of May 20, 2018.This information is current as s-Ectodermal Dystrophy (APECED)

Autoimmune-Polyendocrinopathy-CandidiasiExpression, the Gene Defective in the

AireSelection Are Associated with Normal Thymic Architecture and Negative

Fasler-Kan, Ed Palmer and Georg A. HolländerSaulius Zuklys, Gina Balciunaite, Anni Agarwal, Elizaveta

http://www.jimmunol.org/content/165/4/1976doi: 10.4049/jimmunol.165.4.1976

2000; 165:1976-1983; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/165/4/1976.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2000 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Normal Thymic Architecture and Negative Selection AreAssociated withAire Expression, the Gene Defective in theAutoimmune-Polyendocrinopathy-Candidiasis-EctodermalDystrophy (APECED)1

Saulius Zuklys,2*† Gina Balciunaite,2*† Anni Agarwal,* † Elizaveta Fasler-Kan,† Ed Palmer,‡

Georg A. Hollander3*†

T cell development is tightly controlled by thymic stromal cells. Alterations in stromal architecture affect T cell maturation andthe development of self-tolerance. The monogenic autoimmune syndrome APECED (autoimmune-polyendocrinopathy-candidia-sis-ectodermal dystrophy) is characterized by the loss of self-tolerance to multiple organs. Although mutations in the autoimmuneregulator (AIRE) gene are responsible for this disease, the function ofAIRE is not known. Here we report on the spatial andtemporal pattern of murine Aire expression during thymic ontogeny and T cell selection. Early during development, thymicAiretranscription is critically dependent on RelB and occurs in epithelial cells in response to lymphocyte-mediated signals. In adulttissue,Aire expression is confined to the medulla and the corticomedullary junction, where it is modulated by thymocytes un-dergoing negative selection.Aire may determine thymic stromal organization and with it the induction of self-tolerance. TheJournal of Immunology,2000, 165: 1976–1983.

T he thymus is the primary lymphoid organ for the devel-opment of T cells of theabTCR lineage. Lymphoid cellsdifferentiate in direct physical contact with thymic stro-

mal cells, and this interaction is not only critical for thymocyteselection and maturation but is equally essential for the inductionof a functionally competent thymic stromal compartment. The thy-mic primordium of mice arises bilaterally at day 10 of gestation(E10)4 by fusion of the third pouch endoderm with ectoderm fromthe corresponding branchial clefts. The two tissues are separatedby a thin layer of mesenchyme originating from the cephalic neuralcrest (1). While the relative contribution of each germ layer to themature thymic epithelial compartment remains to be determined, asmall subpopulation of epithelial cells has been characterized thatrepresents a common precursor cell for mature cortical and med-ullary epithelium (2, 3). At day E11.5, hemopoietic precursor cellsseed the thymus anlage although its epithelial cells are not yetcompetent to fully support T cell development (4). This capacity isonly achieved after further maturation when epithelial cells havedifferentiated into distinct stromal cell types (reviewed in Ref. 5).

Finally, the thymic microenvironment is composed of an inte-grated network of epithelial reticular cells and nonepithelial stro-mal cells (i.e., fibroblasts, macrophages, and interdigitating retic-ular cells) each characterized by typical structural, antigenic, andfunctional features. Notably, the organization of the thymic epi-thelium differs from most other epithelial organs in the body:rather than forming a sheet of cells positioned on a basement mem-brane, thymic epithelial cells (TEC) form a three-dimensionalmeshwork (6).

The space between the diverse thymic stromal cells is occupiedby thymocytes at different stages of development as defined bytheir expression of CD4 and CD8 (7). The most immature popu-lation of intrathymic T cell precursors lack CD4, CD8, and CD3expression and are referred to as triple negative (TN). These cellscan be further classified into four independent subpopulations ac-cording to their sequential expression of CD44 and CD25: earlythymic immigrants are CD441CD252 (TN I) and develop via aCD441CD251 (TN II) and a CD442CD251 (TN III) stage toimmature thymocytes with a CD442CD252 (TN IV) phenotype(8). Subsequently, thymocytes begin to express transiently CD8before become CD41CD81 (double positive, DP) cells, a popu-lation that constitutes the majority of thymocytes. At this stage, DPcells express anabTCR that renders them subject to either positiveor negative selection dependent on their Ag specificity. Only aminority (;3%) of the DP cells are positively selected to generatemature single positive (SP) helper (CD41CD82) and cytotoxic(CD42CD81) T cells (9). The developmental progression fromimmature to mature thymocytes occurs sequentially and in distinctthymic microenvironments (10).

The typical architecture of the complex thymic stroma is criti-cally dependent on intercellular communications. Early in ontog-eny, fibroblastoid cells control the differentiation of mesenchymalcells to form a regular epithelial compartment. Depending on cell-cell interactions between developing thymocytes and stromal cells(termed thymic cross-talk), distinct microenvironments are createdthat allow all steps in T cell maturation to occur. Importantly, the

*Pediatric Immunology, The Children’s University Hospital, Basel University, Basel,Switzerland;†Department of Research, Kantonsspital Basel, Basel University, Basel,Switzerland; and‡The Basel Institute for Immunology, Basel, Switzerland

Received for publication April 5, 2000. Accepted for publication June 5, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the Swiss National Science Foundation(3100-043600.95 and 31-55820.98) and the Helmut Horten Foundation.2 S.Z. and G.B. contributed equally to this work.3 Address correspondence and reprint requests to Dr. G. A. Hollander, Pediatric Im-munology, Department of Research, Basel University Medical School, Hebelstrasse20, 4031 Basel, Switzerland. E-mail address: Georg-A.Hollaender@unibas.ch4 Abbreviations used in this paper: E, embryonic day; Aire, autoimmune regulator;APECED, autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy; DP,double positive, i.e., CD41CD81; ISH, in situ hybridization; pTa, pre-TCRa-chain;TEC, thymic epithelial cell; TN, triple negative, i.e., CD32CD42CD82; SP, singlepositive; RT, room temperature.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

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lack of inductive signals from developing T cells prevents the for-mation of the distinct cortical and medullary microenvironments(11, 12). Consequently, the absence of a normal stromal compart-ment hinders the orderly maturation of thymocytes and impedesthe establishment of self-tolerance (13–15). Conversely, diverseexperimental models of autoimmunity, such as Chagas disease,scleroderma, lupus erythematosus, and insulin-dependent diabetes,have been correlated with an obvious disorganization of thymicstromal architecture (16).

Clonal deletion and the induction of anergy represent two majormechanisms that establish self-tolerance among thymocytes. Im-munity to self-Ags is also prevented by the presence of thymus-derived T cells, which play an active role in regulating the auto-reactive potential of cells that have neither been clonally deletednor rendered anergic (17–20). However, the relative importance ofthese mechanisms for the establishment of self-tolerance remainsto be determined for particular Ags.

The cellular and molecular mechanisms responsible for manyforms of autoimmune diseases have yet to be defined due to thefact that these conditions are polygenic and have different envi-ronmental triggers responsible for their clinical emergence. In con-trast, monogenic autoimmune syndromes provide appropriatemodels to gain further in-depth insights into the complex molec-ular processes associated with the loss of self-tolerance. Autoim-mune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED,also known as autoimmune polyglandular syndrome 1; OMIM240300) is a rare autosomal recessive disease with no known HLAassociation. Along with the autoimmune lymphoproliferative syn-dromes 1 and 2 (21, 22), APECED is recognized as one of threeautoimmune disorders known to be caused by a single gene defect.

The gene responsible for APECED has been designatedAIRE(autoimmune regulator) and has been mapped to chromosome21q22.3 (23–25). Mutations in the coding region ofAIREresult intruncated proteins devoid of a normal function. The murineAiregene has recently been cloned and mapped to chromosome 10,revealing a structural organization and sequence homology highlyconserved to its human ortholog (26–28).Aire encodes a predictedprotein of 552 aa that contains structural features that anticipate arole in gene transcription. The protein embodies a proline-rich re-gion, four LXXLL motifs, two plant PHD zinc-finger domains(which are restricted to nuclear proteins including transcriptionalcoactivators and chromatin-modulating proteins of the polycomband trithorax groups), and a SAND domain (a sequence present inSp100, Aire, NucP41/75 and DEAF-1/suppressin) (29–31). More-over, theAIRE gene product is localized to distinct spherical nu-clear structures, further corroborating its putative function as aregulator of gene transcription (32).

The autoimmune manifestations of APECED encompass hypo-parathyroidism, primary adrenocortical failure, and chronic muco-cutaneous candidiasis (29). APECED is characterized by lympho-cytic infiltrations, the presence of a wide variety of tissue-specific,T cell-dependent autoantibodies, and a yet-unidentified defect in Tcell function (29, 33, 34).

To detail its role in thymic function and in T cell toleranceinduction, we investigated the temporal and spatial expression ofAire in embryonic and adult mice and in models of positive andnegative thymic selection. Here we report thatAire expression isrestricted to a distinct subpopulation of TEC, and its expressionrequires thymocyte stromal cell-cell interactions, a critical prereq-uisite for the generation of an appropriate thymic architecture.Moreover,Aire expression is dependent on RelB function, and thenumber ofAire-positive thymic stromal cells correlates with thepresence of thymocytes undergoing negative selection.

Materials and MethodsMice

The BALB/c, C57BL/6, Rag-22/2, Tge26, B6.RAG-22/2 I-Ab, andB6.RAG-22/2 I-Abm12mouse strains were housed at the Animal Facility ofthe Kantonsspital Basel and the Basel Institute for Immunology, respec-tively, according to the Institutional Review Boards. The B6.RAG-22/2

I-Ab and B6.RAG-22/2 I-Abm12mice express the 3BBM74 TCR as a trans-gene on the RAG-2null background (see Ref. 35).

Tissues

Thymic tissue was obtained from adult mice and from embryos after timedpregnancies where detection of the vaginal plug was considered as day 0 ofgestation (E0). Thymic tissue from RelB2/2 mice was kindly provided byDr. Li Wu (Melbourne, Victoria, Australia) and Dr. Philippe Naquet (Mar-seille, France). Thymic tissue was embedded in OCT (Tissue-Tec, Miles,Elkhart, IN) for analysis by immunohistology and in situ hybridization(ISH).

Cell sorting

Thymocytes were obtained by tissue disruption using frosted glass slides,whereas TEC were prepared by enzymatic digestion of whole thymi asdescribed elsewhere (36, 37). After enrichment of low-density cells byPercoll gradient for the sorting of TEC, cell subsets were stained with acombination of CD3, CD4, CD8, CD11c, and CD45 (all from PharMingen,San Diego, CA), as well as mAbs 29 (36), G8.8 (38), and CDR1 (39),respectively. The different lymphocyte and stromal subpopulations weresorted by flow cytometry (FACStar; Becton Dickinson, Mountain View,CA) to a purity of at least 98%.

RNA isolation and RT-PCR analysis

Total RNA was isolated from tissues or sorted cells using TRI reagent(Molecular Research Center, Cincinnati, OH). Sorted cells were supple-mented with 10mg of yeast transfer RNA (Life Technologies, Basel, Swit-zerland) as a carrier. For the generation of cDNA, RNA was treated withRNase-free DNaseI (Roche Molecular Biochemicals, Gipf-Oberfrick,Switzerland) and then reverse transcribed using SuperScript II reverse tran-scriptase with either oligo(dT) (for tissues) or random hexamers (for sortedcells) as primers (Life Technologies). For PCR, various amounts of cDNAwere used with 13Taq PCR buffer (1.5 mM Mg21), 0.5 U Taq, 0.2 mMdNTP (all from Sigma, St. Louis, MO), and 0.4mM of each oligonucle-otide. The following oligonucleotide pairs were designed from publiclyavailable data:Aire, 59-TGC ATA GCA TCC TGG ACG GCT TCC and59-CCT GGG CTG GAG ACG CTC TTT GAG; as well as 59-TCT ACTGAG TGC TGG GAA TGA G, and 59-CAG GAA GAG AAG GGT GGTGTC (see Fig. 3B);Gapdh, 59-ACC ACA GTC CAT GCC ATC AC and59-TCC ACC ACC CTG TTG CTG TA;Whn, 59ATG GAG ACC TTGGGA CTG AC and 59-TGG CTG AGT GGC ATA GGA GA;pTa, 59-ATC ACA CTG CTG GTA TAT GGA and 59-TCA GAG GGG TGG GTAAGA TC; b-actin, 59-GTC GGC CGC TCT AGG CAC CAA and 59-CTCTTT GAT GTC ACG CAG GAT TTC. PCR amplification for cDNA ob-tained from tissues used 25, 26, or 30 cycles, respectively, as indicated forGapdh, 28 cycles forWhnandpTa, and 30 cycles forAire. Amplificationof cDNA from sorted cells used 30 cycles forGapdhandb-actin and 35cycles for Aire. For data shown in Fig. 3B, the relative amount of thefirst-strand cDNAs produced from each sorted stromal subpopulation wasestimated after amplification of a referenceb-actin cDNA fragment. PCRproducts were separated on a 1.7% agarose gel, visualized by staining withSYBR Gold (Molecular Probes, Eugene, OR), and images were analyzedusing the QuantityOne gel-doc system (Bio-Rad, Richmond, CA).

Northern blot analysis

Tissues from 6-wk-old C57BL/6 mice were collected and snap frozen inliquid nitrogen. Total RNA was isolated using TRI reagent, and mRNAwas isolated from total RNA using the Oligotex kit (Qiagen). For eachsample, 1mg of mRNA was electrophoresed in a 1% agarose-formalde-hyde gel, transferred to Hybond N1 membrane (Amersham PharmaciaBiotech, Uppsala, Sweden) by overnight capillary blotting in 103SSC.The membrane was probed with [a32-P]-labeledAire (54–630 bp; acces-sion no. AJ132243) orGapdh (566–1017 bp; accession no. M32599)cDNA fragments. Hybridization was performed for 2 h at 65°C using theQuickHyb buffer (Stratagene, La Jolla, CA) according to the manufactur-er’s instructions. The blot was exposed overnight using a PhosphoImagerScreen (Amersham Pharmacia Biotech), and results were analyzed byImageQuant software (Bio-Rad).

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In situ hybridization

Sense and antisense digoxigenin-labeled (Roche Molecular Biochemicals)cRNA probes were generated by in vitro transcription using cDNA specificfor Aire, Whn, andRag-1as templates. ISH was performed as previouslyreported (40). In short, frozen sections (15mm) were cut from thymictissue embedded into OCT, air dried at room temperature (RT; 20 min),fixed in 4% paraformaldehyde (10 min), washed tree times with PBS, andfinally acetylated (10 min). Prehybridization was performed overnight atRT with hybridization buffer (50% formamide, 53SSC, 13Denhardt’s,100 mg/ml yeast transfer RNA, 100mg/ml salmon sperm DNA) using achamber humidified with 53SSC. The hybridization mixture was preparedby adding 200 ng of digoxigenin-labeled probe per milliliter of hybridiza-tion buffer, which was first heated (85°C, 5 min) and subsequently chilled(4°C). The hybridization mixture was spread over the sections, which werethen covered with siliconized coverslips and sealed with rubber cement(Stanford, Bellwood, IL). Slides were placed in Quadriperm dishes(Heraeus Instruments, Hanau, Germany), and hybridization was performedovernight at 68°C in a 53SSC humidified chamber. Slides were washedinitially in 53 SSC at 72°C and subsequently in 0.23SSC at 70–72°C (60min) followed by 0.23SSC at RT (5 min). Next, slides were rinsed with0.1 M maleic acid, 0.15 M NaCl buffer (pH 7.5), blocked for 1 h in 1%blocking reagent (Roche Molecular Biochemicals) diluted in maleic acidbuffer, and incubated for 1 h with alkaline phosphatase-conjugated anti-digoxigenin Ab (Roche Molecular Biochemicals) diluted in blocking so-lution (1:2500). Slides were washed for 13 10 min and 23 30 min inequilibrated maleic acid buffer (100 mM Tris-HCl, 100 mM NaCl, 5 mMMgCl2, pH 9.5). For color development, slides were placed upside-down ina Quadriperm dish containing nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate substrate (Roche Molecular Biochemicals; nitroblue tet-razolium, 112.5ml; 5-bromo-4-chloro-3-indolyl-phosphate, 87.5ml; di-luted in equilibrated maleic acid buffer). The reaction was performed in thedark at RT and stopped by transferring the slides into PBS containing 1mM EDTA (10 min). The tissue was counterstained with Methylene Green(0.05% in PBS), and slides were coverslipped using Kaiser’s gelatin(Merck, Darmstadt, Germany). Positive signals are represented by dots ofstrong purple color, while unspecific signals are detected as small anddiffuse dots of weaker intensity. These latter signals are also present in theabsence of a specific probe (data not shown).

Immunohistochemistry

Frozen thymic sections (5mm) were fixed with acetone (Merck) at RT for15 min and washed in PBS. Endogenous peroxidase activity was blockedby a 15-min incubation with 3% H2O2, 0.1% NaN3 in PBS. The sectionswere blocked with normal goat serum (10% in PBS) for 30 min. Individualthymic sections were incubated with the appropriate dilution of primary Ab(30 min, RT), washed three times in PBS, and incubated with HRP-con-jugated goat anti-rat Ig for 30 min. After additional washing in PBS, thesubstrate amino-ethyl-carbazole (Sigma) was applied to the sections. Thetissues were washed in PBS, counterstained with Mayer’s Hemalaun(Merck), mounted with Crystal/Mount (Biomeda, Foster City, CA), andcoverslipped.

ResultsAire transcription in thymic tissue

To detail the expression pattern ofAire in different murine tissues,a Northern blot analysis was performed. As shown in Fig. 1A,specific mRNA was detected mainly in thymus, and to a lesserextent in spleen and lymph nodes. At least three distinct bandswere consistently observed in each of these organs, suggesting theexistence of multipleAire transcripts.

To ascribeAire expression to a distinct thymic cell lineage, RT-PCR was performed on cDNA from freshly isolated subpopula-tions of thymocytes, TEC and thymic dendritic cells, respectively.While thymocytes at all maturational stages failed to expressAire,analysis from cDNA obtained from freshly isolated TEC resultedin amplification of Aire transcripts (Fig. 1B). The use ofAire-specific primers with cDNA from MHC class II1 CD11c1 thymicdendritic cells resulted only in a very faint amplification product(see below and Fig. 3B). Thus, stromal cells but not lymphoid cells(at any stage of their intrathymic development) are responsible forAire expression in adult thymic tissue.

Next we determined the pattern ofAire expression during thy-mic ontogeny using RT-PCR. For this purpose, RNA was isolatedfrom thymic tissue of C57BL/6 embryos taken at distinct gesta-tional ages and analyzed forAire, Whn, andpTa transcripts. Thelatter two transcripts were used to monitor the two dominant celltypes present during early thymic ontogeny, i.e., epithelial cellsand immature thymocytes.Whn is a transcription factor typicallyexpressed in all thymic epithelial cells and critical for their growthand differentiation (41, 42). In contrast,pTa is a T lymphoid-specific cell-surface molecule expressed exclusively in early thy-mocytes, where it is essential for the maturational transition of aCD442CD251 (TN III) to a CD442CD252 (TN IV) phenotype(43, 44).

Aire-specific cDNA was detected only after E14, whileWhn-andpTa-specific transcripts were already apparent at E12 and E13,respectively. By day 14, commitment to thymocytes of the TCR

FIGURE 1. Expression ofAire in different murine tissues.A, Northernblot analysis of mRNA from thymus (Th), liver (Li), brain (Br), spleen(Sp), bone marrow (BM), lymph node (LN), stomach (St), skin (Sk), andkidney (K) analyzed with a probe specific forAire and Gapdh, respec-tively. The markers on theright represent the 28S (upper band) and the 18S(lower band) ribosomal RNAs.B, RT-PCR analysis of mRNA fromCDR11G8.81 TEC and from isolated thymocyte subpopulations. Thymictissue was either gently digested for the retrieval of stroma cells or me-chanically separated for the isolation of thymocytes using frosted glassslides. Cell suspensions were stained and sorted by flow cytometry as out-lined in Materials and Methods. Thymocytes were defined by their char-acteristic forward/side scatter and enriched for the phenotypic subpopula-tions of CD32CD42CD82 (TN), CD41CD81 (DP), or either matureCD41CD82 (SP4) or CD42CD81 (SP8) thymocytes. Total thymic RNAnot subjected to reverse transcription was used as a negative control (des-ignated RNA).C, Expression ofAire, Whn,pTa, andGapdhwas assessedby RT-PCR using thymic tissues from embryos at different developmentalages (days post conception, p.c.) and from adult mice.

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ab lineage has already occurred as demonstrated by the transcrip-tion of the pTa gene (Fig. 1Cand Ref. 45). Moreover, thymicepithelial subpopulations with a cortical and medullary phenotypecan be distinguished at this point in time (36). AbundantAireexpression was demonstrated at E16, a time during thymic devel-opment when the formation of DP thymocytes has been initiatedbut TCR-mediated thymic selection has not yet begun. The relativeabundance ofAire transcripts decreased somewhat after day 16 butremained detectable throughout fetal and postnatal life (Fig. 1C).The observed changes inWhnandAire expression during thymicdevelopment are likely accounted for by shifts in the relative fre-quency ofAire-positive epithelial cells. Taken together, these re-sults demonstrate thatAire expression in thymic stromal cellsemerges relatively late during ontogeny but is sustained intoadulthood.

Aire transcription is restricted to a subpopulation of epithelialcells

We next determined the spatial expression ofAire mRNA in fetalthymi using ISH. In E16 tissue,Aire expression was detected insmall but distinct aggregates scattered throughout the entire organ(Fig. 2A). In contrast, ISH using aWhn-specific probe revealed a

network of cells that represented the majority of epithelial cells atthis stage in thymic development (Fig. 2B). Similarly, detection ofa compact network of TEC was also achieved using Abs specificfor cytokeratin 18 (data not shown). Thus, comparison of these twodistinct staining patterns indicates that only a subpopulation ofTEC expressAire-specific transcripts.

Induction of a prototypical thymic microenvironment is aprerequisite forAire expression

Organization and differentiation of thymic stromal cells into dis-tinct microenvironments with a typical cellular architecture aresubject to inductive signals provided by thymocytes (6, 11, 46).In particular, the comparison of two mouse strains, Tge26 andRAGnull mice, has been most informative in revealing the identityof the thymocyte subpopulation critical for the formation of a nor-mal thymic microenvironment: TN II and/or TN III thymocytesprovide signals that induce the three-dimensional organization ofTEC (11). Evidence for this comes from the fact that Tge26 mutantmice, which overexpress the human CD3e chain in high copy num-ber, display a complete arrest in early thymocyte development atthe transition of TN I to TN II of intrathymic T cell development,a point in time that corresponds to a developmental stage beforeE14.5 of normal thymic organogenesis. Consequently, the thymicprimordium ceases to develop a mature three-dimensional networkof TEC (13). In contrast, thymopoiesis in RAGnull mutant mice isblocked later during development, i.e., at the TN III stage, whichrelates to E15.5 in the thymic developmental in wild-type mice.The cortical stroma of RAGnull mice reveals a normal cellularcomposition and a typical architectural organization (13).

To test whether TEC expressAire transcripts before their for-mation of a three-dimensional architecture, thymi from Tge26 andRAGnull mice were analyzed using ISH. At E16 of development,the pattern ofAire expression in RAGnull thymi was similar to thatseen in age-matched thymi from wild-type mice (Fig. 2A, c). Incontrast, thymicAire transcripts could not be detected in E16Tge26 thymi despite the ample presence of TEC (Fig. 2A,e andf).This result was confirmed by RT-PCR analysis of fetal Tge26 andRAG thymic tissue (Fig. 2B). In Tge26 thymi, the absence ofAireexpression correlates with the lack of TN II/III thymocytes, whilethe presence ofAire transcripts in RAGnull thymi correlates withthe presence of thymocytes at these specific developmental stages.These results are consistent with the idea that TN II/III thymocytesinduce the expression ofAire in TEC, which correlates with theformation of a normal thymic architecture.

Aire is expressed in the medulla and at the corticomedullaryjunction

To document the spatial expression ofAire transcripts in a fullydifferentiated thymic microenvironment, sections of adult thymiwere analyzed by ISH (Fig. 3A).Aire expression was localized tothe medulla and the corticomedullary junction. While ISH withinthe medulla revealed a punctate pattern of staining, the pattern atthe corticomedullary junction suggested the formation of a morecontinuous arrangement ofAire-positive epithelial cells. In partic-ular, the slender extensions of the medulla that protrude into thecortex displayed several layers ofAire-positive cells, thus forminga concise boundary at the corticomedullary junction.

Several surface markers have been described that are expressedon medullary epithelial cells (36, 39, 47–50). The Ab designated29 recognizes a subpopulation of TEC thought to represent stromalcells bearing features of “activated” cells (36, 51). Otherwise char-acterized by Ia-specific Abs and lectin UEA-1 binding, this sub-population of 291 epithelial cells has been implicated in toleranceinduction (52–54). To detectAire expression in 291 epithelial

FIGURE 2. Thymic Aire expression at day 16 of embryonic develop-ment is localized to single cells and small aggregates dispersed throughoutthe organ.A, Thymic tissue from wild-type (aandb), RAGnull (c andd),and Tge26 (eandf) was analyzed by ISH using an antisense probe specificfor Aire (a, c, ande) andWhn (b, d, andf), respectively (original magni-fication of all panels,320).B, RT-PCR forAire (35 cycles) andGapdh(26cycles).

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cells, stromal cells were isolated by gentle digestion of thymicfragments. Phenotypically distinct subpopulations were separatedby flow cytometry using a combination of different markers:CD11c1MHCII1 dendritic cells; CD11c1291 dendritic cells; andCD11c2291 epithelial cells. cDNAs obtained from these differentstromal cell populations were amplified by semiquantitative PCRto detectAire expression. As seen in Fig. 3B, specific mRNA waspreferentially and strongly expressed in the population ofCD11c2291 epithelial cells when compared with all other stromalpopulations tested. Taken together, 291 epithelial cells in the me-dulla and at the corticomedullary junction are to a large extentresponsible forAire expression in adult thymic tissue.

To exclude the possibility that the observed absence ofAireexpression in Tge26 (Fig. 2A, e) is simply caused by the lack of291 epithelial cells, Tge26 thymi at E16 that lackAire expressionwere analyzed by immunohistology for reactivity with either a29-specific Ab or the UEA-1 lectin (Fig. 3Cand data not shown).Epithelial cells staining positively for either marker could be easily

detected in thymi of Tge26 mice. These results provide furtherevidence that the lack of inductive signals provided by early thy-mocytes at the TN II/III phenotype—and not the absence of theresponsive epithelial target cells—account for the deficit ofAireexpression in Tge26 thymi.

RelB is essential forAire expression

Medullary epithelial cells mediate negative selection of developingthymocytes (52) and contribute to late thymocyte maturation (55).For its architectural organization, the medulla is dependent on dis-tinct signals provided by postselection thymocytes (12). These sig-nals may likely be mediated by the transcription factor RelB (36)as mice deficient for RelB display a medullary thymic atrophy (56,57) and aberrant clonal deletion of autoreactive thymocytes (15).Both traits may be the direct consequence of a severe decrease ofAg 291, UEA-11 thymic medullary epithelial cells (36).

To test whetherAire expression can be detected in thymi de-void of 291 epithelial cells, mice homozygous for a null mutationof RelB were analyzed by ISH. The comparison with wild-typethymi demonstrated thatAire expression was completely absent inRelBnull thymi (Fig. 4A, a andb). Identical results were obtainedby use of RT-PCR (Fig. 4B). Moreover, ISH with aRAG-1-specificprobe revealed a dramatic change in the organization of the thymicmicroenvironment: in lieu of a centrally located medulla,

FIGURE 3. Aire expression is restricted to a subpopulation of TEC.A,ISH of adult thymic tissue localizesAire expression to cells at the corti-comedullary junction and in the medulla. Counter-staining with MethyleneGreen (original magnification,310). B, RT-PCR analysis forAire expres-sion by distinct subpopulations of thymic stroma cells: MHCII1 CD11c1

dendritic cells and Ag 29-positive stromal cells that are either CD11c2

(medullary epithelial cells) or CD11c1 (dendritic cells).C, Immunohistol-ogy for 29 Ag expression in thymic tissue of Tge26 and RAG-2null mutantmice (original magnification,340).

FIGURE 4. RelBnull mutant mice lack thymicAire expression.A, Thy-mic tissues from RelB-deficient (a) and wild-type mice (b) were analyzedby ISH for Aire expression (original magnification of all panels,310). B,RT-PCR forAire (35 cycles) andGapdh(30 cycles).

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RelBnull thymi displayed multiple small medullary foci dispersedthroughout the RAG1 cortex (data not shown). Thus, the absenceof Aire expression correlated with a homozygous deficiency forRelB and the disruption of a regular thymic architecture despite thepresence of medullary epithelial cells other than 291 stromal cells.

Correlation ofAire expression with thymic selection

A normal thymic microenvironment is a prerequisite for generat-ing a repertoire of T cells restricted to self-MHC molecules andtolerant to self-Ags. Therefore, we sought to correlateAire geneexpression with thymocyte selection asAire has been 1) implicatedin the maintenance of tolerance and 2) localized to the anatomicalsite usually associated with negative selection (58). For this aim, amurine model was analyzed where thymocytes express the3BBM74 TCR transgene on a RAG-2null background (35). Thy-mocytes bearing this TCR are positively selected by I-Ab but neg-atively selected by I-Abm12 (59).

Mice of either the positively selecting or the negatively selectinghaplotype were analyzed by ISH for thymicAire expression. In thepositively selecting (I-Ab1) thymi, Aire expression was limited toa few scattered cells located within the sparse thymic medulla (Fig.5A). In contrast, abundantAire expression at the corticomedullaryjunction and in the extended medulla was seen in thymi undergo-ing negative selection (I-Abm121, Fig. 5B). Under conditions ofnegative selection, abundantAire expression may relate to theTCR-mediated negative selection signals leading to apoptotic celldeath. Alternatively,Aire expression may result from any form ofthymocyte death including death by neglect. To address the rele-vance of death by neglect forAire expression, thymic tissue frommice deficient for the expression of both MHC class I and II mol-ecules was analyzed (Fig. 5C). In these MHCnull mutant mice,almost all thymocytes undergo cell death by neglect due to theabsence of TCR ligands and the ensuing lack of TCR-mediatedsurvival signals. Thymic tissue from MHCnull mice revealedAireexpression restricted to a few scattered cells within the medulla.This pattern was comparable to the expression noted in mice withonly positive thymic selection. Thus, thymicAire expression isdirectly correlated to the presence of apoptotic cell death due tonegative selection and appears to be independent of mechanismsinvolved in cell death by neglect.

DiscussionWe investigated the temporal and spatial pattern ofAire transcrip-tion to elucidate its role for regular immune functions. UsingNorthern blot analysis, abundantAire expression was localized tothe thymus and to a lesser degree to spleen and lymph nodes ofmice. Interestingly, differentAire transcripts were detected amongmRNA from thymus and secondary lymphoid organs, suggestingthe presence of splice variants. However, the pattern of intensityfor the three distinct bands did not appear to be unique for eachtissue tested (Fig. 1A). The detection ofAire variants in Northernblots is in keeping with previous PCR analysis demonstrating al-ternatively spliced transcripts in different tissues. In particular,variants with deletions of exon 6, 8, 10, and/or 11, respectively,have been detected among mRNA from thymic tissue (27, 60). Forexample, the complete deletion of exon 10 fails to cause a changein the reading frame but results in a shortened protein lacking asubstantial part of the proline-rich region; whereas the combinedloss of exons 10 and 11 introduces a stop codon in exon 13 anddeletes the second PHD zinc-finger, a motif present in a number ofchromatin-associated transcriptional regulators (61–63). It re-mains to be determined whether all of these transcripts encodefunctional proteins with distinct capacities.

Previously, little was known concerning the transcriptional reg-ulation of Aire. Our experiments now demonstrate thatAire ex-pression is principally restricted to the subpopulation of Ag 291

medullary TEC located within the medulla and at the corticomed-ullary junction. During thymic ontogeny, 291 stromal cells arefirst morphologically detected around E14 (36) and may initiallyrepresent a population of lineage-committed precursors of medul-lary epithelial cells. The firstAire-specific transcripts are detectedat E14 during thymic ontogeny (Fig. 1C), in parallel with the ap-pearance of TN II thymocytes. Two days later, a network of 291

medullary stromal cells is established concomitant with the emer-gence of more mature thymocytes and the abundant expression ofRelB (36, 64). In adult mice, the subpopulation of 291 TEC rep-resent a network of scattered medullary stromal cells with abun-dant and reticulated cell processes. These cells share many mem-brane markers known to be critical for Ag presentation to T cells(51) and have indeed been implicated in negative thymic selection(53, 54).

The cross-talk between developing thymocytes and epithelialcells is critical for the induction of a typical thymic microenviron-ment (6, 11, 46). However, the molecular nature of the signals that

FIGURE 5. Negative thymic selection correlates with abundant expres-sion of Aire. Thymic tissue from TCR-transgenic mice displaying exclu-sively either positive thymic selection (H-2b, A) or negative selection(H-2bm12, B) were analyzed by ISH and compared with thymic tissue frommice deficient for MHC I and II molecules (C) (original magnification ofall panels,310).

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mediate this stromal organization have yet to be defined.Aire is apossible epithelial target molecule for such a pivotal interaction asits expression correlates with a normal stromal organization. Thecomparison between RAGnull and Tge26 E16 embryos clearlydemonstrates thatAire expression does not occur in a cell-auton-omous manner but is induced after provision of activation signalsmediated by TN II/III thymocytes. However, the molecular natureof the signals responsible for the transcriptional regulation ofAireremain presently unknown. Thus,Aire constitutes to our knowl-edge the first epithelial gene product induced by developing earlythymocytes and associated with the correct establishment of a reg-ular thymic microenvironment.

RelB belongs to the NF-kB family of transcription factors thatare characterized by distinct structural features, interaction witheach other and regulation via the IkB inhibitor (65). RelB expres-sion is detected in thymic tissue during embryo development and,after differentiation into distinct stromal compartments, exclu-sively confined to the medulla (64). In RelB-deficient mice, theoutright absence of 291 thymic stromal cells and the decreasednumber of thymic dendritic cells has been correlated with an ir-regular medullary architecture (56, 57) and the loss of efficientnegative selection (15, 66). Therefore, our observation of a com-plete deficiency ofAire transcripts in RelBnull mice may be ex-plained by the absence of 291 medullary stromal cells. In contrast,it is unlikely that RelB transcription factors constitute a strict re-quirement forAire expression, which, in turn, determines the fate(e.g., growth and differentiation) of 291 epithelial cells becauseTge26 thymi harbor 291 epithelial cells but lackAire expression.Therefore, the conclusion can be drawn thatAire expression is notan unconditional requirement for the generation and maintenanceof 291 medullary epithelial cells. But it remains to be formallydetermined whetherAire transcription is directly RelB dependent.However, the lack of canonical RelB-binding sequences within thefirst 600 bp immediately 59 of theAire start site suggests thatAiretranscription is independent of this transcription factor (28).

The molecular properties of theAire gene product predict afunction as a regulator of gene transcription. In this capacity,Airemay regulate the architectural organization of the thymic micro-environment via transcriptional control of downstream targetgenes. This notion is supported by our data demonstrating the com-plete absence ofAire transcripts and the aberrant organization ofthe thymic microenvironment in Tge26 mutant mice. In these an-imals, an appropriate corticomedullary differentiation is missing,epithelial cells are organized in an abnormal two-dimensionalfashion, and the medullary foci are scattered throughout the entirethymus (11). Importantly, morphological similarities exist betweenTge26- and RelB-deficient mice, as both mutant mice display scat-tered and poorly separated areas of medullary epithelial cellsin lieu of a centrally located medulla (36, 56). Furthermore, inRelBnull mice, the lack of distinct stromal compartments is re-flected by the abnormal spatial distribution of areas where TCRgene recombination occurs (data not shown).

Signals transmitted via the TCR/peptide/MHC ligand interac-tion during later stages of thymocyte development affect the orga-nization of the medullary architecture (46, 67) and determine theselectional fate of developing thymocytes. To correlateAire ex-pression with thymic selection, RAG-2-deficient mice transgenicfor a TCR that is positively selected by I-Ab and negatively se-lected by I-Abm12were studied. BothAire expression and the sizeof the medullary compartment were strikingly different in the pos-itive (I-Ab) and negative (I-Abm12) selecting backgrounds. Inthymi undergoing positive selection,Aire-positive medullary epi-thelial cells were scattered throughout the organ as either singlecells or small densely packed aggregates occasionally located

close to the thymic capsule. In contrast, thymi undergoing onlynegative selection contained a large number ofAire-positive epi-thelial cells in the medulla and at the corticomedullary junction.Aire expression in epithelial cells is associated with TCR-mediatedprogrammed cell death because there are very fewAire-positivecells in MHCnull thymi, which lack TCR ligands and cause thy-mocyte death by neglect.

Taken together, the results observed in fetal and adult thymictissue support the hypothesis that thymicAire expression is regu-lated at two distinct steps duringthymic ontogeny. First,Aireexpression is induced in TEC by the presence of TN II/III thymocytesand confers to the formation of a functional microenvironment capa-ble to effect thymocyte selection. Second,Aire expression by epithe-lial cells is largely modulated at a later stage of thymic developmentwhen DP thymocytes are subjected to selection.

Although the affinity/avidity model of thymic selection predictsthat the interaction of the TCR with its peptide/MHC ligand de-termines the developmental fate of an immature thymocyte, theexact tissue requirements for negative selection are presently apoint of discussion. However, ample evidence exists that medul-lary epithelial cells are capable to delete or anergize self-reactiveT cells (52, 54, 68). Here we have demonstrated thatAire expres-sion 1) correlates with the correct structural organization of thethymic microenvironment, 2) is localized to cells and anatomicalsites known to effect negative selection, and 3) is modulated bythymocytes undergoing negative selection. Because all of thesefeatures are critical for appropriate thymic function, it is conceiv-able thatAire mutations as observed in APECED patients mayaffect thymic T cell selection and the formation of self-tolerance.

AcknowledgmentsWe thank Philippe Naquet (Marseille, France) and Werner Krenger (Basel,Switzerland) for helpful discussions and critical reading of the manuscript;Sandrine Guerin for cell sorting and PCR analysis of thymic stromal cells;Mathias Merkenschlager (London, U.K.) for thymic stromal cDNA; andBarbara Hausmann (Basel, Switzerland) for technical assistance. The BaselInstitute for Immunology was founded and is supported by F. Hoffmann,LaRoche & Company, Basel, Switzerland.

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