Role of AIRE in Thymoma-associated Myasthenia Gravis ?

Role of AIRE in Thymoma-associatedMyasthenia Gravis ?

University Medical Centre Mannheim

Thymoma

AIRE-PHD1

AIRE in mTEC

Alexander Marx, MD

University of Heidelberg

EuroMGParis, 01-12-09

APECED/APS-1 and AIRE

• The Autoimmune regulator (AIRE) is a gene mutated in autoimmune polyendocrinopathy candidiasis ectodermal dystrophy or APECED

• 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)

Thymus

EuroMG, Paris01.12.2009

• Virtually 100% of patients have auto-Ab to type I interferons

AIRE is mainly expressed in keratin-positive medullary thymic epithelial cells (mTEC), less commonly in dendritic cells

Aire

Pan-Cytokeratin (mTEC)

EuroMG, Paris01.12.2009

Regulation of > 1000 genes

Aire

Thyroglobulin

Aire

p450_c17

Pärt Peterson

Aire drives „promiscuous“ expression of „tissue-specific antigens“ (TSA)

and some other genes mainly in medully thymic epithelial cells

AIRE-dependent TSA: Insulin, TPO, casein , -subunit of AChR, p450, S100 A8

EuroMG, Paris01.12.2009

Mathis & Bennoit, ARI, 2009

Functional effect of AIRE-driven autoantigen expressionin medullary thymic epithelial cells (mTECs)

mTEC

Cross-presentation

by dendritic cells (DC)

Promiscuous expression

and improved prentation

of autoantigens & MHCII

DC

Apoptotic mTEC

Tolerance Induction

EuroMG, Paris01.12.2009

-200 0 200 400 600 800 1000 1200

Hassall n

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200A

IRE

n

Significant Correlation between Prevalence of HCs and Prevalenve

of AIRE+ Cells in Normal Human Thymus

r = 0.81p< 0.00001

EuroMG, Paris01.12.2009

qHassFläch NT Ausreisser qAIRE_Fläch NT Ausreisser

1 2 3 4 5 6 7 8 9

Dekade

-2

0

2

4

6

8

10

12

14

16

18

20

22

24

Age-dependent decline of Hassall‘s Corpuscles and

AIRE+ cells in normal human thymus

Age, Decades

Ha

ss

all

‘s C

orp

us

cle

s &

Air

e+

Ce

lls

p

er

Are

a

HCs Aire+ Cells

Mean HCs per area NTOutlier (HCs)Mean Aire+ per area NTOutlier (Aire+)

Aire/HCs ~ 2 - 3 (0 - 40y)

~ 1 -1.5 (41- 90y)

EuroMG, Paris01.12.2009

A AB B1 B2 B3 „C“

„Medullary“ Mixed „Cortical“ TSCC

Among WHO-defined Thymomas Hassall‘s Corpuscles are common only in Type B1 Thymomas („predominantly cortical“)

WHO-Classification: 1999, 2004

Histogenetic Classification: Marino & Müller-Hermelink, 1985; Kirchner, 1992

EuroMG, Paris01.12.2009

>95% of thymomas, including all type A & AB thymomas do not express AIRE* (Ströbel, 2007)

AIRE---

Thymus Type B1§

§ ~50% of type B1 thymomas are Aire+

*AIRE, Autoimmune Regulator

Loss of AIRE and AIRE-related self-antigen expression

in >95 of thymomas

Ströbel et al. J Pathol 2007

Mathis & Bennoit, ARI, 2009

Does AIRE-deficiency in thymomas elicit autoimmune diseases, including APECED ?

mTEC

Cross-presentation

by dendritic cells (DC)

Promiscuous expression

and improved presentation

of autoantigens & MHCII

DC

Apoptotic mTEC

Autoimmunity

NO

NO

ImpairedClonal Deletion

EuroMG, Paris01.12.2009

Minimal overlap of clinical and serological autoimmune features in APS-1 patients and patients with AIRE(-)

thymomas

anti-insulinanti-GAD65anti-Cyp1a2anti-Cyp17anti-Cyp21

ANAanti-AChRanti-striationalanti-TPO

overlap:anti-IFNtype I

MG

EuroMG, Paris01.12.2009

Generation and export of CD4+ T-Cells is the „sine qua non“ of MG development in type AB and B Thymomas (Ströbel; Buckley)

CD

45

RA

FIT

CC

D4

5 R

A F

ITC

NT / Thymitis

CD4 RA CD4 RA16,0 % 26,7 % 2,5 %

CD 8 PE

18,3 %

CD8 RA25,3 %

CD8 RA4,5 %

CD8 RA

CD 4 QR

MG-negative Thymoma (AIRE----)

CD4 RA

MG+ Thymoma(Aire ----)

% C

D4

+C

D2

5+

(g

ate

do

n C

D4

+ C

D8

-ce

lls)

Thymus MG(+) MG(-)

100 101 102 103 104

100

101

102

103

104

CD

4

CD25

Thymus

Thymoma

100 101 102 103 104

100

101

102

103

104

0

8

16p < 0.05

Thymomas

Production of Regulatory T-cells is Impaired

in Type Ab and B Thymomas (Ströbel, Ann Neurol, 2004)

EuroMG, Paris01.12.2009

Thymomas as an human model to study the relationship

between

AIRE expression and generation of FOXP3+ regulatory T

cells

00

200

400

600

800

1.000

Thymus Thymomas

AIRE-1AIRE-2p < 0.05

mol

ecul

es/

-act

in

Thymomas Thymus

MHC levelTreg

Exp

ress

ion

/ -

Act

in

0

2

4 FOXP3

NT MG(+) MG(-) -

* *

Ströbel et al. Ann N Y Acad Sci, 2008

EuroMG, Paris01.12.2009

Auswertung medulläre Areale in B1 Thymomen

H+A+ H-A+ H+A- H-A-

1

Var4

-10

0

10

20

30

40

50

AIRE+ AIRE-

Hassall + 343 222

Hassall --- 34 123

P<0.0001

AIRE & HC Status of Medullary Islands in B1 Thymomas

H+A+H--A+H+A--H--A--

AIRE Expression and Hassall‘s Corpuscles are correlated in B1 Thymoma

Hassall+ B1 Thymoma

Hassall--- B1 Thymoma

EuroMG, Paris01.12.2009

mean FOXP3 pro AIRE+ mean FOXP3 AIREneg

1 2 3 4 5 6 7

Fall No

-1

0

1

2

3

4

5

6

7

8

mean FOXP3 pro AIRE+ Ausreisser Extremewerte mean FOXP3 AIREneg Ausreisser Extremewerte

1

Var9

-2

0

2

4

6

8

10

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, Paris01.12.2009

Why is AIRE Expression Reduced in Thymomas?

EuroMG, Paris01.12.2009

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 LTRANK/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 ?

EuroMG, Paris01.12.2009

NO !

Phenotypic Markers of Thymic Epithelial Cell Subsets

MHC-IIlo

Aire -----MHC-IIhi Aire +/- Kyewski, 2007

Anderson, 2009

MHC-IIlo

DEC205+MHC-IIhi DEC205+

Medullary GenesCortical Genes

153

53

4606 Geneson OncoChip

443

„Compartment-specific“ gene expression profiling of thymomas compared to purified cortical and medullary normal TECs

162Gotter et al.

2004

Hypothetical „Developmental Concept“ of the WHO Thymoma

Classification

MHC-IIlo

Aire ----MHC-IIhi

Aire +

Mutation

B1 ThymomaOther MG+Thymoma Types

EuroMG, Paris01.12.2009

B1 Thymoma

Oversize of

Cortex

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

Mannheim:Philipp Ströbel

Wen-Yu Chuang

Christian Sauer

Djeda Belharazem

Li Li

Norbert Gretz

Alexander Marx

Würzburg etc.

Bei Huang

Andreas Zettl

Andreas Rosenwald

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

Acknowledgments