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A chief goal of T lymphocyte thymic development is to ensure the selec-tion of a T cell repertoire that is both immune competent and self toler-ant. T cells that are reactive to ubiquitous self antigens are deleted in thethymus. How these same central mechanisms might forestall autoimmu-nity against peripheral tissue–restricted antigens, such as insulin, was aperplexing mystery1. Mutations in Aire are responsible for autoimmunepolyenocrinopathy–candidiasis–ectodermal dystrophy (APECED), anautosomal recessive monogenic disorder characterized by organ-specificautoantibodies and multiorgan autoimmune destruction. Patients withAPECED suffer from a variety of clinical pathologies ranging frominsulin-dependent diabetes to hepatitis2–5. Analysis of Aire–/– mice hasshown that Aire is essential in central tolerance by initiating the ectopicexpression of peripheral organ–restricted antigens in a key thymicepithelial population, such that developing autoreactive T cells can bedeleted or otherwise restrained6. Understanding how Aire functionshelped to elucidate the unusual pattern of multiorgan autoimmunity inAPECED patients and shed light on a longstanding immunologicalconundrum: how central tolerance covers peripheral tissue-restrictedantigens.

Despite progress in understanding Aire and its downstream effects,the cellular signals responsible for its induction and regulation havenot been defined. Lymphotoxin is expressed prominently in the lym-phocyte compartment7,8. Its signaling through lymphotoxin-β recep-tor (LTβR), expressed mainly by stromal cells resistant to irradiation,is crucial for the induction of key cytokines, chemokines and otherfactors that organize and maintain the intricate microenvironmentwithin lymphoid tissues7,9. In the periphery, this same pathway orga-nizes tertiary lymphoid structures that sustain chronic inflammationin autoimmune models ranging from diabetes to colitis10–12. Mice

deficient in lymphotoxin-α (Lta–/–) and LTβR (Ltbr–/–), however,show a paradoxical and often overlooked pattern of peripheral organinfiltration, reminiscent of that reported in Aire–/– mice13,14. Given theestablished function of lymphotoxin in organizing lymphoid microen-vironments, and the prominent expression of both lymphotoxin andLTβR in the thymus7,15, we sought to determine the extent to whichlymphotoxin regulates the ‘education’ of thymocytes during develop-ment.

RESULTSCellular infiltration in Lta–/– and Ltbr–/– miceAnalysis of Lta–/– and Ltbr–/– mice 7–9 months of age by hematoxylinand eosin staining of formalin-fixed tissues showed considerableperivascular lymphocytic infiltratration relative to that of age-matched wild-type controls (Fig. 1). Infiltration was greatest in thelung, pancreas, liver and kidney (Fig. 1a). These findings echo the ini-tial descriptions of Lta–/– and Ltbr–/– phenotypes, which also includedinfiltration of the submandibular glands, the fatty tissue of the medi-astinum, the mesenterium and the cortex of the suprarenal glands13,14.The resemblance of this pattern of infiltration to that of Aire–/– mice,and to clinical presentations of APECED, indicates that the etiology isrooted in general autoimmunity.

Lymphocytic infiltrations in organs of Lta–/– and Ltbr–/– mice havereceived little attention since their initial description, and are for themost part dismissed as the passive overflow arising from the lack oflymph nodes in these mice. To confirm that this cellular infiltration isthe result of an active ongoing immune process, rather than passivedisplacement, we isolated infiltrating cells of the lung by dissectionand collagenase digestion, followed by separation. We then analyzed

1The Department of Pathology and Committee in Immunology, The University of Chicago, Chicago, Illinois 60637, USA. 2Institute of Medical Technology,Lenkkeilijankatu 6, University of Tampere, Tampere 33014, Finland. 3Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, San Diego,California 92121, USA. Correspondence should be addressed to Y.-X.F. (yfu@midway.uchicago.edu).

Published online 28 September 2003; doi:10.1038/ni982

Lymphotoxin pathway directs thymic Aire expressionRobert K Chin1, James C Lo1, Oliver Kim1, Sarah E Blink1, Peter A Christiansen1, Pärt Peterson2, Yang Wang1, Carl Ware3 & Yang-Xin Fu1

The autoimmune regulator Aire is a key mediator of central tolerance for peripherally restricted antigens. Its absence in humanpatients results in autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy. The cellular signals that regulate Aireexpression are undefined. We show here that lymphotoxin signaling is necessary for the expression of Aire and its downstreamtarget genes. The failure of Aire induction in the thymi of lymphotoxin-deficient and lymphotoxin-β receptor–deficient micecontributes to overt autoimmunity against self antigens normally protected by Aire. Conversely, stimulation of lymphotoxin-βreceptor by agonistic antibody leads to increased expression of Aire and tissue-restricted antigens in both intact thymi andcultured thymic epithelial cell line. These findings define the essential cross-talk between thymocytes and thymic stroma that is required for central tolerance.

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these cells by flow cytometry for key activation and memory pheno-type markers. Staining for the activation marker CD69 showed a 30-fold increase in the percentage of activated CD4 and CD8 cells inthe lungs of Lta–/– mice (Fig. 1b). The percentage of CD44+ memoryphenotype within the CD4+ T cells was increased 8-fold in Lta–/– lung,whereas the CD44+CD8+ population was increased 15-fold. Ltbr–/–

lung infiltrates showed a similar phenotype (data not shown). Thisincrease in the percentage of activated and memory cells, coupled withthe approximately threefold increase in the absolute number of totalinfiltrating lymphocytes, indicates the infiltration of the lung is anactive ongoing immune process.

Aire expression and the lymphotoxin pathwayTo determine whether lymphotoxin participates in the regulation ofAire, we used real-time PCR to measure Aire mRNA expression inwild-type, Lta–/– and Ltbr–/– thymi from age-matched mice. BothLta–/– and Ltbr–/– thymi showed a 75–80% reduction in their expres-sion of Aire (Fig. 2a). To determine whether this reduction was suf-ficiently severe to decrease downstream expression of targetperipheral tissue–specific antigens, we used real-time PCR forinsulin expression in these thymi. Insulin mRNA expression wasreduced in Lta–/– and Ltbr–/– thymi compared with that of wild-typethymi (Fig. 2b). These reductions in Aire and insulin expression areshared by Ltb–/– mice (data not shown). Thus, LTα expression in thethymus, signaling through LTβR, is required for Aire and ectopicperipheral antigen expression.

Thymic medullary epithelial cells are considered key mediators ofcentral tolerance, especially as they were shown to express manyperipherally restricted antigens1. Subsequent experiments involvingfetal thymus transplantation and immunofluorescence microscopyhave emphasized the importance of Aire expression in thesemedullary epithelial cells for the expression of peripheral antigensand central tolerance6,16. To determine whether the reduction in AiremRNA expression in Lta–/– and Ltbr–/– mice arises from a develop-mental failure to differentiate these key medullary epithelial cells, welooked for these cells in Lta–/– and Ltbr–/– thymi. Aire expression hasbeen colocalized with epithelial cells expressing the marker epithelial

cell adhesion molecule (Ep-CAM), also known as gp40, recognized bythe antibody G8.8 (refs. 1,6). Monoclonal antibody MTS10, whichstains subcapsular and medullary epithelial cells but not corticalepithelial cells, is another marker that colocalizes with Aire expres-sion16. Finally, the lectin UEA-1 defines a subpopulation of medullaryepithelial cells whose absence from thymi of mice deficient in RelB(Relb–/–), a critical NF-κB family member, is thought to contribute tothe autoimmunity that develops early in these mice17. In all cases, wecompared staining with that of appropriate isotype controls or sec-ondary reagents alone. We examined Lta–/– and Ltbr–/– thymi for thepresence of all three cell populations and found they were representedin numbers indistinguishable from those of wild-type (Fig. 2c,d anddata not shown). Furthermore, double staining of these markers withAire in Lta–/– and Ltbr–/– thymi showed a substantial reduction in Aireexpression within these key epithelial cell populations (Fig. 2d anddata not shown). Thus, although Lta–/– and Ltbr–/– thymi showedprofoundly reduced Aire expression, this defect is unlikely to reside atthe level of medullary epithelial cell differentiation.

Induction of Aire expression through LTβR signalingAlthough Aire expression in the thymus relies on the presence of LTαand LTβR signaling, the kinetics of this regulation remained unclear,as did the question of whether the prominent medullary epithelialpopulation in Lta–/– and Ltbr–/– thymi is indeed able to express Airewith the reconstitution of LTβR signaling. To address these questions,were treated young adult wild-type and Lta–/– mice briefly with ago-nistic LTβR monoclonal antibody (3C8)18. We injected mice intraperi-toneally with 3C8 or control rat immunoglobulin and collected thymiafter 8 or 24 h.

In wild-type mice treated with 3C8, we found a 7-fold induction inAire mRNA expression, and a 40-fold increase in ectopic insulinmRNA expression in thymic tissues at 8 h (Fig. 3a). At 24 h, expressionof both genes had returned to baseline. In similarly treated Lta–/– mice,by 8 h Aire mRNA expression increased by fivefold, and ectopic insulinmRNA expression increased by eightfold (Fig. 3b), demonstrating thatLta–/– medullary epithelial populations are competent for Aire induc-tion in response to LTβR signaling. Thus, not only does Aire and

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Figure 1 Infiltration and inflammation in peripheral organs of Lta–/– andLtbr–/– mice. (a) Substantial infiltration in lungs, livers, pancreatic islets and kidneys of Lta–/– and Ltbr–/– mice. Peripheral organs from B6 wild-type(WT), Lta–/– and Ltbr–/– mice 7–9 months of age were fixed in 10% bufferedformalin and paraffin mounted. Sections 4–5 µm in thickness were stainedwith hematoxylin and eosin. (b) T cells infiltrates in the lung show increasedexpression of activation and memory markers. Lung infiltrates from age-matched mice (12 weeks old) were isolated by collagenase digestion. T celland activation markers were analyzed by flow cytometry with markers forCD4, CD8, CD44 and CD69. Numbers by boxes indicate percentages ofboxed populations. Data are representative of three experiments.

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ectopic insulin gene expression respond to LTβR stimulation, but alsothe kinetics of the regulation of their expression are rapid, demon-strating the exquisite sensitivity by which Aire is controlled by the lym-photoxin pathway.

To demonstrate that the induction of Aire and insulin production bystimulation with agonistic LTβR antibody is specific to Aire and itsdownstream targets, we used as a negative control a marker constitu-tively expressed by medullary epithelial cells and not known to beinducible by Aire. Keratin subtypes have been used to characterizemedullary epithelial cell subsets. Specifically, the phenotypeK8–K18–K5+K14+ has been reported to represent the main populationof medullary epithelial cells. Cortical epithelial cells, along with theminor population of medullary epithelial cells have various other per-mutations of K8, K18 and K5, but are all negative for keratin 14 (K14;ref. 19). Thus, we used real-time PCR to examine K14 expression inour induction models. In all conditions of stimulation, we found

ample expression but negligible induction ofK14 (that is, never more than a twofoldchange; Fig. 3a,b,d).

Aire expression in response to in vivo stimu-lation indicates that Aire expression in the thy-mus, probably by medullary epithelial cells, isregulated by LTβR signaling. The in vivo stim-ulation experiment leaves open the possibility,however, that thymic epithelial cells may beresponding not directly to LTβR signaling butto a secondary downstream signal fromanother cell. To establish that thymic stromacan respond directly to LTβR signaling byexpressing Aire, we stimulated the cell line427.1 in vitro with 3C8. Line 427.1 was clonedfrom the thymic stroma of SV40-T transgenicmice, and expresses both cortical andmedullary epithelium markers20. Indeed,intrathymic injected of 427.1 rescues bothpositive and negative selection of thymocytesin selection-deficient hosts21. Initial stimula-tion of 427.1 cultures with 3C8 for 6 and 24 hyielded minimal responses (Fig. 3c). Access tothe Aire promoter is tightly restricted duringdifferentiation by DNA methylation and his-tone deacetylation22. It is possible thatimmortalization and in vitro culture of 427.1in the absence of crucial cytokines has led tothe suppression of its Aire promoter by thesemechanisms. To ensure that the Aire promoteris accessible, we treated 427.1 cultures with thedemethylating agent 5′-azacytidine or withtrichostatin A, a specific histone deacetylaseinhibitor, or with both. Stimulation with 3C8after trichostatin A treatment led to a 40-foldinduction of Aire expression relative to that ofan identically pretreated isotype control, asmeasured by real-time PCR (Fig. 3c). Pre-treatment with 5′-azacytidine resulted in a 26-fold induction of Aire and a 5-fold induc-tion of insulin after 6 h of stimulation with3C8, relative to that of identically pretreatedisotype control samples (Fig. 3d). After 24 h ofstimulation with 3C8, insulin expressionincreased to 15-fold above isotype control,

whereas Aire expression was increased 5-fold (Fig. 3d). To demonstratethat the induction of Aire and insulin expression in 427.1 was a specificresponse, we analyzed K14 expression concurrently in all experimentsand found it was relatively unchanged (Fig. 3d and data not shown).We stimulated the mouse thymoma line EL4 and the mouse sarcomaline MC57 and analyzed them using a protocol identical to that usedwith 427.1, but found they failed to express Aire in response (data notshown), demonstrating the specificity of this process. These findingsindicate that lymphotoxin may regulate Aire expression by thymicmedullary epithelial cells directly, through LTβR signaling.

Autoantibodies in Lta–/– and Ltbr–/– miceHaving found profound reductions in Aire expression in the thymi ofLta–/– and Ltbr–/– mice, and having demonstrated that this reductionin Aire is sufficient to effect a corresponding reduction in downstreamectopically expressed antigens, such as insulin, we investigated further

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Figure 2 Lymphotoxin and LTβR regulate Aire expression in the thymus. (a,b) Real-time PCR analysisof cDNA prepared from DNase-treated RNA extracted from whole thymus. Relative abundance of mRNAof Aire (a) and insulin (Ins1; b) in thymi from 4- to 6-week-old age-matched wild-type (WT), Lta–/– andLtbr–/– mice. Data are representative of three experiments. (c) Tissue immunofluorescence staining forthymic medullary epithelial cell makers G8.8 and UEA-I of thymic tissue sections from age-matchedwild-type (WT) and Lta–/– mice. (d) Immunofluorescence double staining for the thymic medullaryepithelial marker MTS10 and Aire.

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whether this reduction in ectopically expressed insulin is sufficient torepresent a breakdown in tolerance. We assayed for the production ofantibodies to insulin (anti-insulin) in Lta–/– and Ltbr–/– mice byenzyme-linked immunosorbent assay (ELISA). Consistent with abreakdown in tolerance to this representative self antigen, we foundsignificantly higher titers of anti-insulin in Lta–/– and Ltbr–/– mice(Fig. 4). As antibody production in response to simple monovalentprotein antigens requires T cell help, the increase in anti-insulin titersin this case could represent a lapse in T cell tolerance to self antigen.

Breakdowns in self tolerance and autoimmunity do not necessarilylead to full-blown autoimmune disease. To determine the extent towhich Lta–/– and Ltbr–/– mice have autoimmune disease, rather thanmere autoimmunity, we examined these mice for the presence of hall-mark features of established autoimmune disease, such as anti-DNAand anti-immunoglobulin G (IgG) rheumatoid factor. By ELISA, we

found notable increases in both DNA antibody and rheumatoid factorin 5- to 7-month-old age-matched Lta–/– and Ltbr–/– mice (Fig. 4).These findings indicate that by 5–7 months of age in mice, the autoim-munity that results from the defect in Aire-mediated central tolerancehas progressed to a condition of generalized and established autoim-mune disease.

Impaired tolerance in Lta–/– and Ltbr–/– miceAlthough the physiological function of lymphotoxin signaling inorganizing the unfolding of lymphoid structure development isessential, this pathway is also on occasion commandeered to effectthe coordinated destruction of peripheral organs in autoimmunedisease. In mouse models of insulin-dependent diabetes mellitus, forexample, short-term blockade of lymphotoxin by soluble receptorfusion protein prevented diabetes entirely, even after the appearance

of insulitis10,23. In two independent mousemodels of autoimmune colitis, interventionwith LTβR-immunoglobulin substantiallydelayed disease onset, reduced multifactorialdisease activity index values and dampenedleukocyte infiltration12. Similarly, in theabsence of lymphotoxin signaling in theperiphery, the autoimmunity in Lta–/– andLtbr–/– mice characterized above may be apoor reflection of its true severity.

To sidestep the constraint imposed by theabsence of lymphotoxin signaling in theperiphery, and to confirm that the autoim-munity in Lta–/– and Ltbr–/– mice representsa defect within the lymphoid compartment,we transferred 4 × 107 splenocytes from 7-month-old wild-type and Ltbr–/– mice intosublethally irradiated (350 rad) recombina-tion activation gene 1–deficient (Rag1–/–)mice. At 4 weeks after transfer, Rag1–/– recip-ients of Ltbr–/– donor cells showed moderateamounts of inflammatory infiltrationinvolving many organs. The lung showed amainly perivascular pattern of inflammationcomposed of lymphocytes. Similarly, theliver showed a perivascular lymphocyticinflammation concentrated in the portal

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Figure 4 Increased autoantibody production in Lta–/– and Ltbr–/– mice. Autoantibody production,including insulin antibody, DNA antibody and rheumatoid factor, in age-matched (5–7 months) wild-type (WT), Lta–/– and Ltbr–/– mice, measured by serum ELISA. Titers expressed in relative arbitraryunits. Statistical analysis by Student’s t-test.

Figure 3 Induction of Aire expression with agonistic anti-LTβR. (a,b) Wild-type and Lta–/– mice 4 weeks of age were injected intraperitoneally with agonisticanti-LTβR (3C8) or rat immunoglobulin isotype control. Then, 6 or 24 h later, RNA was collected from recipient thymi and real-time PCR was used toevaluate the relative abundance of Aire, Ins1 (insulin) and Krt1-14 (keratin 14) mRNA in wild-type (WT; a) and Lta–/– (b) recipients in response to 3C8stimulation. The ‘fold’ induction was calculated relative to that of rat immunoglobulin (Ig) isotype–treated thymi. (c) 427.1 cells were pretreated with thehistone deacetylase inhibitor trichostatin A (TSA) or the demethylating agent 5′-azacytidine (5′-Aza), or both in combination, before being induced with 3C8 or rat immunoglobulin isotype control. The ‘fold’ induction of mRNA expression of Aire was calculated relative to that of identically pretreated, ratimmunoglobulin–stimulated controls. (d) Relative abundance of Aire, Ins1 (insulin) and Krt1-14 (keratin 14) mRNA in 427.1 cells after 5′-azacytidinepretreatment and 3C8 induction of 6 or 24 hrs. The ‘fold’ induction was calculated relative to that of identically pretreated, rat immunoglobulin–stimulatedcontrols. In c and d: +, treatment; –, no treatment. Data are representative of one of three experiments.

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tracts. The inflammation in the skin involved the dermal region andwas composed mostly of lymphocytes, histiocytes and mast cells. Incontrast, Rag1–/– mice that received wild-type cells showed no evi-dence of inflammation (Fig. 5a). Serum titers of insulin antibodyand DNA antibody were also significantly increased in Rag1–/– recip-ients of Ltbr–/– donor splenocytes (Fig. 5b). Ltbr–/– donor spleno-cytes in Rag1–/– hosts were thus able to recapitulate the findings inLta–/– and Ltbr–/– mice, demonstrating both the lymphoid-autonomous nature of the autoimmunity in these mice and the mag-nitude of autoimmune destruction that can arise from a defect inlymphotoxin regulated central tolerance, notably the pathway bywhich Aire induces ectopic expression of tissue-specific antigen.

DISCUSSIONThe demonstration that Aire is key in promoting the ectopic expressionof a host of peripherally restricted antigens in the thymus provided acrucial insight in understanding how central tolerance covers ubiqui-tous and peripherally restricted antigens alike6. Although it remains tobe determined precisely in which mechanisms of central tolerance Aireis actually involved, Aire is important for the central deletion of high-avidity autoreactive T cells24. Little is known, however, of the mecha-nisms ‘upstream’ that regulate the expression and function of Aireitself. Our finding that Aire and peripherally restricted antigen expres-sion are deficient in Lta–/– and Ltbr–/– thymi emphasizes the impor-tance of the lymphotoxin pathway in regulating Aire expression. Ouradditional findings of an 80–90% reduction of ectopic type II collagenexpression in Lta–/– and Ltbr–/– thymi and a 20-fold induction inresponse to agonist LTβR antibody stimulation in vivo indicate thatlymphotoxin may regulate the ectopic expression of a spectrum of selfantigens linked to autoimmune disease. The deficiency in Lta–/– andLtbr–/– mice of this key component of central tolerance correspondedto the presence of hallmark features of general autoimmune disease inthese mice. Thus, these studies not only show regulation of Aire by the

lymphotoxin pathway but also demonstrate the autoimmunity that canresult from its disruption.

For members of the tumor necrosis factor family, the convergence ofsignals is more often the rule rather than the exception. The findingsdescribed above of severe defects in Aire expression in both Lta–/– andLtbr–/– mice indicate lymphotoxin and its β-receptor are prominent inthe pathway regulating Aire. The deficiency in Aire expression was notabsolute in Lta–/– and Ltbr–/– thymi. Given the promiscuity of receptor-ligand pairings in the tumor necrosis factor family, however, it is stillpossible that LTα and LTβR may be involved even in this marginalexpression, but by binding in each case with alternative partners.Unlike Lta–/– and Ltbr–/– thymi, Relb–/– thymi are completely devoid ofAire and ectopic insulin expression (refs. 16,25 and data not shown).Given evidence that LTβR signaling is mediated by the RelB-p52 NF-κBpathway, the more profound defect in Relb–/– thymi may represent apoint of convergence of signals that regulate Aire18,26,27. This may betoo simplistic, however, as thymic microarchitecture disorganization iswell described in Relb–/– mice, as is the absence of several medullaryepithelial markers associated with Aire production16,17,25. This is incontrast to Lta–/– thymi, in which stroma architecture, medullaryepithelial populations, and CD11c+ dendritic cell numbers and distrib-ution are unperturbed28. Thus, the Aire-deficient phenotype of Relb–/–

mice may alternatively represent a developmental block before theinvolvement of lymphotoxin signaling. Whether thymic architecturedisorganization represents a failure of maturation or survival is unclear,as is its connection to LTβR signaling. Nevertheless, this Aire deficit inRelb–/– mice may contribute to their subsequent T lymphocyte–depen-dent phenotype of aggressive autoimmune inflammation29.

That Aire expression in the thymus is carefully balanced by the inten-sity of LTβR signaling is also indicated by the induction of Aire expres-sion in response to LTβR stimulation by agonistic antibody.Experiments with tgε26 mice, whose overexpression of human CD3εresults in a block in thymocyte maturation at the triple-negative (TNII)

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Figure 5 Autoimmune inflammation in lymphotoxin-deficient mice is lymphocyte autonomous. Splenocytes from age-matched (5–7 months) wild-type (WT)and Ltbr–/– mice were adoptively transferred into (→) sublethally irradiated Rag1–/– recipients. (a) Lung, liver and skin were analyzed for evidence of cellularinfiltration 4 weeks after transfer. (b) At the same time as the experiments in a, recipient serum was analyzed by ELISA for the presence of autoantibodies,such as insulin and DNA. Statistical analysis by Student’s t-test.

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stage (CD3–CD4–CD8–CD44+CD25+), demonstrated that cross-talkbetween post-TNII thymocytes and the epithelium is essential for theexpression of Aire, although the nature and identity of this signal wasunknown25. Given the early and prominent expression of both lym-photoxin and LTβR throughout thymus development, our findings onAire regulation leave open the possibility that lymphotoxin signalingthrough LTβR may be one of the means by which thymocytes maintainthe infrastructure for their own ‘education’15. This hypothesis is espe-cially likely, given our findings that direct LTβR ligation on the thymicepithelial cell line 427.1 leads to its expression of Aire and peripherallyrestricted antigens. Lymphotoxin in developing thymocytes is promi-nently up-regulated after activation30. At the same time, Aire expres-sion is much more abundant in a peptide-independent, majorhistocompatibility complex (MHC)-specific, T cell receptor (TCR)-transgenic model of negative selection, in which selection is indepen-dent of Aire25. Analogous positive-selecting TCR-transgenic mice, orMHC-deficient mice, showed a substantial reduction in Aire25. Thisindicates that the initial baseline Aire expression induced by as-yet-undefined mechanisms may be further modulated by the up-regulationof lymphotoxin on autoreactive cells after their activation by cognateMHC-peptide complexes of Aire-expressing medullary epithelial cells.This interaction not only leads to the eventual deletion the autoreactivecell but also sustains Aire expression in thymic medullar epithelial cells.In this way, Aire expression may respond and adapt to the frequency ofautoreactive T cells encountered, as seen in the TCR-transgenic models,to ensure that central tolerance is both efficient and comprehensive.

The ability of lymphotoxin to induce ectopic expression of periph-erally restricted antigens permits both dissection of the Aire pathwayand the discovery of possible Aire family members. Indeed, the abilityto control the induction of Aire can be a valuable tool for dissecting theregulatory pathway terminating in Aire activation, and the subsequentcascade for the expression of peripheral tissue antigens. Possible path-ways parallel to Aire similarly subordinate to LTβR signaling may bediscovered by induction and screening. Understanding the regulationof Aire and parallel pathways responsible for central tolerance mayprovide new diagnostic possibilities and therapies in the treatment ofautoimmune disease.

METHODSMice. Lta–/– and Ltbr–/– mice were backcrossed to C57BL/6 mice and main-tained in specific-pathogen-free conditions as described14,31. C57BL/6 andRag1–/– mice were purchased from the Jackson Laboratory. Ltbr–/– mice were agift from K. Pfeffer. Animal care and use were in accordance with institutionaland National Institutes of Health guidelines.

Histology. Lung tissues for histologic examination were fixed in 10% bufferedformalin and embedded in paraffin. Sections 4–5 µm in thickness were cutfrom the paraffin blocks and stained with both hematoxylin and eosin. All sec-tions were then examined qualitatively by a pathologist ‘blinded’ to sampleidentity.

Lung leukocyte isolation and flow cytometry. Lung tissues were digested threetimes by being shaken for 30 min at 37 °C in RPMI 1640 medium (LifeTechnologies) containing collagenase VIII (1 mg/ml) and FBS (2%). Lung cellswere passed through Nytex filter, and samples were depleted of red blood cellswith ammonium chloride red blood cell lysis buffer. After samples werewashed, the total number of live cells was determined by trypan blue staining.All antibodies for flow cytometry were purchased from BD Pharmingen.Bronchoalveolar lavage and lung cells were stained for 30 min on ice in PBScontaining 1% FBS plus 0.01% NaN3, and were analyzed by flow cytometry ona FACScan apparatus (BD Biosciences).

Immunofluorescence staining. Thymi from age-matched wild-type, Lta–/– andLtbr–/– mice were embedded in optimal cutting temperature compound

(Sakura) and were ‘snap frozen’. Sections 8–10 µm in thickness were cut fromthe frozen blocks, fixed in acetone, rehydrated in PBS plus 0.1% saponin, andblocked at room temperature for 1 h with 5% goat serum in PBS. AntibodiesMTS10 (BD Bioscience), G8.8 (BD Bioscience), UEA-1-biotin (Sigma) andpolyclonal rabbit anti-Aire were incubated with tissue sections at 4 °Covernight and were detected with appropriate secondary fluorescence reagents.

Real-time PCR. Real-time PCR used cDNA prepared from DNase-treated RNAextracted from whole thymus. For Aire, the following primers were used: forward,5′-CCAGTGAGCCCCAGGTTAAC-3′; reverse, 5′-GACAGCCGTCACAACA-GATGA-3′; probe, 5′-FAM (6-carboxyfluorescein)-TCACCTCCGTCGTGGCA-CACG-TAMRA (N,N,N′,N′-tetramethyl-6-carboxyrhodamine)-3′. For insulin,the following primers were used: forward, 5′-CTTCAGACCTTGGCGTTGGA-3′;reverse, 5′-ATGCTGGTGCAGCACTGATC-3′; probe, 5′-FAM-CCCGGCA-GAAGCGTGGCATT-TAMRA-3′. For keratin 14, the following primers wereused: forward, 5′-TGGGTGGAGACGTCAATGTG-3′; reverse, 5′-ATCTCGTTCAGGATGCGGC-3′; probe, 5′-FAM-ACGCCGCCCCTGGTGTGG-TAMRA-3′. For normalization, primers for glyceraldehyde phosphodehydrogenase(GAPDH) were used: forward, 5′-TTCACCACCATGGAGAAGGC-3′; reverse, 5′-GGCATGGACTGTGGTCATGA-3′; probe, 5′-TET (tetra-chloro-6-carboxyfluo-roscein)-TGCATCCTGCACCACCAACTGCTTAG-TAMRA-3′. Duplex real-timePCR reactions, with GAPDH as an internal control, had a final volume of 25 µlwith 900 nM forward and reverse primers and 200 nM probe, plus 2X TaqmanMaster Mix (Applied Biosystems) containing AmpliTaq Gold polymerase.Reactions were run on the Cepheid SmartCycler. We analyzed expression of genesencoding Aire, insulin, keratin 14 and GAPDH with a concurrently run standardcurve, then normalized the results to GAPDH levels in each sample. The standardcurves for Aire, insulin, keratin 14 and GAPDH had R2 values >0.98.

In vitro stimulation of the 427.1 cell line. The thymic epithelial cell lines 427.1 (a gift from P. Ashton-Rickert, University of Chicago) and MC57G (a gift from P. Ohashi, Ontario Cancer Institute) were maintained in DMEM supplementedwith 10% FBS, 2 mM L-glutamine, 50 µM 2-mercaptoethanol, sodium pyruvateand 1 mM HEPES buffer. The mouse thymoma line EL4 was obtained fromAmerican Type Culture Collection, and was maintained in RPMI medium sup-plemented with 10% FBS, 2 mM L-glutamine and 1 mM HEPES buffer. TheDNA methyltransferase inhibitor 5′-azacytidine (Sigma) was added to culturesat a final concentration of 0.2 µM for 72 h before 3C8 stimulation (0.5 µg/ml).The specific histone deacetylase inhibitor trichostatin A (Sigma) was added tocultures at a final concentration of 100 nM for 24 h before 3C8 stimulation (0.5 µg/ml). Messenger RNA was collected with the MACS mRNA Isolation Kit(Miltenyi Biotec). Real-time PCR analysis was done as described above.

In vivo stimulation of wild-type and Lta–/– mice. Wild-type and Lta–/– micereceived intraperitoneal injections of 50 µg agonistic LTβR antibody or ratimmunoglobulin isotype control in PBS. RNA from thymi was collected at 6 and 24 h after injection. Real-time PCR used cDNA prepared from DNase-treated RNA extracted from whole thymus.

Autoantibody ELISA. For detection of autoantibodies to DNA, ELISA plates(Dynex Technologies) were coated with DNA from herring sperm (250 µg/ml;Sigma). Plates were washed with water and blocked with PBS plus 0.1% BSA.Serum samples were diluted at various concentrations and bound antibodieswere detected with alkaline phosphatase-conjugated goat anti-mouse IgG(Southern Biotechnology Associates). Absorbance was measured at 405 nm witha spectrophotometer (Molecular Devices). For the detection of insulin antibody,ELISA plates were coated with insulin at a concentration of 2 U/ml, and the samedetecting antibody (alkaline phosphatase-conjugated goat anti-mouse IgG) oralkaline phosphatase-conjugated goat anti-mouse IgM was used. For the detec-tion of rheumatoid factor, a panel of purified mouse IgG1, IgG2a, IgG2b andIgG3 was used for coating, and alkaline phosphatase-conjugated goat anti-mouseIgM was used as a detecting antibody (Southern Biotechnology Associates). Forstatistical analysis, P values were determined by Student’s two-tailed t-test.

Splenocyte transfer. Splenocytes were obtained by mechanical disruption ofthe spleens of 5- to 7-month-old age-matched Ltbr–/– and wild-type controlmice. Samples were depleted of red blood cells with ammonium chloride redblood cell lysis buffer. Cells (4 × 107) were injected retro-orbitally into sub-

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lethally irradiated (350 rads) Rag1–/– mice. Mice were killed 4 weeks after trans-fer and analyzed for autoantibodies and tissue infiltration by histology.

ACKNOWLEDGMENTSSupported in part by National Institutes of Health grants (HD-37104, AI 33068and DK-58891). R.K.C. and J.C.L. were supported by a Medical Scientist NationalResearch Service Award (5 T32 GM07281).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 8 July; accepted 28 August 2003Published online at http://www.nature.com/natureimmunology/

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