tant support for this model comes from the finding that transgenes using target gene promoters, such as the insulin promoter-driven MECHANISMS OF AIRE-DEPENDENT TOLERANCE hen egg lysozyme transgene, also show Aire-dependence (Lis- Despite the crucial function of Aire, the molecular mechanism by ton et al., 2004), despite being imbedded in a different chro- which it drives TRA expression in medullary thymic epithelium matin context and using only the core promoter sequence. Like- and lymph node stromal cells remains opaque. The key difficulty wise the insulin promoter-driven ovalbumin transgene, while is the scale of Aire-dependent gene expression, with dependent being initially characterized as Aire-independent (Anderson TRAs numbering in the order of several hundreds to thousands et al., 2005), has now been demonstrated to have partial Aire- of genes (Anderson et al., 2002; Derbinski et al., 2005). In this dependency despite its foreign chromatin context (Su et al., 2008; review, we address the various models that have been proposed Hubert et al., 2011). so far for the elucidation of the mechanism by which Aire regu- Despite the attractive simplicity of the classical transcription lates its target genes. The models can be envisaged in two broad factor model, there are several notable disadvantages. Firstly, the categories: molecular biology models and cell biology models. utilization of KNKA motif as the binding motif in the SAND The molecular biology models utilize the molecular structure of domain instead of KDWK or KNW (K/R) in other SAND- Aire to explain the mechanism of its target gene activation and containing suggests atypical DNA binding activity (Puro- include classical transcription activation model, random tran- hit et al., 2005; Peterson et al., 2008). Secondly, a consensus scriptional activator model, and epigenetic tag recognition model. sequence for DNA binding has not been found among the numer- In each of these models, Aire is proposed to have direct tran- ous target genes. Thirdly, the sheer number of target genes (∼200– scriptional activation properties, driving the expression of target 1000), with highly diverse peripheral expression patterns, make a genes. The cell biology models provide an alternative approach direct binding function unlikely. Finally,the expression of different to the mechanism of Aire target gene regulation and encompass Aire-dependent TRA targets in thymic epithelial cells, peripheral two contrasting models – “developmental retardation” and “dys- lymphnodestromalcells(Lee et al.,2007; Gardner et al.,2008),and regulated death” models – in which both propose Aire to influence modified monocytes (Sillanpaa et al., 2004) is not consistent with the differentiation of medullary thymic epithelial cells to enhance a fixed promoter recognition site. Nevertheless, the classical tran- TRA expression. With new studies revealing insights into both scription factor binding model remains viable, as various modifi- the molecular and cellular impacts of Aire expression, this review cations could explain these discrepancies, such as the expression

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FIGURE 1 | Classical transcription factor model. (A) In this model Aire amplification of Aire-dependent targets may occur when Aire-dependent TRAs binds DNA through sequence-specific interaction with promoter elements are themselves transcriptional activators. For example, Aire-dependent TRA1 within TRA genes, such as TRA1 and TRA2. Gene expression is activated in may in turn drive the expression of TRA3, TRA4, and TRA5. These secondary these genes, but not neighboring genes, either through the direct action of targets are expressed in an Aire-dependent manner despite the lack of an Aire or through the action of recruited binding partners. (B) Possible Aire-binding site in the promoter. of target genes being modulated by the chromatin context in an accessibility of genomic DNA in a way which allows non-specific Aire-independent manner, or the existence of binding partners to gene expression, or permits otherwise illegitimate expression to Aire that modify target recognition (Abramson et al., 2010). One be productive (Abramson et al., 2010). The expression of Aire modification of the model that could explain many of the dis- in the testes (Derbinski et al., 2001) and stem cells (Nishikawa crepancies is one where Aire is responsible for initiating the tran- et al., 2010), and the observation of infertility in Aire knockout scription of a small subset of master transcription factors, which mice (Anderson et al., 2002; Ramsey et al., 2002; Hubert et al., then in turn initiate the expression of larger pools of downstream 2009), generated the plausible model that Aire had a DNA acces- TRAs (Figure 1B). This would enable Aire to drive the expres- sibility function primarily for proliferative processes, which was sion of large numbers of genes without any apparent conservation co-opted for immunological tolerance in the thymic epithelium. within the promoter, and is supported by the finding that diverse However, the infertility observed in Aire knockout mice reflects tissue-specific master transcription factors, such as Pdx1, are autoimmunity against reproductive organs, as it is absent in Aire expressed in thymic epithelial cells in an Aire-dependent manner knockout mice crossed to the Rag knockout background (unpub- (Gillard et al., 2007). lished observation), and TRA expression in the testes is Aire- independent, unlike thymic epithelial cells (Liston et al., 2004). A Aire as a random transcriptional activator primary function for Aire in the cell division process has therefore An alternative to the classical transcription factor model is the been discredited. A modified form of the random transcriptional random transcriptional activator model. Early analysis of the activator hypothesis, however, is still viable, with the observa- cohort of genes expressed as TRAs suggested that genes were tion that Aire-dependent TRAs tend to be found in chromosomal being turned on at low levels in a random manner (Derbin- clusters (Derbinski et al., 2005). It is therefore feasible that Aire ski et al., 2001), similar to the way “leaky” expression of genes recognizes sequence-specific DNA regions (as in model 1),or chro- is observed in the testes (Derbinski et al., 2001) and stem cells matin tags (as in model 3, below) in order to open up transcription (Miyamoto et al., 2002; Zipori, 2004). In this model, Aire would of a small genomic region (Figure 2). Without genome-wide contribute to the loosening of chromatin structure or general knowledge of chromatin boundaries and insulators it is difficult

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FIGURE 2 | Random gene expression model. In this model Aire FIGURE 3 | Epigenetic tag recognition model. Under the epigenetic tag contributes to random activation of genes by loosening up the chromatin model, Aire is recruited to a through binding to sequence-independent structure to increase the general accessibility of TRA genes. This would in epigenetic tags, such as unmethylated histone 3. Following demethylation turn allow the recruitment of transcriptional activators to bind genes that and Aire binding, Aire either directly enhances transcription or operates via would otherwise be physically restricted. the recruitment of other transcriptional activators.

to draw firm conclusions on the existence of these “regional” et al., 2008) and harbors a high density of the human mutations effects. detected in APS1 patients (Bjorses et al., 1998; Chakravarty et al., 2009). While the importance of the PHD1 domain in the function Aire as an epigenetic tag recognition factor of Aire has been demonstrated (Koh et al., 2008; Org et al., 2008), A third model for the activity of Aire is based on recent biochemi- it may not reflect Aire’s targeting mechanism as the PHD1 domain cal analysis of Aire, indicating a capacity to bind modified histones has also been implicated in direct DNA binding (Kumar et al., (Koh et al., 2008; Chignola et al., 2009). Unlike the classical tran- 2001; Purohit et al., 2005). An attractive feature of this model is a scription factor model, this epigenetic tag model does not require putative explanation for the large number of diverse target genes, sequence-specific recognition of target genes, but instead requires as many genes throughout the genome would exhibit similar his- a common histone modification. In this scenario Aire would bind tone modification. Indeed, the range of Aire-dependent genes is the modified histones and activate the transcription of nearby actually far smaller than would be predicted from a common his- genes (Figure 3), probably through the recruitment of a complex tone tag, and the over-expression of a H3K4 demethylase, able to of transcriptional activators and mRNA elongation/processing extend the Aire-binding signature, does not extend the range of factors (Abramson et al., 2010). In support of this model, studies Aire target genes (Koh et al., 2010). The key weakness with this have shown that Aire is able to bind to unmethylated K4 on histone model is explaining how the specificity of target gene expression 3(Koh et al., 2008; Org et al., 2008). Furthermore, Aire has been is achieved, for example how the core insulin promoter is able shown to interact with the transcription co-factors CBP (CREB to be recognized when placed in a different chromatin context in binding protein; Pitkanen et al., 2000), PIAS1 (protein activator the form of a transgene (Liston et al., 2004; Hubert et al., 2011). of activated STAT1; Ilmarinen et al., 2008), and P-TEFb (positive Another weakness is the inability of this model to explain why only transcription elongation factor b; Oven et al., 2007), indicating an small numbers of genes are activated in any given Aire-expressing ability to drive transcription after tethering to the locus (Bjorses epithelial cell (Gillard et al., 2007; Villasenor et al., 2008), unless et al., 2000; Pitkanen et al., 2000, 2005; Oven et al., 2007; Ilmarinen Aire activity itself is limiting. Overall, the biochemical data that et al., 2008). Aire binds epigenetic tags are convincing, but it is yet to be deter- In support of this model is the finding that binding to mined whether this is the function that primarily drives TRA unmethylated K4 on histone 3 is dependent on the PHD1 domain expression, or whether it is a mechanism of Aire used to increase (Chakravarty et al., 2009; Chignola et al., 2009; Koh et al., 2010), the stability of interaction with a promoter it binds due to DNA which is evolutionarily conserved among gnathostomes (Saltis sequence recognition (Ruthenburg et al., 2007).

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CELL BIOLOGY MODELS FOR AIRE ACTIVITY “Dysregulated death” model “Developmental retardation” model An alternative model, that of “dysregulated death,” proposes that In contrast to the direct activity models described above, sev- Aire expression dysregulates normal cell biology of the thymic eral innovative alternatives have been proposed that postulate an epithelial cell, resulting in the partial differentiation toward one effect of Aire on the cell biology of medullary thymic epithelial or more alternative epithelial fates in a terminal process (Figure 4; cells. The first model is the “developmental retardation” model, Gillard and Farr, 2006; Dooley et al., 2008). The terminal nature where it was proposed that Aire expression keeps thymic epithe- of Aire expression may even aid tolerance by facilitating the cross- lial cells in an immature state (Gillard et al., 2007). This model presentation of TRAs to dendritic cells (Gray et al., 2007). Key was supported by data reporting Aire-dependent expression of support for the “dysregulated death” model comes from numer- Nanog, Oct4, and Sox2, which are candidates for expression ous studies which demonstrate changes to thymic epithelial cell by epithelial progenitor cells in the thymus and are critical for biology in the absence of Aire. Beyond the well documented maintaining their multipotentiality (Gillard et al., 2007). This decrease in TRA expression, Aire-deficiency can result in mor- model was also supported by evidence demonstrating that Aire- phological changes, altered antigen presentation capacities, and deficient mice present with a reduced and altered medullary enhanced apoptosis (Anderson et al., 2005; Gray et al., 2007; compartment (Gillard et al., 2007) with fewer terminally differ- Dooley et al., 2008; Yano et al., 2008; Hubert et al., 2011). This entiated epithelial cells in the absence of Aire (Yano et al., 2008). model has the capacity to explain many of the unique charac- Furthermore, Aire has been found to be actively expressed in teristics of Aire-dependent TRA expression, such as the broad early embryogenesis (Nishikawa et al., 2010). Under this model, repertoire of genes that are activated and the retained ability the expression of Aire in the thymic epithelium would prevent to activate transgenes utilizing these promoters, as conserved full differentiation into mature epithelial cells, thereby allow- transcriptional pathways would be utilized. The observation that ing Aire-expressing cells to differentiate into alternative epithe- diverse tissue-specific master transcription factors, such as Pdx1, lial fates, a process which would initiate the expression of TRAs are Aire-dependent supports this hypothesis (Gillard et al., 2007). (Farr et al., 2002). However this latter property would predict that Aire-dependent Ultimately, the “developmental retardation” model for Aire TRA expression should cluster TRAs from the same target organ function is incompatible with more recent findings that Aire- in the same Aire-expressing cells, while this phenomenon is not expressing thymic epithelial cells are concentrated within the observed in practice (Gillard and Farr, 2006; Derbinski et al., mature CD80hi subpopulation, have a low level of proliferation 2008; Venanzi et al., 2008; Villasenor et al., 2008). The key dis- and a high level of apoptosis (Gabler et al., 2007; Gray et al., advantage of cell biology-based models is the lack of a known 2007; Irla et al., 2008; White et al., 2010). A study utilizing 5- molecular mechanism, as the described molecular properties bromo-2-deoxyuridine (BrdU) incorporation instead reveals that of Aire are more consistent with a role in transcription than Aire-expressing epithelial cells are terminally differentiated and in cell physiology. A putative explanation would involve Aire highly apoptotic (Gray et al., 2007). However, this new evidence inducing “dysregulated death” via transcriptional activity, how- does not negate the possibility of a cell biology model for Aire ever again a molecular target is not available to explain such expression of TRAs. behavior.

FIGURE 4 | Dysregulated death model. In the dysregulated death lineages, resulting in the expression of genes outside the normal model, Aire expression within mTECs results in a terminal mTEC transcriptome. The premature apoptosis of the dysregulated degeneration of the normal mTEC biology. Increased plasticity cell may facilitate antigen sharing with dendritic cells for results in partial dedifferentiation or redifferentiation into alternative presentation.

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Table 1 | Explanatory power of proposed models for Aire activity.

Classical transcription Randomized Epigenetic tag Altered cell factor gene expression recognition biology

DNA binding activity ++ −/+− −/+ Chromatin tag binding activity − + ++ −/+ Recruitment of functional molecular partners ++ ++ ++ −/+ Broad range of target genes −/+ ++++++ Differential target gene set in different cell types −/+ −+−/+ Ability to enhance expression of transgenes ++ −/+− ++ Altered cell biology of Aire-expressing cells −−/+−/+++ Stochastic expression of targets −++−/+−

−, incompatible; −/+, compatible; +, weak support; ++, strong support.

CONCLUDING REMARKS and the cellular biology of Aire-expressing cells, there is as yet The failure of thymic deletion in Aire-deficient mice has revealed no single coherent model for Aire activity which adequately the significant role of Aire in promoting clonal deletion of accounts for all the observed phenomena (refer to Table 1). autoreactive organ-specific T cells. The main unresolved issue is The provision of new data is required to definitively support the mechanism of transcriptional activation of Aire-dependent a consensus model, whether it be one of the four main mod- TRAs. Despite large advances that have come from the study els outlined here, a hybrid model or a novel model yet to be of Aire-dependent TRA targets, the molecular biology of Aire proposed.

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Frontiers in Immunology | Immunological Tolerance May 2011 | Volume 2 | Article 14 | 6 Danso-Abeam et al. Models of Aire-dependent transcription

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Pitkanen, J., Doucas, V., Sternsdorf, T., Ramsey, C., Winqvist, O., Puhakka, (2008). Mechanisms of an autoim- Satoh, R., Kawamoto, H., Mouri, Y., Nakajima, T., Aratani, S., Jensen, L., Halonen, M., Moro, A., Kampe, munity syndrome in mice caused by and Matsumoto, M. (2008). Aire K., Will, H., Vahamurto, P., Ollila, O., Eskelin, P., Pelto-Huikko, M., a dominant mutation in Aire. J. Clin. controls the differentiation program J., Vihinen, M., Scott, H. S., and Peltonen, L. (2002). Aire defi- Invest. 118, 1712–1726. of thymic epithelial cells in the Antonarakis, S. E., Kudoh, J., cient mice develop multiple features Venanzi, E. S., Melamed, R., Mathis, D., medulla for the establishment of Shimizu, N., Krohn, K., and Peter- of APECED phenotype and show and Benoist, C. (2008). The variable self-tolerance. J. Exp. Med. 205, son, P.(2000). The autoimmune reg- altered immune response. Hum. immunological self: genetic varia- 2827–2838. ulator protein has transcriptional Mol. 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Frontiers in Immunology. didiasis and autoimmune polyen- changes in the gene expression pro- Wirnsberger, G., Hinterberger, M., and Copyright © 2011 Danso-Abeam, docrine syndrome type I. J. Exp. file of human monocyte-dendritic Klein, L. (2011). Regulatory T-cell Humblet-Baron, Dooley and Liston. Med. 207, 291–297. cell-lineage. Mol. Immunol. 41, differentiation versus clonal dele- This is an open-access article subject to a Purohit, S., Kumar, P. G., Laloraya, M., 1185–1198. tion of autoreactive thymocytes. non-exclusive license between the authors and She,J. X. (2005). Mapping DNA- Su, M. A., Giang, K., Zumer, K., Jiang, Immunol. Cell Biol. 89, 45–53. and Frontiers Media SA, which permits binding domains of the autoim- H., Oven, I., Rinn, J. L., Devoss, J. J., Yano, M., Kuroda, N., Han, H., Meguro- use, distribution and reproduction in mune regulator protein. Biochem. Johannes, K. 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