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  • 1. The Journal of Immunology Lt r Signaling Does Not Regulate Aire-Dependent Transcripts in Medullary Thymic Epithelial Cells1 Vera C. Martins, Thomas Boehm, and Conrad C. Bleul2 Thymic medullary epithelial cells (mTECs) play a major role in central tolerance induction by expressing tissue-specific Ags (TSAs). The expression of a subset of TSAs in mTECs is under the control of Aire (autoimmune regulator). Humans defective for AIRE develop a syndrome characterized by autoimmune disease in several endocrine glands. Aire has been proposed to be regulated by lymphotoxin receptor (Lt r) signaling and there is evidence that, additionally, Aire-independent transcripts may be regulated by this pathway. Given the potential clinical importance of Aire regulation in mTECs for the control of autoimmunity, we investigated the relation between Lt r signaling and TSA expression by whole genome transcriptome analysis. In this study, we show that the absence of Lt r has no effect on the expression of Aire and Aire-dependent TSAs. Also, the lack of Lt r signaling does not disturb regulatory T cells or the distribution of dendritic cells in the thymus. However, mTECs in Lt r-deficient mice show an aberrant distribution within the thymic medulla with disruption of their three-dimensional architecture. This is predicted to impair the interaction between mTECs and thymocytes as shown by the reduced surface uptake of MHCII by mature thy- mocytes in Lt r-deficient mice. We propose that the physiological medullary architecture ensures negative-selection by supporting lympho-epithelial interaction through a large epithelial cell surface distributed evenly across the medulla. The Journal of Im- munology, 2008, 181: 400 – 407. T he adaptive immune system relies on a diverse repertoire velop autoimmune polyendocrine syndrome-type 1, also known as of Ag receptors, each generated by a random process of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. somatic gene rearrangement. The randomness of the pro- This is a syndrome characterized by autoimmune processes in para- cess, however, poses the potential problem of generating Ag thyroid and adrenal glands associated with mucocutaneous candidia- receptors that are self-reactive. Cells expressing self-reactive re- sis (5). Mice deficient for Aire show reduced expression of TSAs in ceptors must be negatively selected or suppressed to preserve self- mTECs and develop autoimmunity against several endocrine organs, tolerance. The thymus is the lymphoid organ responsible for the resembling the phenotype of the human disease (4). generation of the diverse and self-tolerant repertoire of T cells. Treating autoimmune diseases that may be caused by subopti- After positive selection in the thymus cortex, developing thymo- mal deletion of self-reactive thymocytes in the thymus, i.e., a de- cytes reach the thymic medulla, interact with APCs, and are neg- fect in central tolerance induction, in man is a major challenge in atively selected if the TCR-peptide-MHC interaction occurs with the clinic. In respect to central tolerance induction, it has been very high avidity. High affinity interactions below a given thresh- proposed that signaling through the lymphotoxin receptor (Lt r) old have been proposed to direct the cells to a regulatory T cell up-regulates the expression of Aire and Aire-dependent TSAs (6). (Treg)3 fate (reviewed in Ref. 1). If this hypothesis proved to be true, treatment with agonistic Abs Medullary thymic epithelial cells (mTECs) are a fundamental com- specific for LT R might be a clinically important approach that ponent of the thymic stroma. They play a unique role by expressing could lead to the mitigation of autoimmune diseases. However, our tissue specific Ags (TSAs) (reviewed in Ref. 2) and contribute to own experiments have shown no evidence for a direct regulation of negative selection of self-reactive thymocytes through direct presen- Aire by Lt r signaling (7). tation and cross-presentation by dendritic cells (DCs) (3). The only known regulator for the promiscuous expression of TSAs in mTECs Lt r-deficient mice have an abnormal thymic medulla, produce is Aire (autoimmune regulator) (4). Humans defective for AIRE de- autoantibodies against several organs (6, 7), and develop lympho- cytic infiltrations (6, 8). Furthermore, these mice are also defective for the development of secondary lymphoid organs (8). Therefore, Max-Planck-Institute-for-Immunobiology, Department for Developmental Immunol- it is not clear whether the accumulation of lymphocytes in periph- ogy, Freiburg, Germany eral tissues results from an aberrant distribution caused by the Received for publication August 17, 2007. Accepted for publication April 30, 2008. secondary lymphoid organ defect or arises from impaired central The costs of publication of this article were defrayed in part by the payment of page tolerance induction in the thymus. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. In this study, we address four open questions related to the 1 V.C.M. was supported by a fellowship from Fundacao para a Ciencia e Tecnologia, ¸˜ ˆ importance of Lt r signaling for the establishment of central Portugal. tolerance in the thymus: first, is the autoimmune phenotype in 2 Address correspondence and reprint requests to Dr. Conrad C. Bleul, Max-Planck- Lt r-deficient mice caused by a defect in central tolerance? Institute-for-Immunobiology, Stuebeweg 51, Freiburg 79108, Germany. E-mail address: Second, what transcriptional changes occur in mTECs in the 3 absence of Lt r signaling and are these similar to those seen in Abbreviations used in this paper: Treg, regulatory T cell; TSA, tissue-specific Ag; mTEC, medullary thymic epithelial cell; Lt r, lymphotoxin receptor; DC, dendritic Aire-deficient mice? Third, what cellular component(s) is (are) cell; wt, wild type; KO, knockout; Hprt, hypoxanthine-guanine phosphoribosyltrans- associated with the breakdown of central tolerance in the Lt r- ferase; qPCR, quantitative PCR; Teff, T effector cell; RTE, recent thymic emigrant. deficient mice? Finally, is the interaction between lymphocytes Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 and stroma altered in the absence of Lt r signaling?
  • 2. The Journal of Immunology 401 Materials and Methods deficient/wt analysis, differences above a 2-fold change in expression lev- Mice els were considered. Gene symbols were used to identify common up- or down-regulated transcripts in Aire-deficient and Lt r-deficient mTECs. C57BL/6, Lt r-deficient (knockout, KO), and nude (C57BL/6 background; TSA status was attributed as follows: all probe set identifiers of transcripts The Jackson Laboratory) mice were kept under specific pathogen-free con- with an at least 2-fold change in expression levels were loaded into Af- ditions in the mouse facility of the Max Planck Institute for Immunobiol- fymetrix Netaffx Analysis Center ( ogy. Lt r-deficient mice were backcrossed to C57BL/6 at least six gener- index.affx) and individually analyzed for their expression profile in the ations and genotyped as described (8). Experimentation and animal care Unigene database (Unigene ID Build 167, 28 Oct 2007) using the direct was in accordance with the guidelines of the Max-Planck-Institute for link from the Affymetrix Netaffx Analysis Center. Genes were considered Immunobiology. tissue specific if they were expressed in five or less tissues (from a total of 47) in the UniGene database. “Embryonic tissue” was only considered if Autoantibody production by Western blotting this was the only tissue where the transcript was detected. Transcripts that had been removed from the Unigene database were not considered for Western blotting was conducted according to standard protocols. In brief, analysis (52 transcripts among the decreases and 45 transcripts among the whole organ extracts from liver, lung, stomach, and pancreas from Rag- increases in LT RKO; 9 transcripts among the decreases and 7 transcripts 1-deficient mice were prepared in 50 mM Tris/1% NP40/0.5% deoxylic among the increases in AireKO). acid/0.1% SDS/137.5 mM NaCl/10% Glycerin/1 mM EDTA lysis buffer supplemented with a mixture of proteinase inhibitors (complete, mini; Quantitative real-time PCR Roche) and run in a 10% acrylamide gel. Following electrophoresis, pro- teins were blotted onto nitrocellulose membranes that were subsequently Quantitative PCR (qPCR) was conducted with a LightCycler System (Roche) cut in strips. Each strip was incubated with diluted serum from one mouse using a LightCycler-Fast-Start DNA Master SYBR Green I kit. Primers were and developed by incubation with goat anti-mouse IgG-HRP (Dako) fol- designed to span intron-exon boundaries and yield 150 –200 bp fragments. The lowed by ECL detection (Amersham Biosciences). specificity of products was assessed by melting curve analysis and gel elec- trophoresis. For quantitative analysis, the Fit Points Method of the Light Cy- Thymus transplants and analysis for lymphocytic infiltrates cler Software version 3 was used. Results were normalized to expression levels of hypoxanthine-guanine phosphoribosyltransferase (Hprt) and the relative Newborn Lt r-deficient or wild-type (wt) thymi were transplanted under amount of each transcript was determined between wt and Lt r-deficient the kidney capsule of adult (5–9 wk of age) nude mice (C57BL/6 back- mTECs. Primers used were: Hprt: 5 -ggttaagcagtacagcccca-3 ; 5 -caagggcat ground) and hosts were sacrificed 14 –17 wk later. Testis/ovary, salivary atccaacaacaaact-3 ; Aire: 5 -gtagcagcctaaagcctgtg-3 ; 5 -acacggcacactcatcc gland, stomach, liver, adrenal gland, and pancreas were dissected out and tcg-3 ; Insulin2: 5 -ccaccagccctaagtgatcc-3 ; 5 -tagagagcctccaccaggtg-3 ; Sal- cryosectioned with every fifth or sixth section collected. Sections were ivary protein1: 5 -aaatgataacagtaccgggg-3 ; 5 -tgcaaactcatccacgttgt-3 ; Rgs13: fixed in 75% acetone/25% methanol and stained with H&E according to 5 -tggagcacagtgatgagaac-3 ; 5 -gatgatagcttcccgtgttg-3 ; N cadherin: 5 -agatac standard protocols. tgtggagcctgatg-3 ; 5 -gttgtcagcagatttaaggc-3 ; Keratin77: 5 -agcatcatcgatgcc Flow cytometry, cell sorting, and purification of mTECs gtacg-3 ; 5 -tctggatgttacggttgagc-3 ; Keratin2– 6a: 5 -gatcgaccacgttaagaa gc-3 ; 5 -atgtcctgtttggccttctg-3 ; Collagen3 1: 5 -cccagcaaaacaaaaaccac-3 ; Thymi of five to six mice (4 – 6 wk old) were finely minced and stirred in 5 -agcatttgacctgtattggg-3 ; and Ccl11: 5 -tcacggtcacttccttcac-3 ; 5 -ctttgccca RPMI 1640/20 mM HEPES/2% FCS medium for 10 min at room temper- acctggtcttg-3 . Comparing array hybridization and qPCR experiments, a linear ature, followed by three incubations of 10 min at 30°C in RPMI 1640/0.2 relationship exists between the fold changes (FC): for up-regulated genes mg/ml collagenase IV/20 mM HEPES/2% FCS/25 g/ml DNase and su- FC(array) 0.93 FC(qPCR) 3.25; for down-regulated genes: FC(ar- pernatant of all these fractions was discarded. Three incubations of 25 min ray) 0.15 FC(qPCR) 1.75. This indicates that, in comparison to the qPCR at 30°C were performed in the same medium supplemented with dispase, results, the degree of down-regulation is underestimated by the array. followed by two incubations of 15 min at 37°C with 0.05% Trypsin/0.5 mM EDTA (pH 8.0)/0.3% BSA/25 g/ml DNase. Cells from the latter five Immunohistochemistry fractions were collected, stained, and sorted as CD45-negative, G8.8-pos- The 8 m cryosections from Lt r-deficient and wt thymus (4 wk old) were itive, and CDR1-negative. The forward- and side-scatter gate was set to fixed (75% acetone/25% methanol) and incubated with Ab, followed by exclude thymocytes. For analysis and cell sorting of thymocytes and secondary Ab and/or streptavidin detection when required according to splenocytes, single-cell suspensions were prepared and stained with CD4- standard protocols. Abs were diluted in 1 PBS/0.5% BSA and slides were FITC (GK1.5) or -allophycocyanin (L3T4), CD8-PE-Cy7 or -FITC (53- mounted with Fluoromount-G (Southern Biotechnology Associates). The 6.7), CD25-PE (PC61.5), CD62L-allophycocyanin (MEL-14), CD69-bi- following reagents and Abs were used: Ulex europaeus agglutinin1 otin (H1.2F3), and MHCII-PE (AF6 –120.1) purchased from eBioscience (UEA-1; Vector Laboratories), Troma-1 (anti-cytokeratin 8; a gift from Dr. or BD Pharmingen and Foxp3-allophycocyanin (FJK-16s, staining kit) R. Kemler, Max Planck Institute for Immunobiology, Freiburg, Germany), from eBioscience. anti-cytokeratin 5 (MK5; Covance), MHCII I-Ab/d (BD Biosciences), Gene expression analysis MIDC-8 (Serotec), anti-mouse Aire (kindly provided by Dr. H. Scott, Walter and Elizabeth Hall Institute, Melbourne, Australia), donkey anti-rat A total of 10 g total RNA from Lt r-deficient and C57BL/6 mTECs was IgG Cy3 (Dianova), goat anti-rat IgG Alexa488, and streptavidin Alexa647 preamplified and biotinylated in two independent experiments. In brief, (Molecular Probes). RNA was extracted using TRI reagent (Sigma-Aldrich) according to the manufacturers’ recommendations. RNA quantification and quality was Proliferation/Treg assay measured in a Bioanalyzer machine (Agilent). Total mRNA was reverse- transcribed using an oligo(dT)-T7 promoter primer for the first strand syn- A total of 50,000 CD4 CD25 (effector T cells, Teff) Lt r-deficient or wt thesis. The second strand was synthesized and the cDNA was purified by splenocytes were sorted in triplicates and incubated for 72 h at 37°C in phenol-chloroform extraction. GeneChip IVT labeling kit (Affymetrix) was RPMI 1640/10% FCS/2 mM L-glutamine/2 mM HEPES/1 mM sodium used to synthesize the biotin-labeled cRNA, which was then purified and pyruvate/2-ME in the presence of 1 g/ml anti-CD3 (3C11) Ab and 50,000 quantified. Biotin-labeled cRNA was hybridized to Affymetrix GeneChip T cell depleted and irradiated (300 rad) wt splenocytes. Where indicated, Mouse Genome 430 2.0 Arrays for each genetic background according to CD4 CD25 (Treg) Lt r-deficient or wt splenocytes were sorted into the the manufacturers’ protocol. same wells in the indicated proportions. Tritium (1.5 Ci/ml) was added to the cultures for the last 6 h of incubation. Tritium incorporation was mea- Comparison of the Lt r-deficient/wt and Aire-deficient/wt sured in a Trace 96 (Berthold) automatic filter counting system. transcriptomes and TSA identification Results Raw data from Aire-deficient mTEC transcriptome analyses were down- Lt r signaling is required for central tolerance induction loaded from Raw data were loaded in Gene- Spring software (Silicon Genetics) for normalization and Aire-deficient vs Lt r-deficient mice present with signs of autoimmunity, namely wt data were analyzed separately from Lt r-deficient vs the corresponding lymphocytic infiltrations in several organs (6, 8) and autoantibody wt data. The U74Av2 chip (Affymetrix) used for the Aire-deficient tran- scriptome analysis samples 12,000 genes that are represented within the production against liver, lung, pancreas, salivary gland, and stom- 39,000 genes (Mouse Genome 430 2.0 chips; Affymetrix) sampled by the ach (6, 7). Autoantibodies in Lt r-deficient mice are directed Lt r-deficient transcriptome analysis. For the Lt r-deficient/wt vs Aire- against several distinct tissue Ags as shown by Western blotting
  • 3. 402 Lt r DOES NOT REGULATE Aire analysis using liver extracts (Fig. 1A). The patterns of autoanti- body production were considerably diverse among individual mice, indicating that the absence of Lt r signaling leads to a set of self-reactive lymphocytes that differs between individuals. To understand the basis for the loss of immunologic tolerance in Lt r-deficient mice, we first addressed whether this was due to a defect in peripheral or central tolerance induction mechanisms. Lt r-deficient mice have a serious defect in the development of the secondary lymphoid organs (8). These mice lack lymph nodes and Peyer’s patches and have an abnormal organization of the spleen (8). Moreover, Lt r-deficient mice show an aberrant structure in the three-dimensional arrangement of mTECs (7) that play an im- portant role in central tolerance induction (2). Because both central and peripheral lymphoid organs are affected, organ infiltrates could be caused by a lack of central tolerance due to the defect in the thymus or be a consequence of aberrant distribution of lympho- cytes in a mouse without lymph nodes. To address this problem, we transplanted wt or Lt r-deficient newborn thymi (both C57BL/6 background) under the kidney capsule of C57BL/6 nude recipients and analyzed them 14 –17 wk later for signs of autoim- munity. Ovary/testis, salivary gland, stomach, liver, adrenal gland, and pancreas were harvested, sectioned, stained, and analyzed for the presence of mononuclear infiltrations. Recipients receiving thymi from Lt r-deficient donors developed lymphocytic infil- trates in liver, pancreas, and salivary gland (Fig. 1, B and C), while only one of the recipients from a wt thymus developed infiltrations in the liver (Fig. 1B). These findings indicate that central tolerance induction is impaired in Lt r-deficient mice. Lt r signaling does not control Aire expression In agreement with a central tolerance defect in Lt r-deficient mice, previous reports suggested that Lt r signaling positively regulated the expression of Aire and of both Aire-dependent as well as Aire- independent TSAs (6, 9). Furthermore, administration of an ago- nistic anti-Lt r mAb was shown to increase TSA expression (6, 9). These findings opened up the clinically relevant possibility of treating autoimmune diseases by increasing TSA expression. To investigate the validity of this hypothesis in more detail we puri- fied wt and Lt r-deficient mTECs by cell sorting (Fig. 2A) and performed a whole transcriptome analysis. The microarray analy- sis for the wt vs Lt r-deficient comparison covered a total of 39,000 transcripts, including the 12,000 transcripts surveyed in the earlier Aire-deficient transcriptome analysis (4). If Lt r signaling positively regulated the expression of Aire and its target genes, one would expect overlap between the genes differentially regulated in Aire-deficient and Lt r-deficient mTECs. However, this is not the case. Because of the use of different arrays, the datasets from Aire- deficient vs wt (4) and Lt r-deficient vs wt (this paper) could not be FIGURE 1. Autoimmunity in Lt r-deficient mice results from a defect directly compared. Rather, we generated lists of differentially regu- in central tolerance. A, Western blot analysis with Rag2KO whole liver lated genes, determined the FC for each gene and then compared these extract stained with sera from Lt r-deficient (KO, lanes 1–13) and wt lists. To avoid missing TSAs, which are expected to be expressed at (lanes 14 –17) mice aged between 3 and 12 mo. B and C, Newborn Lt r- deficient (LT RKO) and wt thymi (one per mouse) were transplanted un- very low levels, we did not filter the results with respect to expression der the kidney capsule of nude (C57BL/6 background) mice, and recipients levels. For Aire-deficient mTECs, 256 genes were down-regulated were analyzed for the presence of infiltrates 14 –17 wk later by examination and 432 genes up-regulated at least 2-fold among 12,000 investigated of H&E-stained sections. B, Each circle depicts the spectrum of disease in transcripts (Supplemental Tables III and IV)4; using the same crite- individual transplant recipients, and black regions represent the presence of ria, 553 down-regulated genes and 526 up-regulated genes were mononuclear infiltrates in the corresponding organ. C, Representative detected in the Lt r-deficient mTEC comparisons among 39,000 H&E-stained sections from thymus transplant recipients showing extensive investigated transcripts (Table I and Supplemental Tables I and II). lymphoid infiltrates only in mice that received Lt r-deficient thymi (KO, Interestingly, only minimal overlap was found among these sets of right), compared with recipients of wt thymi (left). genes (Table II); only 18 down-regulated and 6 up-regulated genes were found to be in common between the Aire-deficient and the Lt r-deficient mTEC comparisons. Six of these down-regulated transcripts and one of the up-regulated were classified as tissue 4 The online version of this article contains supplemental material. specific. Overall, the percentage of genes differentially expressed
  • 4. The Journal of Immunology 403 Lt r signaling because the transcriptome comparisons show only minimal overlap. Lt r-deficient mTECs have an architectural defect Importantly, the transcriptome comparison of Lt r-deficient to wt mTECs revealed reduced expression of several transcripts in- volved in epithelial cell polarization, membrane compartmental- ization and cell-cell contact formation. Examples are Psd93 (discs large homologue 2, involved in the formation of the immunolog- ical synapse) (10), Advillin, Filaggrin, Keratins 10, 77, and 222, Myosin, N-cadherin (11), Neurexin I, and Integrin- 6 among oth- ers (data not shown and Fig. 2B). The differential expression of several transcripts detected by the array analysis was confirmed by qPCR (Fig. 2B). Because a considerable number of differentially expressed tran- scripts pointed at a deregulation of structural components, we an- alyzed the medullary thymus architecture of Lt r-deficient and wt mice by multicolor immunohistochemistry and confocal micros- copy visualizing mTECs to an extent that was not possible previ- ously (Fig. 3, A and B). The architectural defect in Lt r-deficient mTECs affects the entire medullary epithelium (Fig. 3A). Instead of being spread throughout the medulla, mTECs clump and the three-dimensional network is disrupted, leaving large medullary areas devoid of mTECs (Fig. 3A). UEA-1 and MHCIIhigh cells, which were shown to express high levels of TSAs (12), are par- ticularly affected, and one can observe that they are mainly posi- tioned at the periphery of the LT RKO medulla instead of cover- ing the whole area (Fig. 3, A and B). The general organization of the thymus into cortex and medulla is, however, preserved because the distribution of thymocytes is maintained, with CD4 CD8 double-positive thymocytes localizing to the cortex and CD4 CD8 and CD4 CD8 single-positive thymocytes to the medulla (Fig. 3C). Given the functional importance of Aire for central tolerance, we investigated its expression in the LT RKO thymus and found isolated mTECs that expressed the Aire protein (Fig. 3, D and E). These cells are equally distributed throughout the wt medulla but are restricted to the periphery of medullary FIGURE 2. Signaling through Lt r is not involved in the transcription regions in the Lt r-deficient thymus, leaving large areas devoid of of Aire and Aire-dependent TSAs in mTECs. A, Purification strategy used Aire-expressing mTECs. Collectively, the data indicate that in the for sorting wt and Lt r-deficient CD45-negative CDR1-negative G8.8-pos- absence of Lt r, there is a reduced density and disturbed distribu- itive mTECs. The graphs show the results of a representative sort followed tion of mTECs that are known to express high levels of TSAs, by reanalysis by the same instrument. Plots are gated for CD45-negative suggesting that autoimmunity in these mice might arise as a result cells and set to exclude thymocytes by forward and side scatter. R3 con- of impaired interaction with the TSA-expressing mTECs. Because tains 39.7% of all events in the left plot and 93.3% in the plot to the right. DCs are also important players in the induction of central toler- B, Quantitative RT-PCR analysis for the indicated transcripts on cDNA isolated from purified mTECs of Lt r-deficient (ko) and wt mice as shown ance, we sought to investigate whether their distribution is affected in A. Samples were normalized for the expression of Hprt. Diagrams show by the absence of Lt r signaling. However, the distribution of arbitrary units with the wt sample set to 1. thymic DCs in the medullary thymic microenvironment appears to be unaffected in Lt r-deficient mice (Fig. 3F). in Aire-deficient mTECs is twice the number in Lt r-deficient Lt r does not influence the production and function of mTECs, 5.7 vs 2.8% ( p 0.0001, 2 test of association, Yates CD4 CD25 Treg value 189.25); this difference is even more pronounced for genes The phenotype observed in Lt r-deficient mTECs has similarities deregulated at least 4-fold, 0.93 vs 0.19% (Table I). Thus, Aire to the one in aly/aly mice (7, 13), which are defective for NF- B- deficiency has a much greater effect on transcriptional programs inducing kinase (14), a factor downstream of the Lt r (15). The than Lt r deficiency. This is also true for TSA gene expression, transplantation of aly/aly thymi into nude recipients also induces which dominates the Aire differences in the category of genes with autoimmunity (16). Because aly/aly mice show an impaired pro- at least 4-fold changes (Table I). duction of CD4 CD25 Treg (16), we sought to analyze whether Consistent with the previous results, no change was observed in this was also the case in the Lt r-deficient mice. Treg are produced the mRNA levels for Aire, Insulin2, and Spt1 in Lt r-deficient in the thymus and can also be generated in the periphery upon Ag mTECs as confirmed by qPCR (Fig. 2B). Contrary to a recent stimulation. CD4 CD25 Foxp3 cells are the best characterized report (9), Collagen type 2 was not down-regulated in Lt r-defi- Treg population and are known to have an important influence on cient mTECs in our analysis (data not shown and Supplemental immune responses (reviewed in Ref. 1). Table II). Collectively, our analysis showed no evidence for the We determined the percentage of CD4 CD25 Foxp3 Treg in proposed regulation of Aire and Aire-dependent transcripts by the thymus of Lt r-deficient and wt mice by flow cytometry (Fig.
  • 5. 404 Lt r DOES NOT REGULATE Aire Table I. Tabulation of the number of differentially expressed transcripts for wt to KO transcriptome comparisonsa Lt r-Deficient vs wt mTEC Trancriptome Aire-Deficient vs wt mTEC Transcriptome Comparison (39,000 Transcripts) Comparison (12,000 Transcripts) Down-regulated ( 2-fold) Non-TSA 385 (0.99%) 189 (1.58%) TSA 168 (0.43%) 67 (0.56%) Up-regulated ( 2-fold) Non-TSA 442 (1.12%) 411 (3.43%) TSA 84 (0.21%) 21 (0.18%) Down-regulated ( 4-fold) Non-TSA 13 (0.03%) 44 (0.37%) TSA 12 (0.03%) 36 (0.30%) Up-regulated ( 4-fold) Non-TSA 40 (0.10%) 28 (0.23%) TSA 9 (0.02%) 3 (0.03%) a Absolute number and fraction (in parentheses) of differentially expressed transcripts identified in mTEC transcriptome comparisons. 4A) and found no difference between them in terms of fraction Lt r-deficient thymocytes with the phenotype of recent thymic among CD4 cells and in absolute numbers (Fig. 4, A and B). emigrants (RTE) interact less with the thymic stroma Although no significant differences were detected concerning the Not only the reduced expression of TSAs in Aire-deficient mice numbers of Treg in the thymus, it was possible that the produced but also the restricted positioning of Aire-expressing mTECs at the cells were not fully functional. Therefore, we tested their capacity periphery of the medulla could lead to the reduced access of po- to suppress the proliferation of Teff in vitro. In this assay, Treg activity was investigated by measuring the ability of increasing tentially autoreactive thymocytes to TSA-containing microenvi- numbers of CD4 CD25 Treg splenocytes to suppress the prolif- ronments, resulting in insufficient negative selection. The above eration of the Teff. Both wt and Lt r-deficient CD4 CD25 results indicate that the mTEC network does not homogeneously splenocytes proliferated to a similar extent (Fig. 4C). Addition of cover the whole medulla and that the most affected mTECs are the CD4 CD25 splenocytes (known to contain Treg) to the ones expressing Aire and MHCII at high levels. We, therefore, CD4 CD25 effector cell cultures inhibited proliferation in a hypothesized that if the available surface area of MHCIIhigh dose-dependent manner irrespective of whether the responding or mTECs is reduced in Lt r-deficient thymi, the frequency and du- the added cells were Lt r-deficient or wt (Fig. 4C). ration of contacts with thymocytes might also be reduced. As a Table II. Transcripts coregulated in Aire-deficient and LT R-deficient mTECsa LT RKO vs wt AireKO vs wt Probe Set ID Gene Symbol Norm wt Norm KO FC Norm wt Norm KO FC Up-regulated transcripts 1454842_a_at B3galnt2 0.01 0.03 0.41 0.60 1.37 0.44 1427455_x_at Cr1 0.75 1.53 0.49 0.56 1.43 0.39 1435759_at Ctps2 0.01 0.02 0.50 0.48 1.39 0.35 1451612_at Mt1 0.02 0.04 0.50 0.56 1.33 0.42 1424822_at Slain1 0.01 0.04 0.37 0.24 0.58 0.42 1417162_at Tmbim1 0.11 0.22 0.49 0.64 1.32 0.48 Down-regulated transcripts 1415927_at Actc1 0.11 0.04 2.87 0.55 0.18 3.04 1436998_at Ankrd43 0.02 0.01 2.00 1.69 0.48 3.54 1421129_a_at Atp2a3 0.24 0.09 2.73 1.42 0.47 2.98 1417649_at Cdkn1c 0.82 0.40 2.07 1.94 0.86 2.25 1417853_at Clca1 0.13 0.03 4.47 1.61 0.80 2.02 1419731_at Cyp2b19 0.10 0.05 2.08 1.59 0.59 2.71 1424295_at Dppa3 0.33 0.05 6.69 2.17 0.67 3.26 1416552_at Dppa5 0.12 0.05 2.53 1.40 0.51 2.75 1421802_at Ear1 0.32 0.16 2.03 1.38 0.55 2.52 1429147_at Gas2 0.02 0.01 2.00 0.74 0.18 4.11 1448881_at Hp 0.15 0.07 2.12 1.72 0.55 3.16 1456004_x_at Iapp 0.11 0.05 2.19 1.88 0.50 3.76 1452166_a_at Krt10 1.52 0.63 2.40 1.74 0.70 2.49 1433923_at Krt77 0.43 0.15 2.81 1.20 0.60 2.01 1435382_at Ndn 0.10 0.05 2.04 1.12 0.31 3.67 1451054_at Orm1 0.36 0.18 2.03 1.79 0.38 4.70 1417553_at Plac1 0.04 0.02 2.12 1.37 0.68 2.01 1434793_at Wdr78 0.07 0.02 2.89 1.34 0.59 2.29 a Gene symbols were used to identify common transcripts. Normalized values were obtained in GeneSpring by normalizing the raw data per chip and per gene. Norm, Normalized values. TSAs are in bold.
  • 6. The Journal of Immunology 405 FIGURE 4. The Lt r-deficient thymus produces normal numbers of functional CD4 CD25 Foxp3 Treg. A, The gating strategy used to iden- tify Treg in the thymus is shown. Thymocytes were first gated as CD4 CD8 cells (plots to the left) and the expression of CD25 and Foxp3 in gated CD4 cells is shown in the plots to the right. Numbers indicate the percentage of cells falling into the respective gate or quadrant. B, Fraction in CD4 CD8 cells and absolute numbers of Treg per Lt r-deficient (KO) and wt thymus. Each symbol represents a single mouse, and the mean is represented by a bar. C, Splenocytes from KO or wt mice were sorted as CD4 CD25 (Teff) and stimulated for 72 h with anti-CD3 and irradiated splenocytes depleted for T cells. CD4 CD25 cells (Treg) splenocytes were sorted onto the indicated wells, in the given ratios. The graph repre- sents the mean and SD of triplicate wells. The three last bars are controls: wt CD4 CD25 stimulated only with anti-CD3 Ab (Teff without APC), Teff cultured without anti-CD3 (Teff without aCD3), and irradiated spleno- cytes depleted for T cells cultured with anti-CD3 (APC plus aCD3). Pro- liferation of the cells was measured as cpm. A representative experiment of three independent experiments is shown. measure for contact frequency and duration we determined the amount of epithelial-thymocyte MHCII transfer. MHC molecules can be transferred from the APCs to lymphocytes upon MHC/TCR interaction (reviewed in Ref. 17). This transfer of MHC molecules also occurs in the thymus and developing thymocytes have been shown to acquire MHC molecules expressed by the stroma com- partment (18). Indeed, it has been shown that the amount of MHC on the surface of thymocytes can be used as a tracing system for thymocyte-stromal cell interactions (18). The level of MHCII at the surface of thymocytes is far below the levels usually detected FIGURE 3. There is an architectural defect in Lt r-deficient mTECs whereas DCs are evenly distributed throughout the medulla. A–E, Confocal microscopy ( 200 or 630 (for E) original magnification) of immuno- stained 4-wk-old thymus sections from LT RKO (KO) and wt mice. A, in green, CD8 in blue (double-positive thymocytes appear as cyan) and Thymi were stained with Abs specific for keratin 8 (K8, in red) to identify UEA-1-positive mTECs appear in red. The medulla is outlined by a white cortical TEC. The mTEC were identified by staining with anti-keratin 5 (K5 in line. D, K5 staining of mTECs appears in red, UEA-1 staining in blue and green). UEA-1-positive mTECs are represented in blue. B, Cortical TECs, in staining with anti-Aire mAb is shown in green. E, Presents higher magni- red, were stained as in A, UEA-1-positive cells are represented in green and fication images ( 630) of the thymi shown in D. F, Sections were immu- MHCIIhigh cells are stained in blue. C, Thymocytes are stained for CD4 nostained for mTECs using anti-K5 in green and DC by MIDC8, in red.
  • 7. 406 Lt r DOES NOT REGULATE Aire idence to suggest that the autoimmunity in Lt r-deficient mice is caused by the combined effects of dysregulation of Aire-indepen- dent TSAs and aberrations in medullary architecture. We show that in the absence of Lt r, mTECs express reduced amounts of tran- scripts involved in epithelial cell polarization and appear to inter- act less with thymocytes while thymic DCs and Treg seem unaf- fected. Thus, the molecular mechanisms underlying autoimmune processes in mice lacking Lt r appear to be fundamentally differ- ent from those in Aire and NF- B-inducing kinase-deficient mice. Central tolerance induction, i.e., negative selection of poten- tially self-reactive thymocytes in the thymus, is a coordinated pro- cess that depends on the interaction between thymocytes and two key stromal components in the thymus, namely thymic epithelium and bone marrow-derived DCs (2). For this interaction to be pro- ductive, self-Ags must be presented as peptide-MHC complexes that are accessible to thymocytes. Thymocytes are thought to be deleted if they are reactive to the presented self-Ags, or allowed to undergo further development if they recognize self-Ags with low affinity. Although it is conceptually easy to understand how toler- ance to widely expressed Ags is maintained, this is more difficult in the case of Ags that are specific to organs like the brain or the eye. The expression of a given TSA is restricted to a very small number of mTECs (20), resulting in very low overall levels of expression. Still, it has been described that mTECs are competent mediators of negative selection of TSA-reactive thymocytes, al- though some uptake and cross-presentation by DCs also occurs (3). Because the expression of TSAs is so restricted, the proper pre- sentation of these rare Ags is of critical importance because it must ensure that all thymocytes are exposed to all TSAs, each expressed FIGURE 5. Lt r-deficient RTEs interact less with the thymic stroma. by a minute number of mTECs, before exiting into the periphery. The histogram plots show MHCII-specific staining on the surface of dou- Thus, tolerance induction might fail either when TSAs are not ble-positive thymocytes (A) and thymocytes with the RTE phenotype (B). presented by peptide-MHC complexes (4, 21) or when such com- Lt r-deficient (black line) and wt (gray line) thymocytes were stained, plexes cannot be contacted by thymocytes. analyzed by FACS and compared with thymocytes stained with an isotype Autoimmunity in Lt r-deficient mice has been proposed to be control (shaded histogram). Results of repeat experiments are represented by symbols in the graphs to the right. The mean fluorescence intensity of caused by the lack of Lt r signaling-mediated regulation of Aire MHCII staining in Lt r-deficient (KO) thymocytes for each experiment expression (6). If this were true, significant overlap between the was normalized to that of wt thymocytes. Each symbol represents one changes in the expression profiles of Aire- and Lt r-deficient experiment. mTECs would be expected. We show here that this is not the case. In a non-exclusive scenario, Lt r signaling might control a unique set of TSAs, independent of Aire, as proposed previously (9). Al- on DCs or epithelial cells (data not shown). However, in compar- though this might be true for a small number of TSAs, it does not ison to an isotype control, thymocytes could be shown to have appear to be the sole mechanism, as it is the case for Aire. The surface MHCII, confirming that FACS analysis is sensitive enough absence of one single TSA was shown to induce organ-specific to detect these low levels of MHCII (18) (Fig. 5B). Interestingly, autoimmunity (21). However, this does not appear to be the case of we could not detect differences in the levels of MHCII on the the Lt r-deficient mice, which develop a generalized autoimmune surface of double-positive thymocytes between wt and Lt r-defi- phenotype, where several organs are targeted and a polyclonal rep- cient mice (Fig. 5A) (the mean fluorescence intensity in the KO is ertoire of self-reactive immunoglobulins is produced. The pattern 94 5% of the wt). This is in accordance with the histological of autoantibody production indicates that some common targets results that show no abnormalities in the cortical epithelium, where exist, while individual mice also have unique targets. This is con- CD4 CD8 double-positive thymocytes reside (Fig. 3, A and B). sistent with a scenario where some self-Ags are missing and others However, the thymocytes with the RTE phenotype have a lower are presented in a suboptimal fashion to developing thymocytes, amount of MHCII on their surface (mean fluorescence intensity in resulting in a random element of autoantibody production. the KO is 66 12% of the wt) in the absence of Lt r signaling Could the autoimmune phenotype observed in Lt r-deficient (Fig. 5B), supporting our hypothesis that lympho-epithelial inter- mice be caused by structural abnormalities in the medulla? Several actions depend on the proper mTEC architecture. observations support this interpretation. First, we find that auto- immunity is transplantable and, thus, resides in the stromal com- Discussion partment of the thymus. Second, the multicolor immunohisto- Autoimmune diseases like rheumatoid arthritis, systemic lupus chemistry data show areas that clearly lack mTECs but are packed erythematodes, scleroderma, inflammatory bowel disease, and oth- with lymphocytes; third, there is evidence that thymocytes take up ers cause significant morbidity and mortality. Although many of reduced amounts of surface MHCII from thymic stroma cells, sug- the pathomechanisms leading to autoimmune disease have been gesting reduced lympho-epithelial interaction. correlated with the breakdown of peripheral tolerance, recent years Taken together, our results show that Lt r signaling in the thy- have seen a revival of the important role of central tolerance in the mic stroma is required for central tolerance induction in an Aire- prevention of autoimmunity (2, 19). In this study, we present ev- independent manner. Instead, Lt r signaling is required for
  • 8. The Journal of Immunology 407 mTECs to acquire their proper three-dimensional conformation, Projection of an immunological self shadow within the thymus by the aire pro- tein. Science 298: 1395–1401. which optimizes the accessibility of self-peptide MHC to maturing 5. Bjorses, P., J. Aaltonen, N. Horelli-Kuitunen, M. L. Yaspo, and L. Peltonen. thymocytes. Central tolerance induction in the thymus requires an 1998. Gene defect behind APECED: a new clue to autoimmunity. Hum. Mol. ideal microenvironment where self-Ags are optimally displayed, Genet. 7: 1547–1553. 6. Chin, R. K., J. C. Lo, O. Kim, S. E. Blink, P. A. Christiansen, P. Peterson, promoting the interaction between the thymocytes and the thymic Y. Wang, C. Ware, and Y. X. Fu. 2003. Lymphotoxin pathway directs thymic epithelium. Aire expression. Nat. Immunol. 4: 1121–1127. While this manuscript was under review, two other reports were 7. Boehm, T., S. Scheu, K. Pfeffer, and C. C. Bleul. 2003. Thymic medullary epi- thelial cell differentiation, thymocyte emigration, and the control of autoimmu- published that support our conclusions. Venanzi et al. (22) use a nity require lympho-epithelial cross talk via LT R. J. Exp. Med. 198: 757–769. whole-transcriptome approach where mTECs from Aire- and Lt r- 8. Futterer, A., K. Mink, A. Luz, M. H. Kosco-Vilbois, and K. Pfeffer. 1998. The deficient were analyzed and only a minimal overlap was detected, lymphotoxin receptor controls organogenesis and affinity maturation in periph- eral lymphoid tissues. Immunity 9: 59 –70. showing that the role of Aire and Lt r in mTECs is independent 9. Chin, R. K., M. Zhu, P. A. Christiansen, W. Liu, C. Ware, L. Peltonen, X. Zhang, and does not involve lack of negative selection. As their transgenic L. Guo, S. Han, B. Zheng, and Y. X. Fu. 2006. Lymphotoxin pathway-directed, autoimmune regulator-independent central tolerance to arthritogenic collagen. approach (3) does not address mTEC-mediated deletion of CD4 J. Immunol. 177: 290 –297. cells, the result might be explained by the intact deletion mediated 10. Ludford-Menting, M. J., J. Oliaro, F. Sacirbegovic, E. T. Cheah, N. Pedersen, by DCs, which we observe to be unaffected in Lt r-deficient mice. S. J. Thomas, A. Pasam, R. Iazzolino, L. E. Dow, N. J. Waterhouse, et al. 2005. A network of PDZ-containing proteins regulates T cell polarity and morphology The second publication (23) detected a defect in negative selection during migration and immunological synapse formation. Immunity 22: 737–748. when using a different transgenic approach (3); this system ad- 11. Miyatani, S., K. Shimamura, M. Hatta, A. Nagafuchi, A. Nose, M. Matsunaga, dresses CD8 T cells that are deleted by direct Ag presentation by K. Hatta, and M. Takeichi. 1989. 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Kogishi, mocytes and cells capable of presenting self Ags. T. Serikawa, and T. Honjo. 1999. Alymphoplasia is caused by a point mutation in the mouse gene encoding Nf- b-inducing kinase. Nat. Genet. 22: 74 –77. 15. Matsushima, A., T. Kaisho, P. D. Rennert, H. Nakano, K. Kurosawa, D. Uchida, Acknowledgments K. Takeda, S. Akira, and M. Matsumoto. 2001. Essential role of nuclear factor We thank Dr. R. Kemler (Max Planck Institute for Immunobiology, (NF)- B-inducing kinase and inhibitor of B (I B) kinase in NF- B activation Freiburg, Germany) for providing the anti-cytokeratin 8 (Troma-1) Ab and through lymphotoxin receptor, but not through tumor necrosis factor receptor I. J. Exp. Med. 193: 631– 636. Dr. Hamish Scott (Walter and Elizabeth Hall Institute, Melbourne, Aus- 16. Kajiura, F., S. Sun, T. Nomura, K. Izumi, T. Ueno, Y. Bando, N. Kuroda, H. Han, tralia) for providing the Aire Abs. We thank A. Wuerch and J. Wersing for Y. Li, A. Matsushima, et al. 2004. 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