Transcripts in Their Tumors Patients with Thymomas and Autoantigen

Clinical and Serologic Parallels to APS-I in Patients with Thymomas and Autoantigen Transcripts in Their Tumors

This information is current as Anette S. B. Wolff, Jaanika Kärner, Jone F. Owe, Bergithe of September 28, 2021. E. V. Oftedal, Nils Erik Gilhus, Martina M. Erichsen, Olle Kämpe, Anthony Meager, Pärt Peterson, Kai Kisand, Nick Willcox and Eystein S. Husebye J Immunol 2014; 193:3880-3890; Prepublished online 17

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Supplementary http://www.jimmunol.org/content/suppl/2014/09/17/jimmunol.140106 http://www.jimmunol.org/ Material 8.DCSupplemental References This article cites 67 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/193/8/3880.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Clinical and Serologic Parallels to APS-I in Patients with Thymomas and Autoantigen Transcripts in Their Tumors

Anette S. B. Wolff,* Jaanika Ka¨rner,† Jone F. Owe,‡,x Bergithe E. V. Oftedal,* Nils Erik Gilhus,‡,x Martina M. Erichsen,{ Olle Ka¨mpe,‖ Anthony Meager,# Pa¨rt Peterson,† Kai Kisand,† Nick Willcox,** and Eystein S. Husebye*,{

Patients with the autoimmune polyendocrine syndrome type I (APS-I), caused by mutations in the autoimmune regulator (AIRE) gene, and myasthenia gravis (MG) with thymoma, show intriguing but unexplained parallels. They include uncommon manifestations like autoimmune adrenal insufficiency (AI), hypoparathyroidism, and chronic mucocutaneous candidiasis plus autoantibodies neutralizing IL-17, IL-22, and type I IFNs. Thymopoiesis in the absence of AIRE is implicated in both syndromes. To test whether these parallels extend further, we screened 247 patients with MG, thymoma, or both for clinical features and organ-specific autoantibodies charac- teristic of APS-I patients, and we assayed 26 thymoma samples for transcripts for AIRE and 16 peripheral tissue-specific autoantigens Downloaded from (TSAgs) by quantitative PCR. We found APS-I–typical autoantibodies and clinical manifestations, including chronic mucocutaneous candidiasis, AI, and asplenia, respectively, in 49 of 121 (40%) and 10 of 121 (8%) thymoma patients, but clinical features seldom occurred together with the corresponding autoantibodies. Both were rare in other MG subgroups (n = 126). In 38 patients with APS-I, by contrast, we observed neither autoantibodies against muscle Ags nor any neuromuscular disorders. Whereas relative transcript levels for AIRE and 7 of 16 TSAgs showed the expected underexpression in thymomas, levels were increased for four of the five TSAgs most frequently targeted by these patients’ autoantibodies. Therefore, the clinical and serologic parallels to APS-I in patients with thymomas http://www.jimmunol.org/ are not explained purely by deficient TSAg transcription in these aberrant AIRE-deficient tumors. We therefore propose additional explanations for the unusual autoimmune biases they provoke. Thymoma patients should be monitored for potentially life-threatening APS-ImanifestationssuchasAIandhypoparathyroidism. The Journal of Immunology, 2014, 193: 3880–3890.

uch is being learned about pathogenetic pathways sufﬁciency (AI), and chronic mucocutaneous candidiasis (CMC). from two human autoimmune syndromes and from the Many patients develop other autoimmune manifestations, such as unexpected parallels between them. In autoimmune premature ovarian insufﬁciency (POI), vitiligo, alopecia, autoim-

M by guest on September 28, 2021 polyendocrine syndrome type I (APS-I), the autoimmunity against mune hepatitis, keratitis, enamel dysplasia, and intestinal malab- endocrine and ectodermal targets results from recessive mutations sorption (Table I) (4, 5). Phenotypes vary widely, even within in the autoimmune regulator (AIRE) gene (1, 2). Expressed mainly families; some patients are first recognized in adulthood (4, 5). in medullary thymic epithelial cells (mTECs), wild type AIRE Numerous autoantibodies recognize organ-specific autoantigens is one factor that normally ensures that mTECs promiscuously (6–10), and often correlate with the clinical manifestations in express peripheral tissue-specific autoantigens (TSAgs) that APS-I patients. For example, autoantibodies to NACHT leucine-rich- then induce self-tolerance in thymocytes maturing nearby (3–5). repeat protein 5 (NALP-5) correlate with hypoparathyroidism (6), According to current hypotheses, potentially autoreactive T cells 21-hydroxylase (21OH) with AI (10) and side-chain cleavage en- escape this negative selection in AIRE-deficient thymi, emigrate, zyme (SCC) with POI (9). Remarkably, at diagnosis almost 100% and cause autoimmune damage to target tissues (3, 4). of these patients have autoantibodies neutralizing type I IFNs, Starting in infants or young children, the typical diagnostic triad especially IFN-a2, IFN-a8, and the related IFN-v (11–13). of APS-I comprises hypoparathyroidism, autoimmune adrenal in- Moreover, autoantibodies against the Th17-mediators IL-17A,

*Department of Clinical Science, University of Bergen, 5021 Bergen, Norway; Address correspondence and reprint requests to Dr. Anette S.B. Wolff, Department of †Molecular Pathology Group, Institute of Biomedicine and Translational Medicine, Clinical Science, University of Bergen, Laboratory Building, 8th floor, 5021 Bergen, University of Tartu, 50090 Tartu, Estonia; ‡Department of Clinical Medicine, University Norway. E-mail address: [email protected] of Bergen, 5021 Bergen, Norway; xDepartment of Neurology, Haukeland University { The online version of this article contains supplemental material. Hospital, 5021 Bergen, Norway; Department of Medicine, Haukeland University Hos- pital, 5021 Bergen, Norway; ‖Department of Medicine, Solna, Karolinska University Abbreviations used in this article: AADC, aromatic L-amino acid decarboxylase; Hospital, Karolinska Institutet, SE-171 76 Stockholm, Sweden; #Biotherapeutics Group, AChR, acetylcholine receptor; AI, adrenal insufficiency; AIRE, autoimmune regula- The National Institute for Biological Standards and Control, South Mimms, Potters Bar, tor; APS-I, autoimmune polyendocrine syndrome type I; APTase, adenosine Hertfordshire EN6 3QG, United Kingdom; and **Department of Clinical Neurology, triphosphatase; CMC, chronic mucocutaneous candidiasis; EOMG, early-onset my- Weatherall Institute for Molecular Medicine, University of Oxford, Oxford OX3 9DU, asthenia gravis (onset before 45 y); GAD65, glutamic acid decarboxylase 65; IA-2, United Kingdom insulinoma-associated tyrosine phosphatase–like protein; LOMG, late-onset MG; MG, myasthenia gravis; mTEC, medullary thymic epithelial cell; NALP-5, NACHT Received for publication April 29, 2014. Accepted for publication August 7, 2014. leucine-rich-repeat protein 5; 17OH, 17-hydroxylase; 21OH, 21-hydroxylase; POI, This work was supported by grants from the Regional Health Authorities of Western premature ovarian insufficiency; qPCR, quantitative PCR; SCC, side-chain cleavage Norway, the European Union Framework Programme 7 Project Euradrenal (Grant enzyme; TEC, thymic epithelial cell; TH, tyrosine hydroxylase; TPH-1, tryptophan 201167), the Bergen Medical Research Foundation, the Estonian Research Agency hydroxylase type 1; TPO, thyroid peroxidase; Treg, regulatory T cell; TSAg, tissue- (Grant IUT2-2), the Center of Excellence of Translational Medicine of the Tartu specific autoantigen. University, the Sir Jules Thorn Charitable Trust, and the U.K. Medical Research Council. The funding sources have not been involved in the study design, the execu- Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 tion of the study, or the writing process. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401068 The Journal of Immunology 3881

IL-17F, or IL-22, involved in mucous membrane defenses against for autoantibodies against 21OH, 17-hydroxylase (17OH), SCC, GAD65, Candida albicans, are prevalent (14, 15) and correlate with the tryptophan hydroxylase type 1 (TPH-1), aromatic L-amino acid decarboxylase CMC (14). (AADC), tyrosine hydroxylase (TH), and NALP-5 (7, 13). The threshold for positivity was set as the mean of the indices for all healthy blood donors There are intriguing parallels and differences in the autoimmune tested (n = 57–150) +3 SD. We assayed autoantibodies against thyroid per- associations with thymic epithelial neoplasms. Overall, myasthenia oxidase (TPO) with Immulite 2000 solid-phase chemiluminescence immu- gravis (MG) occurs in $30% of all patients with thymomas, noassays (FDA Clears Siemens, Malvern, PA), against titin by ELISA (DLD especially of the thymopoietic types B2 and B3, plus characteris- diagnostika, Hamburg, Germany), and against AChR by RIA (IBL Interna- tional, Hamburg, Germany) (18). Anti-TH and anti-TPO autoantibodies were tic autoantibodies against the acetylcholine receptor (AChR), titin, analyzed only for patients from whom we had thymoma samples. and other muscle Ags (16–19). Other autoimmune features in thymoma patients (with or without MG) are also sharply focused, AIRE mutations especially on hemopoietic targets (in ∼5%), causing various bone Both AIRE alleles were sequenced using standard protocols and primers as marrow aplasias (20–23) and possibly hypogammaglobulinemia. At described elsewhere (35). ∼ diagnosis, 70% have autoantibodies neutralizing type I IFNs (24, RNA extraction from thymomas and real-time PCR 25), similar to those in APS-I, and again with an IgG4 bias (26). Occasional thymoma patients have been reported with autoimmune Tissue samples were homogenized in Trizol (Thermo Scientific, Waltham, endocrine (27–29) or ectodermal (30) manifestations, or even CMC MA) using AutoMACS with M-tubes (Miltenyi Biotech, Bergisch Gladbach, Germany), followed by RNA extraction according to the man- plus autoantibodies neutralizing IL-17 and IL-22 deficiencies in ufacturer’s protocol. RNA concentrations were measured with NanoDrop Th17 and Th22 cells (14), similar to those observed in APS-I (4, 5, (Thermo Scientific, Waltham, MA); 5 mg of total RNA was reverse-

14). Whereas autoantibodies against AChRs are clearly pathogenic transcribed using Superscript III (Invitrogen), 10 mM dNTP Mix, Ribo- Downloaded from in MG, the organ-specific Abs in APS-I are useful diagnostic Lock RNase inhibitor and random hexamers (Thermo Scientific, Waltham, MA). Real-time quantitative PCR (qPCR) was performed using Applied markers, and typically directed against intracellular TSAgs, as are Biosystems ViiA 7 Real-Time PCR System with 384-Well Block (Life those against titin and ryanodine receptor in many late-onset MG Technologies) and Maxima SYBR Green /ROX qPCR Master Mix (LOMG; onset after age 45) and most thymoma patients (16–19). (Thermo Scientific, Waltham, MA). Every sample was run in three parallel The most obvious link between these disparate syndromes is reactions in two separate series of experiments; their results were broadly consistent and have been combined. We detected reliable signals for all that, in nearly all thymomas, the neoplastic thymic epithelial http://www.jimmunol.org/ transcripts tested except NALP-5. cells (TECs) fail to express AIRE detectably, implying reduced –DDCt Every transcript signal was expressed as 2 (where Ct represents the expression of TSAgs such as AChR, insulin, and glutamic acid threshold cycle), and normalized relative to the value for b-actin in the decarboxylase 65 (GAD65) (31, 32). The currently prevailing hy- same sample, and then to its (b-actin-normalized) KRT8 value. For Table V pothesis is that AIRE-deficient thymi or thymomas fail to express and Figs. 2 and 3, the resulting AIRE or TSAg values were next expressed relative to that in one control infant thymus. Primers are listed in target autoantigens and consequently generate and export non- Supplemental Table II. tolerant T cells that eventually cause autoimmune damage in the periphery (3). An alternative explanation suggests biased selec- Statistics tion or active autoimmunization against certain common targets in We evaluated differences in autoantibody prevalences between patients aberrant thymic tissue (33). To distinguish between these hy- and controls using Pearson x2 tests and program SPSS version 15, and by guest on September 28, 2021 potheses, we assessed whether the clinical and serologic similar- in transcript values between different groups using nonparametric one-way ities between APS-I and thymoma patients extend to APS-I– ANOVA (Kruskal–Wallis) with Dunn’s multiple comparison tests and Mann–Whitney U tests (Fig. 3; GraphPad Prism, La Jolla, CA). For TSAg typical organ-specific autoantibodies, and whether these correlate transcript values, the threshold for significance was set at p=0.01. Dif- with underexpression of AIRE and TSAg transcripts in their ferences between thymoma and thymus remnant expression of AIRE were thymomas. These tumors must hold clues to autoimmunizing evaluated using paired t tests. We also calculated z-scores to show the mechanisms, which are otherwise difficult to study in humans. number of SDs by which each thymoma TSAg signal differed from the corresponding mean of the five infant control thymi, whether above the mean (positive z-scores) or below (with minus signs). Materials and Methods Patients Results All samples were taken with informed consent and ethics committee ap- APS-I–typical clinical manifestations in patients with MG or proval in each referral center (Bergen, London, Oxford, and Tartu). The demographics of the patients and controls are shown in Supplemental thymomas Table I. Over 200 of the 247 patients with MG, thymomas, or both were Among the 121 patients with thymomas (with or without MG), 10 seen (and many followed for long periods) by a single U.K. neurology team (8%) had a total of 15 APS-I–typical manifestations, including AI (34); all clinical information was taken from hospital and autopsy records. An additional 43 patients were assessed similarly in Bergen. They are in non-MG patient P2 (Tables I, II). Several of these disorders grouped into those with: thymoma (with or without MG), LOMG, early- occurred together, even including asplenia or nail dystrophy (in onset MG (EOMG; onset before age 45), and ocular MG. The 38 patients patients P1 or P3). Both are unusual in autoimmune patients such with APS-I were from the Norwegian panel detailed previously (35). as these who had no genomic AIRE mutations (asterisked in Thymus and thymoma samples Table II and Supplemental Table III), as is the CMC (plus IL-22 autoantibodies) that severely afflicted P8-10. Many of the APS-I– Thymoma samples were snap-frozen as blocks from 26 patients and stored 2 typical manifestations appeared long after the thymomas, for ex- at 80˚C until use (34). Nearly all thymomas were encapsulated and could . be clearly separated from any adjacent thymic remnants (n = 5), which ample, in 6 of 29 U.K. cases with intervals 5 y (21%) versus were often minimal or absent in older or steroid pretreated cases. At least only 5 of 70 (7%) of patients with shorter intervals (p=0.051). one remnant showed follicular hyperplasia, and so did $38 of the 48 Of the 121 MG patients without thymomas at diagnosis, only EOMG thymi removed. Most MG thymomas resemble disorganized infant one with LOMG (P11) showed clear APS-I–typical features, thymic cortex (17, 34); therefore, pediatric thymi seem the most suitable and available controls. whereas another (P12) had pernicious anemia, thyroid disease, and a strong family history of thyroid autoimmunity (Table II). Assays for autoantibodies Although this study is retrospective, all patients were assessed All samples were tested in the same laboratory with radioligand binding assays similarly and thoroughly by the same experts in each referral against in vitro transcribed and translated proteins (Promega, Fitchburg, WI) center. Thus, the EOMG and LOMG groups are ideally matched 3882 APS-I AUTOANTIBODIES AND MANIFESTATIONS IN THYMOMA PATIENTS

Table I. APS-I–like manifestations given as number (%) in the different patient groups

Thymoma

APS-I with MG No MG LOMG EOMG Manifestation (n = 38) (n = 114) (n =7) (n = 63) (n =55) Any APS-I–like manifestation 38 (100) 9 (8) 1 (14) 2 (3) 0 (0) Hypoparathyroidism 26 (68) Addison disease 25 (66) 1 (14) Gonadal failure 8 (21) Autoimmune thyroiditis 5 (13) 2 (2) 2 (3) Type I diabetes 3 (8) 1 (1) Alopecia 12 (32) 5 (4) Vitiligo 6 (16) 2 (2) 1 (2) Nail pitting 5 (13) 1 (1) Keratitis 3 (8) Enamel dysplasia 12 (32) Malabsorption 8 (21) Autoimmune hepatitis 3 (8) Pernicious anemia 2 (3) Dry eyes or mouth 2 (5) 1 (2)

Squamous carcinoma 1 (3) Downloaded from Chronic mucocutaneous candidiasis 28 (74) 3 (3) 1 (2) Hypogammaglobulinemia 1 (14) Pure red cell aplasia 2 (2) Asplenia 2 (5) 1 (1) See Table II and Supplemental Table III for more details on the patients.

“disease controls” for the thymoma patients—for their geographic In the LOMG, EOMG, and ocular MG subgroups, by contrast, http://www.jimmunol.org/ origins and the assessors. none of these autoantibodies were more common than in the APS-I–typical organ-specific autoantibodies in patients with controls, even in P11, who had several APS-I–like manifestations MG or thymomas (Table II). Because the above clinical and serologic associations thus appear to be with the thymomas rather than the MG, and In the thymoma patients with or without MG, we found signifi- although we had only seven non-MG thymoma patients, we group cantly higher prevalences of autoantibodies against 21OH, 17OH, all the thymoma patients together from this point forward. SCC, TPH-1, and GAD65 than in the healthy controls or the other MG subgroups (Fig. 1, Table III); against TPH-1 and GAD65, MG-specific autoantibodies in APS-I patients prevalences were approximately half of those found in the APS-I None of the APS-I patients had detectable autoantibodies against by guest on September 28, 2021 patients. Many of the binding signals were in the same range as in either AChR (0 of 38 versus 114 of 114 of the MG/thymoma APS-I (Fig. 1), as also against AADC and NALP-5 in occasional patients; p , 0.001) or titin (p , 0.001; not shown), nor did cases. Autoantibodies against AADC, TPH, and NALP-5 were any of them have any obvious MG-like neurologic features. considered APS-I–specific (7). In total, 49 of 121 thymoma patients (40%) had $1 APS-I– Transcript levels for AIRE and TSAgs in thymomas and related organ-specific autoantibodies, and they tended to occur adjacent thymic remnants together with each other (Table IV), though often not with the To test the hypothesis that these autoantibodies correlate with corresponding clinical manifestations (Table II). For example, the decreased AIRE and TSAg expression, we assayed AIRE and 16 one patient with AI (P2) tested negative against 17OH or 21OH, TSAg transcripts in the available thymoma, remnant or control but positive against GAD-65. She was one of the seven thymoma thymus blocks. To adjust for content of any TEC subtype (36, 37), patients without MG, of whom two also had GAD65 autoanti- which varies greatly between thymomas (17, 34), we normalized bodies, and one gave low signals versus 17OH and 21OH. Notably, the qPCR by the keratin-8 (KRT8) signals; these correlated APS-I–type autoantibodies were found in more patients with dis- strongly, but inversely, with the thymocyte content estimated ease durations .5 y (13 of 25 [52%]) than with more recent onset when the tumors were first processed (34) (not shown). They (20 of 65 [31%]), although not quite significantly (p=0.061). were broadly consistent in the duplicate blocks available from five In the serial samples available from these patients, the auto- thymomas and one remnant, which have each been averaged. antibodies showed minor variations at different time points (Supple- Relative AIRE transcript levels were low in almost all thymoma mental Fig. 1); some of them might reflect changes in immuno- subtypes, but there were large individual differences (Fig. 2A). suppressive drug doses, although similar variations occur in APS-I Values were high in one of the two available type B1 thymomas, where these drugs are used only rarely (35).The autoantibodies in line with previous reports (31, 32). As expected, AIRE ex- in the thymoma patients showed no significant correlations with pression was much lower in the thymomas than in the adjacent MG status or thymoma histology (where the proportions of autologous thymic remnants in all five available pairs (one was thymocytes/TECs varied greatly). World Health Organization non-MG; Fig. 2B). typing was available for 71 MG thymomas; we found $1 APS-I– Data from these paired thymomas or thymic remnants also illus- like autoantibody in 2 of the 12 (17%) patients with types A or AB trate the variability between different patients and different versus 20 of the 56 (36%) with types B2 and B3 (p = 0.20). Type TSAgs (Table V). TDRD6 and H+/K+ adenosine triphosphatase B1 is the only subtype reported to express AIRE (in ∼50%) (32). (ATPase) transcript values were lower in most of the thymomas Among four such patients, P5 had high-index autoantibodies than in the remnants, as expected. In contrast, the steroidogenic against 21OH and 17OH (Table II), but unfortunately we did not enzyme transcripts mostly showed similar or even higher values have access to his thymoma. in the thymomas. h ora fImmunology of Journal The

Table II. Thymoma and MG patients with APS-I–like clinical features and autoantibodies

Thymoma APS-I–type Autoantibodies

Patient WHO Duration Clinical Features (Age in Other (Sex, MG Onset Age [y]) MG Status Classification (y)a Years at Onset or Death)b 21OH 17OH NALP5 SCC GAD65 TPH-1 AADC IFN-as IL-17/22 Autoantibodies P 1 (F, 40)c MG B2, invasive $8 Urt (42), T (48), ++ ++ ++ – IL-12, ANA As (50), V (56), SLE (59), NMT (56)d P 2 (F) Not MG B2 (50) $14 Hypo-g (35), TC ++ – nd (41), AI, Urt, TLP (50) P 3 (M, 46)c MG “Malignant” $4, $23 T1D (43), T, Alo (50), + + ++ ++ ++ nd IL-12 recurred (56) V, Nail dystr,e SPS (65) P 4 (F, 41)c MG B2 Alo (38) ++ ++ – IL-12 P 5 (M, 28) MG B1 ∼3 Alo (5, 31), myocardial ++ ++ ++ – IL-12 infarct (42) P 6 (F, 50) MG B1 Alo (51) + ++ nd IL-12 P 7 (F, 64) MG B3 Alo (45), RBC + + – nd IL-12 6 aplasia (74) P 8 (F, 46)c MG B3 $12 CMC (58, 73) + ++ ++ + ++ ++ IL-12 P 9 (M, 27)c MG B2, metastatic $16 CMC (44), NMT ++ ++ ++ (50, 59) P 10 (F, 35) MG B2/B3 ∼9.5 CMC (44, 47) ++ ++ IL-12 6 P 11 (M, 74)c LOMG None foundf V (60), PA (70), CMC, ++ ++ thyroid ++ ANA T, dry eyes, pulm fibr (74)f P 12 (F, 53) LOMG None found PA (48), T (52), strong ++ nd IL-12 family history of T aDelay between first signs of thymoma and of APS-I–type features. bTwo ages indicate both onset and death. cNo genomic AIRE-mutations. dCommon APS-I manifestations are marked in bold. eNoted at autopsy. fLost to follow-up at 74 years of age. +, autoantibody positive but index , 200; ++, autoantibody index . 200; AI, adrenal insufficiency; Alo, alopecia; ANA, anti-nuclear Abs, As, asplenia; CMC, chronic mucocutaneous candidiasis; F, female; hypo-g, hypogamma-globulinemia; IFNs, autoantibodies to type 1 IFNs; M, male; Nail dystr, nail dystrophy; NMT; neuromyotonia; PA, pernicious anemia; pulm fibr, pulmonary fibrosis; SLE, systemic lupus erythematosus; SPS, stiff-man syndrome; T, thyroid autoimmunity; T1D, type 1 diabetes mellitus; Th17, autoantibodies to IL-17A, IL-17F, or IL-22; TLP, tongue lichen planus; Urt, urticaria; V, vitiligo.

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Downloaded from from Downloaded http://www.jimmunol.org/ by guest on September 28, 2021 28, September on guest by 3884 APS-I AUTOANTIBODIES AND MANIFESTATIONS IN THYMOMA PATIENTS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. APS-I–related autoantibodies in MG subgroups, APS-I patients, and controls against the indicated autoantigens. Thresholds for positivity are marked by horizontal lines.

The 26 thymomas (including two non-MGs) showed the most ATPase and AChR-a clearly followed the AIRE expression pat- striking variability in TSAg transcripts, even when AIRE expres- tern: highest in control thymi, intermediate in remnants, and low sion was low (black circles in Fig. 3). Values for AADC, H+/K+ in thymomas (Fig. 3). We also noted signiﬁcantly lower values for

Table III. Prevalences (%) of APS-I–type organ-speciﬁc autoantibodies in the different cohortsa

Autoantibody APS-I Thymoma 6 MG LOMG EOMG Healthy Controls AI/APS-IIb 21OH 55** 13* 1.6 3.6 3.5 86 17OH 24** 8.8* 0 1.8 0 11 SCC 40** 11** 3.2* 7.3* 0 6.5 AADC 41** 6.2 0 3.6 4.4 2.2 NALP-5 35** 3.5 1.8 1.8 1.6 NA TPH-1 28** 14** 0 1.8 0 NA GAD65 29** 15* 4.8 1.8 3.4 21 aNumbers of sera assayed for the various autoantibodies varied slightly. We tested sera from $37 patients with APS-I, $113 patients with thymomas 6 MG, $56 patients with LOMG, $55 patients with EOMG, and $57 controls. We tried to test the earliest bleeds from each patient, but many with thymomas were already taking immunosuppressive drugs for their MG. bData from 426 Norwegian AI/APS-II patients fully characterized by Erichsen et al. (1). APS II is deﬁned as AI plus thyroid disease, type I diabetes, or both. *p , 0.05, patient group versus control; **p , 0.01, patient group versus control. NA, not analyzed. The Journal of Immunology 3885

Table IV. Percentages of patients without and from 1 to $3 APS-I–type phosphatase–like protein (IA-2) [1], SOX9 [11], and TPH-1 [3] organ-speciﬁc autoantibodies (Supplemental Table III). Thus, these TSAgs appear AIRE-independent, despite their frequent recognition by autoantibodies in Thymoma thymoma and APS-I patients (Fig. 1, Table III). Transcript values No. of APS-I With MG No MG LOM EOMG showed no obvious correlations with MG status or thymoma Autoantibodies (n = 38) (n = 114) (n =7) (n = 63) (n = 55) histology. 0 8 57* 57 89* 82* Correlating APS-I–type features and autoantibodies with TSAg 12921291115transcript values in thymomas 211161404 $3526000For manifestations that could be correlated with TSAg transcripts, *p , 0.0001, when comparing all thymoma patients (6 MG) with MG patients there were only six informative patients (Supplemental Table III). without thymoma (EOMG plus LOMG) using x2 test. In the two with alopecia (P6 and P7), TH transcript levels were similar or even higher than in control thymi (z-scores, 13.4 and 0.8). In three of the four patients with autoimmune thyroid dis- insulin, HDC, and TDRD6 in thymomas (p , 0.01; Kruskal– ease (P1, P13, and P14), it presented later than their MG and Wallis test) and for GAD65 when compared with the pooled their thymomas. Interestingly, two of them gave elevated transcript control and remnant thymi (p , 0.01; Mann–Whitney U test; values for TPO (z-scores, 2.3 and 3.4), the target that correlates Fig. 3). best with autoimmune thyroiditis, whereas values were low for Surprisingly, even when AIRE values were low, we found higher

both TPO and TG in P15, whose thyroiditis had presented several Downloaded from TSAg transcript values per epithelial cell in many thymomas (num- years earlier. bers with z-scores . 3 are shown in brackets) than in any of the The autoantibodies likewise failed to show consistent negative control thymi (Fig. 3) for: 21OH [10], 17OH [1], TG [3], TPO [5], correlations with TSAg transcript values. In 14 of 26 patients, we TH [1], HDC [2], TDRD6 [2], insulinoma-associated tyrosine detected a total of 31 autoantibodies against informative targets, excluding AChR (Supplemental Table III). Transcript values gave

positive z-scores in 14 of these 31 instances (for 21OH, TPO, and http://www.jimmunol.org/ SOX9 they were .3 in five instances or more). Although AIRE values overlapped the controls in P19 and P20, together they had autoantibodies to five TSAgs, despite positive z-scores for three of these TSAgs (and AChR-a). Conversely, AIRE was low in P21, but she had adrenal autoantibodies despite z-scores .2.5 for 17OH and 21OH. Overall, these findings provide no clear support for general TSAg underexpression in thymomas as the main cause of the

associated autoimmunity. by guest on September 28, 2021

Discussion This wide-ranging study unexpectedly shows APS-I-typical organ- specific autoantibodies in over 40% of thymoma patients, and APS-I-typical clinical manifestations in 8%, but often not in the same individuals. This study confirms and extends an earlier observation of “thymocopying” of APS-I development in a patient with a type B thymoma (27). Despite having no AIRE mutations, some thymoma patients developed major APS-I manifestations such as AI, CMC, or asplenia, but at much lower frequencies than in APS-I. However, APS-I patients showed no clinical or serologic signs of MG. Surprisingly, some of the autoantigens targeted in thymoma patients showed the expected underexpression in their AIRE-deficient tumors, but many did not, including several adrenocortical, gonadal, and neuroectodermal targets, some of which are considered APS-I–specific. Thus, our results highlight differences and similarities in the pathogenesis in these two syndromes. These results also question current hypotheses that a lack of AIRE-regulated TSAg expression in thymomas is the major cause of the unusually biased autoimmunity. Clinical and serologic parallels implicating thymic aberrations in autoimmunization The overlapping autoantibody reactivities were largely confined to patients with thymomas rather than LOMG or EOMG, even FIGURE 2. Relative AIRE expression in thymoma types and adjacent thymic remnants. (A) AIRE expression in blocks from different World though thymoma and LOMG patients are well known to share Health Organization thymoma types using keratin-8 (KRT-8) to adjust for several other autoantibodies, especially against internal muscle epithelial cell content, and a pediatric control sample as calibrator. (B) autoantigens (18), type I IFNs, or IL-12 (25). Thymoma patients AIRE expression in paired thymomas or thymic remnants for five infor- without MG are difficult to collect; although their numbers in mative patients. Expression was calculated as for (A). *p , 0.05. this study are too small to exclude differences in prevalences of 3886 APS-I AUTOANTIBODIES AND MANIFESTATIONS IN THYMOMA PATIENTS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Relative transcript signals for APS-I target autoantigens (normalized to keratin-8) and shown as fold change compared with one pediatric control sample. The bars for each group represent the medians. Gray squares and black circles indicate samples with AIRE expression .0.1 or ,0.1, respectively. Black horizontal lines above indicate where the thymomas differed signiﬁcantly from remnants or from control groups (one-way ANOVA [Kruskal–Wallis] with Dunn’s multiple comparison correction), and the red horizontal lines where they differed signiﬁcantly from the combined remnants plus controls (Mann–Whitney U tests). ** p , 0.01.

APS-I–type autoantibodies or manifestations deﬁnitively, the pres- autoantibodies—not only by their prevalence in both APS-I and ent results—and the contrasts with LOMG—suggest that the thymoma patients, but also by their rarity in numerous other au- thymoma alone, rather than the associated MG, is responsible for toimmune diseases, even where peripheral type I IFNs, dendritic this serologic and clinical overlap. Thymic aberrations are further cells, or Th17 or Th22 cells are involved in pathogenesis (14, 15, implicated by the type I IFN-, IL-17– and IL-22–neutralizing 25, 38). Conversely, most of the “standard autoantibodies” com- The Journal of Immunology 3887

Table V. Tissue-speciﬁc autoantigen transcript values in paired thymoma divided by thymic remnant Downloaded from http://www.jimmunol.org/

Each number is the TSAg value in the patient’s thymoma block divided by the value in the autologous thymic remnant. Red indicates transcript values .4.00 (.4-fold higher in thymomas than in thymic remnants), green indicates transcript values ,0.25 (.4-fold lower in thymomas than in thymic remnants), and yellow indicates intermediate transcript values (0.26–4.00). monly found in sporadic and systemic autoimmune diseases are been missed in many others in whom prethymectomy follow-up uncommon in thymoma patients (22, 32, 39, 40). was much shorter, as possibly in a previous study in which only 28 by guest on September 28, 2021 Thymic aberrations are also implicated in autoimmunization patients were tested for autoantibodies (32). Asplenia might have by our observations in patients with the 22q11.2 deletion (or Di escaped notice because it requires imaging. Moreover, the im- George Syndrome) who have primary thymus aplasia. Approxi- munosuppressive therapy often used for MG can cause repressed mately 15% have similar autoantibodies against SCC, 17OH, and autoimmune T cell–mediated tissue destruction, and their glu- 21OH, again with no signs of AI or POI (41, 42). Interestingly, cocorticoid treatment might have compensated for unsuspected MG has been reported in rare patients with hypomorphic RAG1 AI. There might also be genetic differences in responsiveness to mutations who also have disorganized AIRE-deficient thymi certain TSAgs. (43), or IFN-a autoantibodies (44), which again implicates aber- If there is some true uncoupling of autoimmunization of the rant AIRE-deficient thymopoiesis in autoimmunization in these B cell and pathogenic T cell responses in patients with thymomas, disparate syndromes. it might reflect several immunologic differences from APS-I. Normally, thymic AIRE plays its key roles when the T cell rep- Clinical and immunologic differences between APS-I and ertoire is being established in the fetus and infant. Indeed, its thymoma patients deletion in mice after postnatal day 3 does not cause autoimmunity It is widely accepted that the autoimmune endocrine tissue (47), nor does thymectomy, even in children. Whereas APS-I damage in APS-I is T cell–mediated (45). Whereas autoanti- patients have genomic AIRE mutations from conception, the bodies against AChR are directly pathogenic in MG, those AIRE-deficiency arises only in the tumors (31, 32) and decades against intracellular targets are valuable as diagnostic markers. later in life in thymoma patients. Second, APS-I patients also lack In APS-I, the autoantibodies against steroidogenic enzymes functional AIRE expression in spleen and lymph nodes, where it correlate well with AI and POI, against TH with alopecia (46) could play important roles in maintaining peripheral tolerance and against TPH-1 with malabsorption (7); curiously, GAD65 (48, 49). Those checkpoints presumably remain intact in patients autoantibodies correlate with malabsorption in APS-I instead of with thymomas, and they could be sufficiently potent to restrain type 1 diabetes (7). some of the potentially autoaggressive T cells exported by these Although the APS-I–typical manifestations appear to be tumors, again making the observed parallels seem the more uncoupled from the autoantibodies in our thymoma patients, both remarkable. were probably underestimated for several clinical reasons. There Moreover, most thymomas show additional aberrations not to are often delays of many years between detection of autoanti- be expected in APS-I thymi, which are not available for study. bodies and onset of the corresponding clinical feature in APS-I These might also contribute to loss of tolerance, as they include (35). Similarly, additional manifestations appeared $15yafter absence of HLA class II on the neoplastic TECs and of muscle-like initial presentation in several thymoma patients (e.g., P1-P3, P8, thymic myoid cells (17, 34). Interestingly, in both thymomas and and P9; Table II, Supplemental Table III), but they might have Aire2/2 mouse thymi, there are few (if any) Hassall’s corpuscles, 3888 APS-I AUTOANTIBODIES AND MANIFESTATIONS IN THYMOMA PATIENTS around which AIRE+ TECs are normally frequent and Foxp3+ against AADC and GAD65 clearly conformed to current thinking. regulatory T cells (Tregs) are positively selected (17, 50–52). Overall, our data question the generality of current hypotheses that There are also changes in Tregs in both syndromes (51, 53–56). the similar AIRE deficiency in APS-I and thymomas predisposes These clinical factors, plus the late onset of AIRE deficiency purely by impairing their expression of AIRE-dependent TSAgs/ localized in aberrant thymoma microenvironments, probably con- self-tolerance induction. We therefore propose that AIRE deficiency tribute substantially to the apparent differences from APS-I. might also create dangerous, Treg-deficient microenvironments where available AIRE-independent autoantigens bias selection TSAg transcripts in thymomas or actively autoimmunize (33), as in paraneoplastic syndromes. Currently favored hypotheses assume that minimal or undetect- To us and others (66), this proposal explains these unusual early able TSAg expression by the AIRE-deficient neoplastic TECs dominant responses more neatly than current hypotheses, which (31, 32) is the main cause of the associated autoimmunity. Al- apply better to those against other TSAgs like GAD65. Once again, though it is much easier to test TSAgs for Aire-dependence in key evidence has come from observations in patients. Aire2/2 mouse TECs (3, 57) than in humans, their AIRE-deficient Our findings also have implications for patient management. thymomas seem a practical alternative. Although we recognize They argue for reconsideration of thymectomy in APS-I children, that the TSAg transcript values alone might not fully reflect pro- to halt the continuing supply of autoaggressive T cells. Further- tein expression levels, other options were not available to us. more, in thymoma patients, subclinical adrenal insufficiency can We also recognize that other biologic factors might have affected mimic MG, with muscle weakness and fatigue (67). If not rec- the AIRE and TSAg signals we detected. For example, thymoma ognized, it could lead to an acute or even fatal Addisonian crisis.

TECs apparently derive from progenitors with combined cortical Downloaded from and medullary markers (34); the normal counterparts of these Acknowledgments progenitors are rare, and they have scarcely been studied. Because We thank the late Prof. J. Newsom-Davis and Sister E. Goodger, the patients thymoma TECs appear clonal (58), AIRE and TSAg expression for their invaluable help, Elisabeth Halvorsen for handling patient samples can vary between tumors, as they both do between individual and coordinating the APS-I biobank, Laura Tomson and Maire Pihlap for mTECs (59, 60). They can vary within thymomas, too, although excellent technical assistance with qPCR, and Prof. R. Klein for autoantibody this was not obvious in the available duplicate samples. Expres- results on tissue sections. http://www.jimmunol.org/ sion might also change as thymomas evolve over time or react to changing blood supply or hormonal influences (34), which might Disclosures explain the correlations we noted with thymoma durations. Some The authors have no financial conflicts of interest. of these issues might be clarified by testing single TECs from thymomas and pediatric thymi. Nevertheless, some TSAgs were clearly underexpressed in thy- References momas, notably AChR-a, H+/K+ ATPase, AADC, insulin, GAD65, 1. Finnish-German APECED Consortium. 1997. An autoimmune disease, HDC, and TDRD6. These data partially fit with previous re- APECED, caused by mutations in a novel gene featuring two PHD-type zinc- finger domains. Nat. Genet. 17: 399–403. by guest on September 28, 2021 ports of AIRE-dependent, but variable, expression of insulin, 2. Nagamine, K., P. Peterson, H. S. Scott, J. Kudoh, S. Minoshima, M. Heino, AChR-a, IA-2, and H+/K+ ATPase (32, 33, 60, 61). 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