Role of AIRE in Thymoma-associated Myasthenia Gravis ? Alexander Marx, MD University Medical Centre Mannheim University of Heidelberg AIRE-PHD1 Thymoma AIRE in mTEC EuroMG Paris, 01-12-09

APECED/APS-1 and AIRE The Autoimmune regulator (AIRE) is a gene mutated in autoimmune polyendocrinopathy candidiasis ectodermal dystrophy or APECED Thymus Several autoimmune (endocrine) diseases (highly variable) Hypoparathyroidism & Addison’s disease Type 1 diabetes mellitus Gonadal dysfunction (e.g. premature ovarian failure) Autoimmune hepatitis, Alocecia, Autoimmune Thyroid Disease... Mucocutaneous Candidiasis (often first & leading symptom) Virtually 100% of patients have auto-Ab to type I interferons EuroMG, Paris 01.12.2009

Regulation of > 1000 genes AIRE is mainly expressed in keratin-positive medullary thymic epithelial cells (mTEC), less commonly in dendritic cells Aire Pan- Cytokeratin (mTEC) Regulation of > 1000 genes EuroMG, Paris 01.12.2009

and some other genes mainly in medully thymic epithelial cells Aire drives „promiscuous“ expression of „tissue-specific antigens“ (TSA) and some other genes mainly in medully thymic epithelial cells Aire p450_c17 Aire Thyroglobulin Pärt Peterson AIRE-dependent TSA: Insulin, TPO, casein b, a-subunit of AChR, p450, S100 A8 EuroMG, Paris 01.12.2009

Functional effect of AIRE-driven autoantigen expression in medullary thymic epithelial cells (mTECs) Apoptotic mTEC mTEC DC Cross-presentation by dendritic cells (DC) Promiscuous expression and improved prentation of autoantigens & MHCII Tolerance Induction Mathis & Bennoit, ARI, 2009 EuroMG, Paris 01.12.2009

Significant Correlation between Prevalence of HCs and Prevalenve of AIRE+ Cells in Normal Human Thymus r = 0.81 p< 0.00001 EuroMG, Paris 01.12.2009

Age-dependent decline of Hassall‘s Corpuscles and AIRE+ cells in normal human thymus HCs Aire+ Cells Aire/HCs ~ 2 - 3 (0 - 40y) ~ 1 -1.5 (41- 90y) Hassall‘s Corpuscles & Aire+ Cells per Area Mean HCs per area NT Outlier (HCs) Mean Aire+ per area NT Outlier (Aire+) Age, Decades EuroMG, Paris 01.12.2009

Among WHO-defined Thymomas Hassall‘s Corpuscles are common only in Type B1 Thymomas („predominantly cortical“) WHO-Classification: 1999, 2004 A AB B1 B2 B3 „C“ „Medullary“ Mixed „Cortical“ TSCC Histogenetic Classification: Marino & Müller-Hermelink, 1985; Kirchner, 1992 EuroMG, Paris 01.12.2009

AIRE--- >95% of thymomas, including all type A & AB thymomas do not express AIRE* (Ströbel, 2007) Thymus Type B1§ § ~50% of type B1 thymomas are Aire+ AIRE--- *AIRE, Autoimmune Regulator

Loss of AIRE and AIRE-related self-antigen expression in >95 of thymomas Ströbel et al. J Pathol 2007

Does AIRE-deficiency in thymomas elicit autoimmune diseases, including APECED ? Apoptotic mTEC mTEC NO DC Cross-presentation by dendritic cells (DC) NO Promiscuous expression and improved presentation of autoantigens & MHCII Impaired Clonal Deletion Autoimmunity Mathis & Bennoit, ARI, 2009 EuroMG, Paris 01.12.2009

MG overlap: anti-IFN type I Minimal overlap of clinical and serological autoimmune features in APS-1 patients and patients with AIRE(-) thymomas anti-insulin anti-GAD65 anti-Cyp1a2 anti-Cyp17 anti-Cyp21 overlap: anti-IFN type I MG ANA anti-AChR anti-striational anti-TPO EuroMG, Paris 01.12.2009

Generation and export of CD4+ T-Cells is the „sine qua non“ of MG development in type AB and B Thymomas (Ströbel; Buckley) MG+ Thymoma (Aire ----) MG-negative Thymoma (AIRE----) NT / Thymitis CD4 RA CD4 RA CD4 RA 16,0 % 26,7 % 2,5 % CD45 RA FITC CD 4 QR 18,3 % CD8 RA 25,3 % 4,5 % CD45 RA FITC CD 8 PE

Production of Regulatory T-cells is Impaired in Type Ab and B Thymomas (Ströbel, Ann Neurol, 2004) EuroMG, Paris 01.12.2009

Thymomas as an human model to study the relationship between AIRE expression and generation of FOXP3+ regulatory T cells Ströbel et al. Ann N Y Acad Sci, 2008 Expression / b-Actin 2 4 FOXP3 NT MG(+) MG(-) - * EuroMG, Paris 01.12.2009

AIRE Expression and Hassall‘s Corpuscles are correlated in B1 Thymoma Hassall+ B1 Thymoma AIRE & HC Status of Medullary Islands in B1 Thymomas Hassall--- B1 Thymoma AIRE+ AIRE- Hassall + 343 222 Hassall --- 34 123 P<0.0001 EuroMG, Paris 01.12.2009

In B1 Thymomas the number of FoxP3+ cells (likely Tregs) is NOT correlated with the presence of Aire+ cells FoxP3 per Aire+ cell FoxP3 per Aire-- cell Case # AIRE is probably NOT involved in the generation of Tregs in humans EuroMG, Paris 01.12.2009

Why is AIRE Expression Reduced in Thymomas? EuroMG, Paris 01.12.2009

NO ! Factors known to be associated with thymic Aire deficiency in thymic epithelial cells (TECs) Germ line mutation of Aire gene (APS I, APECED) Abnormal hyper-methylation of Aire promoter Lack of LTb, RANK/RANKL, TRAF, RelB / NFkB signalling Defective T-cell maturation in various SCIDs, Omen Syndrome, chronic cyclosporine treatment (> secondary defects of TECs) Defective TEC maturation in thymoma ? NO ! EuroMG, Paris 01.12.2009

Phenotypic Markers of Thymic Epithelial Cell Subsets MHC-IIlo DEC205+ MHC-IIhi DEC205+ MHC-IIlo Aire ----- MHC-IIhi Aire +/- Kyewski, 2007 Anderson, 2009

„Compartment-specific“ gene expression profiling of thymomas compared to purified cortical and medullary normal TECs 4606 Genes on OncoChip 153 53 Gotter et al. 2004 162 443 Cortical Genes Medullary Genes

Hypothetical „Developmental Concept“ of the WHO Thymoma Classification Mutation Other MG+Thymoma Types B1 Thymoma B1 Thymoma Oversize of Cortex MHC-IIlo Aire ---- MHC-IIhi Aire + EuroMG, Paris 01.12.2009

Summary AIRE deficiency in thymomas does not correlate with the MG Status of thymoma patients the efficiency of intrathymomatous CD4+ thymopoiesis the capacity to generate FoxP3+ Tregs AIRE deficiency in thymomas is an indicator of TEC immaturity and likely of functional insufficiency of TECs in A, AB, B2 and B3 thymomas The massively increased cortex/medulla ratio in B1 thymomas might be a structural basis for tolerance failure in AIRE+ B1 thymomas

Acknowledgments Mannheim: Philipp Ströbel Wen-Yu Chuang Christian Sauer Djeda Belharazem Li Li Norbert Gretz Alexander Marx Würzburg etc. Bei Huang Andreas Zettl Andreas Rosenwald Email: alexander.marx@umm.de