do Pujol Borrellnology Division, VHIR

Biology Physiology and Immunology, Faculty of Medicine, Universitat Autònoma de Barcelona

RG-VHIR ENCOUNTER on RARE DISEASES

“From the analysis of tolerance in thyroid autoimmudiseases to the generation of an approach to rare dof the immune system"

18th Jan 2012

Marta Ruiz - IGTPEva Martínez-Cáceres - BSTMaria del Pilar Armengol - IGTPManel Juan Otero - BSTMarco A. Fernández -IGTPMarta Vives - IGTPRaquel Planas - IGTPRoger Colobran- IGTP-VHIRManuel Hernández - HVHMonica Martinez Gallo - HVHPere Soler Palacin - HVHAndrea Martin - HVH

Dolores Jaraquemada, Dept of ImmuUABBruno Kyewski, Dept Immunology, GCancer Research Center

c de San i Teixits

Universitat Autònoma de BarcelonaFacultad de Medicina

m: onal, MICNNe Salud Carlos III

Autoimmune diseaseMost entities have a complex pattern of inheritance, HLA, CTLA, PTPN2TREX1, CD25 etcA few paradigmatic are mendelian (APECED, IPEX,…)Many are rare

mmunodeficienciesMost entities are mendelianThe most common have a complex pattern of inheritanceMost are rare

All result from a failure of the mechanisms of toleranceThey affect almost every organ and tissues:‣ Skin: pemphigus, pemphigoid, vitiligo‣ Stomach: Atrophic gastritis → Pernicious anemia‣ Liver: Chronic active hepatitis, Primary biliary cirrhosis‣ Endocrine glands: Type-1 diabetes, Thyroid autoimmune disease, Addison dis‣ CNS: Multiple sclerosis, onconeural autoimmune syndromes,‣ PNS: Guillain-Barré‣ Muscle: Polymyositis, Myasthenia gravis‣ Joints: Rheumatoid arthritis, Ankylosing Spondilitis,‣ Eye: Autoimmune uveitis and retinitis‣ Systemic: SLE, vasculitis, systemic sclerosis‣ etc, etc

Incidence 100.000

Prevalence 100.000

Female(%) Age og Onset±SD

Geograpethnic

toimmune diseases ND 2,482 90 48 GD 59 HT

betes 10-15 192 45 10 (6-13)* Y

disease ND 14 93 33

Anemia ND 150 67 Y

us 0,7 7 66 71

a gravis 0,41 5 73 40

ND 400 52

lerosis 3 58 64 37 (35-65) Y

tive Hepatitis ND 0,4 88

iary Cirrhosis 0,9 3,5 89

ease 3,9 14 94 59 (43-75)

clerosis 1,4 4,4 92 50 (35-65)

d Arthritis 40 1,008 75 58 (42-74)

upus Erythematosus 7,3 24 88 40 (30-50) Y

3 7%

✓A failure of immunotolerance‣A complex system that limits the tendency of the

immune system to react with self componentss instructives transferablehas memory

✓Levels of tolerance‣Periphery, dependent on the secondary lymphoid

organs and the ignorance to peripheral tissue‣Central, dependent on the immune system primary

organs

Exit to citculatioperipheral tissu

immunoprivilegedsubjected

ImmunoloIgnoran

Resting immature DC

Active mature DC

g

XXLymph Node

Tolerogenic DC

Tregs

Ag

Ag

p

X Negatively selected T lymphoc

Treg

Anergic lymphocytePositively selected lymphocyteTransitional B lymphocyte

Selected T lymphocyte

Target O

, ghyroid with a MoAb to HLA-R

Immunological ignorance Immunological ignorance broken?

y

ol-Borrell R, Hanafusa T, Chiovato L, Bottazzo GF. (1983) Lectin induced expression of DR antigen ohuman cultured follicular thyroid cells. Nature 304:71-3nafusa, T., Pujol-Borrell, R., Chiovato, L., Russell, R.C., Doniach, D., and Bottazzo, G.F. 1983. Aberrexpression of HLA-DR antigen on thyrocytes in Graves' disease: relevance for autoimmunity. Lance2:1111-1115.

ttazzo GF, Pujol-Borrell R, Hanafusa T, Feldmann M. (1983) Role of aberrant HLA-DR expression anantigen presentation in induction of endocrine autoimmunity. Lancet 2:1115-9

o they play a significant pathogenic role?Evidence of proliferation, apoptosis (affinity maturation) & Ig gene revision → true functional GCs (TLO)Antigen specificity: Tg & TPOngol et al. Amer J. Pathology 2001; 159: 861-873.

HEV

d of an unbiased approach!!

IgG

TPO Tg

ch signals are driving the immune esponse from the target tissue?

TB20

9

TB37

7

TB37

3

TB42

3

TB39

4

TB42

6

TB41

3

TB44

23m 9m 10m 40m 48m 36m

Type II84

Viral Response

iol M, Armengol Barnils MP, Colobran Oriol R, Sánchez Pla A, Borràs Serres F-E, Lucas-Martin A, nez-Cáceres EM, Pujol-Borrell R. Analysis of the cumulative changes in Graves' disease thyroid glans to IFN signature, plasmacytoid DCs and alternatively activated macrophages as chronicity determirs J Autoimmun 2011; 36:189–200

__________he immune response builds up in the target organ wit

meome of the changes that determine chronicity may belated with the antigen presentation and IFNs pathwanderneath the inflammatory changes there are parative pathways activated ➡ M2nbiased approaches held the promise of contributing veal chronicity determinants → Biomarkershe autoimmune response is probably driven from the rget organ at least after 30 months

Thymus

hymic selection

X

Bone Marrow

Bone sele

X Negatively selected T lymphocyte

Treg

Anergic lymphocyte

Positively selected lymphocyteTransitional B lymphocyte

b-actin

GAPDH

H-YAlbumin

Tg

TPO

Insulin

GAD 67

GAD 65

Glucagon

MBP

MOG &

PLP

Bcristallin

S100

AChR

AChR

AChR

AChR &

Retinal S

g p y

Kyewski & J Derbinski Self representation in the thymus: an extended view Nature Rev Immunol 4: 688 698 20

______ ______________ ____ ___ _____ __ _______ ___________ __ __ _______ _____ __ __ _____

____ _______ ____ _______ _______ ______ __ ___ ____________ _______ _________ ______ _______ __________ __ ___ _____________ __ __ _______ __ __

abetes susceptibility gene was maped to insulin promoterBell GI et al Diabetes 33: 176-183, 1984Davies, JL et al Nature 371: 130-136, 1994. RR IDDM2 1.9-5.0VNTR, ACAGGGGTSYGGGG Class I (26-63 repeats) Class III (141-209 repeats),inked to a 1127 PstI siteotective alleles determine higher insulin expression in the thymus

Pugliese A et al 1997Vafiadis P et al 1997

idual variability of expression in the thymus may confer susceptdisease

White bars protective allele; black bars, predisposing allelesTotal insulin transcript to AIRE transcript correlation

ter, L, Ferrer-Francesch, X, Sospedra, M, Caro, P, Juan, M and Pujol-Borrell, R Insulin alleles ammune regulator (AIRE) gene expression both influence insulin expression in the thymus. J Aut312-8 (2005)

HR and central tolerance in GravesBasedow disease

alleletransquan

b-actin

GAPDH

H-YAlbumin

Tg

TPO

Insulin

GAD 67

GAD 65

Glucagon

MBP

MOG &

PLP

Bcristallin

S100

AChR

AChR

AChR

AChR &

Retinal S

Hypoparathyroidism

T1D

Thyroid AutoimmuneDisease

Chronic Active HepatitisVitiligo

Addison Disease

Moniliasis

APECED (Autoimmune Polyendocrinopathy candidiasis ectodermal dystroplso known as APS-1 (Autoimmune Polyendocrine Syndrome-type 1) is a rec

mendelian diseasePositional cloning → AIREAIRE is a transcription factor expressed in mTECsAIRE correlates with transcription gene expressio

Giraud M et al. PNAS 2012;109:535-540

✓APECED - AIRE → Promiscuous gene expression and central tolerance

✓IPEX - Foxp3 - Tregs✓C2 and other C genes plus fas and fasL, lup

like syndrome

y2011Pere Soler PalacinXavier de GraciaConsuelo ModestoManuel HernandezMonica Martinez-Gallo

Formerly Physiciens andClinical ScientistsTeresa EspañolIsabel CaragolDragomira DetkovaManuel Hernandez

Genes Prevalence New cases Carriers

<1/500,000 2 4

~1/100,000 5 10~1/100,000 5 10

E1C1 ~1/200,000 3 4

1/50,000-100,000 7 14

<1/500,000 2 4

<1/500,000 2 4

D 28 50fibrosis <1/2,000-3,500 13000

emis

ne Chr exons nt aa Associated disease Disease incidence

10 3 2226 (3) 555 Familial Hemophagocytic Lymphohistiocytosis type 2 (FHL2)

17 32 8515 (16) 1091 Familial Hemophagocytic Lymphohistiocytosis type 3 (FHL3)

) 6 2 1359 (2) 288 Familial Hemophagocytic Lymphohistiocytosis type 4 (FHL4)

11 2 4099 (9) 1043 Spectrum of Severe Immunodeficiencies

11 2 2475 (4) 527 Spectrum of Severe Immunodeficiencies

X 19 4543 (17) 659 X-linked agammaglobulinemiatype 1

X 4 1583 (4) 129 Lymphoproliferative syndrome X-linked type 1 (Duncan disease)

10 9 3982 (8) 335 Autoimmune Lymphoproliferative Syndrome (ALPS)

X 13 4508 (13) 570 Chronic Granulomatous Disease (CGD)

e 21 14 7730 (12) 545Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED)

< 1/25.000

Gene Chr Nº exons

nt sequenced (amplicons)

Nº amino acids Associated disease Disease

incidence

nding protein 19 19 5959 (10) 593 Familial Hemophagocytic Lymphohistiocytosis type 5 (FHL5)

e 3) 19 24 9139 (17) 1124 Severe Combined Immunodeficiency Disease (SCID)

2 receptor, X 8 4337 (6) 369

-X-linked severe combined immunodeficiency (XSCID),-X-linked combinedimmunodeficiency (XCID)

X 12 4715 (9) 502 Wiskott-Aldrich Syndrome (WAS)

X 7 3926 (7) 497X-linked familial hemophagocytic lymphohistiocytosis / X-linked lymphoproliferative disease

ing protein 2) 3 7 6186 (6) 480

-Monocytopenia and MycobacterialInfection (MonoMAC) syndrome-Syndrome associated with defects in dendritic cells, monocytes, and B and NK lymphoid cells (DCML)

Clinical and laboratory presentation of Familial Hemophagocytic

LymphoHistiocytosis (FHLH) in a patient witha novel homozygous deletion in PRF1 gene.

munology Division and Molecular Medicine Program, 2Pediatric Infectious Diseaseand Immunodeficiencies Unit, 3Pediatric Oncology and Hematology Division Hospital Univ. Vall d’Hebron i Vall d’Hebron Institut de Recerca

4Cell Biology, Physiology and Immunology and 5Pediatric DeptsUniversitat Autònoma de Barcelona

Martínez-Gallo1,4, A. Martín-Nalda2, R. Colobran1, F. Caracseghi2,rnandez-Gonzalez1,4, C. Díaz de Heredia3, P. Soler-Palacin2,5, J.L. Dapena3

eeks-old baby girlrile urinary tract infectionnomegalyressive liver failure

ent died a few days after from multiorganic failure, with severe lactic acidosi

dmission:ncytopenia: Anemia (Hb: 7-8g/dl)

Neutropenia (0.5x109/L)Trombocytopenia (13.9x109/L)

w plasma fibrinogen: 1.90 g/Lh plasma ferritin: 51336 ng/mlh plasma triglycerides: 152 mg/dL

H: 4339 UI/mlemophagocytosis was detected in a bone marrow aspiration sample

OCYTE ETS % x 109/L Ref.

values

3+) 89.6 2868 60-85%D4+ 26 0.802 41-68%D8+ 64 2.135 9-23%in CD4% 90 in

2519+) 5.29 0.169 4-26%

16+) 4.5 0.147 3-23%

Effector to Target ratio

common symptoms: Fever (91%)Progressive hepatosplenomegaly (84-90%)

Neurologic symptoms (47%)Skin manifestations non-specific (43%)Often triggered by infectionsViralEBV, CMV…

H is a rare autosomal recessive disorder of immune dysregulation associatedntrolled T cell and macrophage activation and high IFN-, TNG- , GM-CSF, IL-6, I, IL-18, sIL-2Rence of FHLH is 0,12/100,000 children born per year.l unless a HSCT is performed.0% Symptoms of HLH in the first year of life

ratory findings: CytopeniasTrombocytopenia, anemia, neutropeniaHigh serum ferritin levelHypofibrinogenemia / hipertrigliceridemiaLiver disfunctionLactate dehydrogenaselactic acidHemophagocytosisLow o absent NK cell activity

High serum levels of CD25s

MACPF domain

RF1r10

exon1

exon2 exon3

p.Gly477fs X479

4483delG

Rattus norvegicus

Mus musculus

Homo sapiens

New variant

LDFGDVLLATGGPLRLQVWDQDSGRDD

LDFGDVLLATGGPLRLQVWDQDSGRDD

LDFGDVLLATGGPLRLQVWDQDSGRDD

LDFGDVLLATGGPLX

479

481

LDFGDVLLATGGPLRLPVWDQDSGRDDQ481P variant

g

5´… GCC ACA GGG GGG CCC CTG AGG …3´

5´… GCC ACA GGG GGC CCC TGA GGT …3´

STOPCODON

A T G G P L R

A T G G P

d type

ent

PRF1 exon2 exon3

Patient

Father

Mother

4483delG

477 479

GRANZYME B PERFORIN

Rab27a

Munc13-4

microtubules

Syntaxin11Munc 18-2

FHL2

FHL3

FHL5FHL4

T

GS

LH1 10% Cr9q21-22LH2 20-50% PRF1LH3 30-40% UNC13DLH4 n=10 STX11LH5 10% STXBP2

etic basis of FHLH:

CHS

LYST

Low TRECS

PBL phenotype

IL2RG (50%)IL7RA (10% ) JAK3 (10%)

RAG1RAG2DCLRE1C (2%)ADA (8%)

Molecular analysis of candidate genes

BTK (50%)

T‐B+ Ig IgT‐B‐ T‐B+NK+

✓Develop strategies to identify new genes causing immunodeficiencies

✓Develop semi-automated methods for screening the known mutations

✓To better understand autoimmunity throughstudy of “immune dysfunctional genetic phenotypes”

✓To identify better biomarkers for therapeuticresponses in Graves’ disease