Scurfy Mouse (outside)Scurfy Mouse (outside)

• X-linked recessive inheritance• Lethality at 21-25 days• Wasting syndrome • Exfoliative dermatitis• Small, thickened ears

Wild type Scurfy

Scurfy Mouse (inside)Scurfy Mouse (inside)

• Severe autoimmune disorder• Hepatosplenomegaly• Enlarged lymph nodes• Multi-organ lymphocytic infiltrates• Elevated cytokines (GM-CSF, IL-2, -4, -5,-6,-7,

-10, IFN-, TNF)

Wild type Scurfy

CD4CD4++ T cells are hyper-active T cells are hyper-active in Scurfy micein Scurfy mice

Resting

3H-T

hy C

PM

150000

Scurfy

NLC

50000

100000

0Stimulated

Clark LB, et al., J. Immunol. 1999

Scurfy T cells are insensitive Scurfy T cells are insensitive to inhibitorsto inhibitors

CsA (ng/mL)

3H-T

hy C

PM

60000

10000

NormalIC50 3 ng/mL

ScurfyIC50 105 ng/mL

Cyclosporin A dose vs. IL2 response

100010 1001

20000

30000

40000

50000

0

Clark LB, et al., J. Immunol. 1999

IIPPEEXX

IImmune deficiency/dysregulationmmune deficiency/dysregulation

PPolyendocrinopathyolyendocrinopathy

EEnteropathy nteropathy (Often have Ab against gut epithelium)(Often have Ab against gut epithelium)

XX-linked inheritance-linked inheritance

IPEX – Outside (Clinical Findings)IPEX – Outside (Clinical Findings)• First described in 1982 by Powell et al. First described in 1982 by Powell et al. as a syndrome of diarrhea, as a syndrome of diarrhea, polyendocrinopathy, and fatal infection in polyendocrinopathy, and fatal infection in infancy.infancy.

• Neonatal onset diabetes mellitusNeonatal onset diabetes mellitus

• HypothyroidismHypothyroidism

• Enteritis (diarrhea/villous atrophy)Enteritis (diarrhea/villous atrophy)

• Hemolytic anemia & thrombocytopenia.Hemolytic anemia & thrombocytopenia.

• DermatitisDermatitis

• Dermatitis (eczema)Dermatitis (eczema)

• Death by 1-2 years of ageDeath by 1-2 years of age

IPEX – Inside (Autopsy IPEX – Inside (Autopsy Findings)Findings)

• Pancreas: islets of Pancreas: islets of Langerhans absent Langerhans absent with lymphocytic with lymphocytic infiltratesinfiltrates

• Intestine: Intestine: villous atrophy withvillous atrophy withlyphocytic infiltrateslyphocytic infiltrates

• Liver: CholangitisLiver: Cholangitis

• Spleen: enlargedSpleen: enlarged

• Lymph nodes:Lymph nodes: Follicular hyperplasia Follicular hyperplasia

• Thyroid: Thyroid: Lymphocytic infiltrates Lymphocytic infiltrates

• Lungs:Lungs: Consolidation/InflammationConsolidation/Inflammation

• Thymus: AtrophyThymus: Atrophy

Mapping of the Scurfy Mapping of the Scurfy genetic regiongenetic region

X Chromosome ~500 genes

FOXP3 FOXP3 Encodes a Novel Encodes a Novel Forkhead/ Forkhead/

Winged-Helix ProteinWinged-Helix Protein

PROLINE-RICH DOMAIN ZINC-FINGER LEUCINEZIPPER

FORKHEAD DOMAIN

N CNLS

Mouse and human scurfy gene Mouse and human scurfy gene products are highly conservedproducts are highly conserved

(86% identity)(86% identity)

What does FOXP3 do?What does FOXP3 do?

1 – Rheostat of the immune response

2 – Plays an essential role in the development and function of CD4+CD25+ Regulatory T cells

3 – FOXP3 may function as a transcriptional repressor of cytokine promotors

Khattri R, et al., J. Immunol. 2001

Hori S, et al., Science 2003Khattri R, et al., Nat. Immunol. 2003 Fontenot JD, et al., Nat. Immunol. 2003

Schubert L, et al., J. Biol. Chem. 2001

5 Habits of Highly 5 Habits of Highly Effective Transcription Effective Transcription

FactorsFactors

1.1. Nuclear import – may be regulatedNuclear import – may be regulated2.2. Interaction with partners (homo- or Interaction with partners (homo- or

heterodimerization) heterodimerization) 3.3. DNA bindingDNA binding4.4. Transcriptional enhancement or Transcriptional enhancement or

suppression at specific gene promotorssuppression at specific gene promotors5.5. Down regulation (nuclear export / Down regulation (nuclear export /

degradation)degradation)

FOXP3 is a rheostat of the FOXP3 is a rheostat of the immune responseimmune response

0 1x 5x0

*

**

Immune ResponseImmune Response

FOXP3 Expression Level (fold)

*

Nu

mb

er o

f C

D4+

CD

25+ T

reg

(% o

f C

D4+

cel

ls)

5-10%

15-20%

1 -- FOXP3 may function as a transcriptional repressor of cytokine promotors

2 – PBMC from patients with IPEX show poor up-regulation of IFN- production in response to activationChatilla TA, et al., JCI 2000Neives D, et al., Arch. Dermatol. 2003

Schubert L, et al., J. Biol. Chem. 2001

What is known about FOXP3 What is known about FOXP3 mediated gene transcriptionmediated gene transcription

CD4CD4++CD25CD25++ Regulatory T Regulatory T CellsCells

• Make up 5-10% of the normal CD4+ T cell population

• Characterized by expression of CD4 and CD25 on thecell surface at baseline. Cytotoxic T-Lymphocyteassociated Antigen-4 (CTLA-4), Glucocorticoid-InducedTumor-necrosis factor receptor-Related protein (GITR),Transforming Growth Factor (TGF), and Interleukin-10(IL-10) have all been reported to play a role in Treg function but are not specific to Treg cells

• Require activation and cell contact to repress proliferationof other T cells but do not appear to proliferate Themselves after activation.

GoalsGoals

• To better define the clinical phenotype of IPEXTo better define the clinical phenotype of IPEX

• To understand the molecular structure and To understand the molecular structure and function of FOXP3:function of FOXP3:• What does it do – transcriptional regulation?What does it do – transcriptional regulation?• How is it regulated?How is it regulated?• What does it regulate?What does it regulate?

• To use the Scurfy mouse and naturally occurring To use the Scurfy mouse and naturally occurring human mutations as models to study the human mutations as models to study the development and function of Regulatory T cellsdevelopment and function of Regulatory T cells

FOXP3 MutationsFOXP3 Mutations

Zn - finger

FKH (DNA binding)Zip

ATG

A38S C424Y

R397WR397WR397L

delGA(TGA)1317-1318delT(TGA)13115-1316I363V

F371C

AG

+4AG

A384TA384T A384TA384T

R347P

del251E

C565T splicingC565T splicing

L76frameshift

1 2 3 4 5 6 7 8 9 10 11 PolyA

FOXP3FOXP3 Mutations in IPEX Mutations in IPEX

a Newly identified mutationb The indicated amino acid change differs from that originally reported but is the sequence that results from the reported nucleotide change.

A.

B.

PROLINE-RICH DOMAIN ZINC-FINGER LEUCINEZIPPER

FORKHEAD DOMAIN

N C

3 4a 5 A 6 7a B 8a

CD 9a

E

F14

13a

Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 7 Exon 8 Exon 9 Exon 10 Exon 11 PolyA

1a 2a 101112a

Nucleotide Amino Acid Ref

1. 112G>Ta A38S2. 210G>Ta Q70H 3. 227delT L76fsX128 84. 303_304delTTa F102fsX1015. 543C>T Splicing 206. 750_752delGGA E251del 57. 776A>Ca H259P8. 1010G>Aa R337P9. 1099T>Ca F367L10. 1150G>A A384T 6,711. 1189C>T R397W 712. 1190G>Ta R397L 13. 1271G>Aa C424Y14 1293_1294delCT P431fsX457 615. AAUAAA>AAUGAA Polyadenylation 16

Nucleotide Amino Acid Ref

A. 747_749delAAG K250del 21 B. IVS9+459A>G 5C. 1040G>A R347H 21D. 1087A>G I363V 8E. 1113T>G F371Lb 7F. 1290_1309del/insTGG G430fsX452b 7

15

Human FOXP3 Promotor Structure

-1 1~4500 bp ~6000 bp

• Is there a tissue-specific repressor that limits FOXP3 expression to the CD4+CD25+ population (similar to the CD4 or Btk promoters) or are there tissue-specific enhancers that lead to expression only in this cell type?

Human FOXP3 Promoter Structure

-1 1~4500 bp ~6000 bp

SummarySummary1.1. We have identified 16 different mutations We have identified 16 different mutations

in patients with IPEX syndrome.in patients with IPEX syndrome.

• Patients with FOXP3 mutations have a Patients with FOXP3 mutations have a more severe phenotype than those more severe phenotype than those without mutations.without mutations.

• We have identified domains of the FOXP3 We have identified domains of the FOXP3 protein important for its function.protein important for its function.

• We have identified a deletion in the We have identified a deletion in the FOXP3 promoter that leads to low FOXP3 FOXP3 promoter that leads to low FOXP3 mRNA expression levels.mRNA expression levels.

A Better Picture of IPEXA Better Picture of IPEX25 kindreds (31 patients) with the IPEX phenotype:Clinical data obtained from 24 kindreds by questionnaire to PMD:

-FOXP3 mutations found in 14 kindreds (17 patients) – “IPEX”-No FOXP3 mutation in 10 kindreds (10 patients) – “IPEX-like”

IPEX: 100% have enteropathy diarrhea, often bloody100% have skin disease – eczema (most), erythroderma80-90% have one endocrinopathy – usually IDDM or

thyroid60% have glumerolonephropathy – often mild50% have autoimmune hematologic problems: hemolytic anemia, thrombocytopenia, or neutropenia.90+% have failure to thrive and ~40% have developmental delay or neurologic abnormality (seizures, etc).50-60% have one or more major infections (sepsis, osteo, etc.)

Treatment Options: CsA, FK506, Steroids, BMT

Skin Disease in IPEXSkin Disease in IPEX

The Expanding IPEX PhenotypeThe Expanding IPEX Phenotype

Kindred I-9:Kindred I-9:

• The original IPEX kindred. A large family with The original IPEX kindred. A large family with several affected males, some who lived into several affected males, some who lived into adulthood. adulthood.

• Mutation in the first Polyadenylation site – Mutation in the first Polyadenylation site – leads to decreased mRNA levels. Variations in leads to decreased mRNA levels. Variations in mRNA processing may account for variable mRNA processing may account for variable phenotype.phenotype.

• Immune activation (severe Immune activation (severe illness/vaccinations) caused rapid worsening of illness/vaccinations) caused rapid worsening of disease in some patients.disease in some patients.

Kindred I-11:Kindred I-11:

• 12 year old male with 12 year old male with Diarrhea starting at 6 Diarrhea starting at 6 m/o, bloody stools ~ 1 year ago – Dx’d with m/o, bloody stools ~ 1 year ago – Dx’d with Crohn’s disease by biopsyCrohn’s disease by biopsy

• Autoimmune Autoimmune Hyperthyroidism diagnosed at 5 Hyperthyroidism diagnosed at 5 y/o and ablated with Iy/o and ablated with I125125 therapy therapy

• Severe eczema over last 2 years with Severe eczema over last 2 years with numerous superinfections numerous superinfections

• Neutropenia over last 1-2 years with anti-Neutropenia over last 1-2 years with anti-neutrophil antibodiesneutrophil antibodies

• Elevated IgE (1080 gm/dl)Elevated IgE (1080 gm/dl)

• Mutation in the first base of Exon 5 (Splicing?)Mutation in the first base of Exon 5 (Splicing?)

Quantitative RT-PCR of Quantitative RT-PCR of FOXP3FOXP3 Expression in IPEX-like PatientsExpression in IPEX-like Patients

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

I/L-7 I/L-9 I/L-10 I/L-4 I-9 NC

Rel

ativ

e ex

pres

sion

of

FOX

P3

0.00

0.50

1.00

1.50

2.00

2.50

I/L-7 I/L-9 I/L-10 I/L-1 I/L-6 I-9 NC

Rela

tive

expr

essi

on o

f FO

XP3

PBMC

PHA-induced T cells

A

B

Scurfy Mouse - Scurfy Mouse - ImmunologyImmunology

• Mediated by CD4 Mediated by CD4 ++ T cells – adoptive transfer T cells – adoptive transfer

• Overproduction of cytokines IL-2, 4,5,6,10; Overproduction of cytokines IL-2, 4,5,6,10; IFNIFN, TNF, TNF, GM-, GM-

CSFCSF

• Mac-1 Mac-1 ++, , B220B220++

• Activation antigens CD69, CD25, CD80, CD86Activation antigens CD69, CD25, CD80, CD86

• Scurfy T cells Scurfy T cells sensitivity to tyrosine kinase sensitivity to tyrosine kinase Inhibitors (genistein, herbimycinA) and to Inhibitors (genistein, herbimycinA) and to cyclosporin Acyclosporin A

Scurfy Mouse - Scurfy Mouse - ImmunologyImmunology

• Mediated by CD4 Mediated by CD4 ++ T cells – adoptive transfer T cells – adoptive transfer

• Overproduction of cytokines IL-2, 4,5,6,10; Overproduction of cytokines IL-2, 4,5,6,10; IFNIFN, TNF, TNF, GM-, GM-

CSFCSF

• Mac-1 Mac-1 ++, , B220B220++

• Activation antigens CD69, CD25, CD80, CD86Activation antigens CD69, CD25, CD80, CD86

• Scurfy T cells Scurfy T cells sensitivity to tyrosine kinase sensitivity to tyrosine kinase Inhibitors (genistein, herbimycinA) and to Inhibitors (genistein, herbimycinA) and to cyclosporin Acyclosporin A

Expression of Foxp3Expression of Foxp3

• SpleenSpleen strongstrong

• ThymusThymus strongstrong

• CD4+ CD8-CD4+ CD8- strongstrong

• Cd4- cd8+Cd4- cd8+

• B220B220Barely detectableBarely detectable

All Forkhead/HNF3 Proteins All Forkhead/HNF3 Proteins Possess “Winged-Helix” Possess “Winged-Helix”

DomainDomain

• ~110 amino acid domain~110 amino acid domain• Involved in DNA bindingInvolved in DNA binding• Forkhead/hnf3 proteins act as Forkhead/hnf3 proteins act as

transcriptional regulatorstranscriptional regulators– (Examples of activators and repressors)(Examples of activators and repressors)

• Large gene family:Large gene family:– Found in all species; Involved in many Found in all species; Involved in many

different processes (eg., Development, different processes (eg., Development, organogenesis, tumorigenesis)organogenesis, tumorigenesis)

Scurfy gene/mutationScurfy gene/mutation• Proximal X-chromosomeProximal X-chromosome• Tightly linked (but not synonymous) Tightly linked (but not synonymous)

to Waspto Wasp• Novel forkhead geneNovel forkhead gene• 2 base pair deletion, frameshift2 base pair deletion, frameshift• Exon 8 (proximal to forkhead Exon 8 (proximal to forkhead

domain)domain)• Early termination – truncated proteinEarly termination – truncated protein• Loss of functionLoss of function

ScurfinScurfin(transcriptional regulator)(transcriptional regulator)

• 47 KD protein47 KD protein• 429 a.a. (mouse)429 a.a. (mouse)• 431 a.a. (human)431 a.a. (human)• 3 a helices3 a helices• 2 loop regions (“wings”)2 loop regions (“wings”)• Zinkfinger domainZinkfinger domain• Forkhead domain (116 a.a. Forkhead domain (116 a.a.

downstream of ZF mediates downstream of ZF mediates protein – DNA contactprotein – DNA contact

Scurfin - mutatedScurfin - mutated

• Failure to control T cell functionFailure to control T cell function

• Mutations of :Mutations of : ScurfinScurfin• CD95, CD95 ligandCD95, CD95 ligand• CTLA-4CTLA-4• TGF-TGF-

• Scurfy mouse resemblesScurfy mouse resembles• Knockout of CTLA-4 and TGF-Knockout of CTLA-4 and TGF-

SyndromeSyndrome WASWAS XLT/IXLTXLT/IXLT XNPXNP

XLT/ XLT/ thalassemia thalassemia

traittrait IPEXIPEX

genegene WASPWASPprot. – prot. –

WASPWASPprot.+prot.+

WASPWASPL270PL270P GATA-1GATA-1 FOXP3FOXP3

Thrombo-Thrombo-cytopeniacytopenia yesyes yes / yes /

(yes)(yes) nono yesyes yesyes

anemiaanemia (yes)(yes) (yes)(yes) nonoyes yes (Hgb (Hgb

synthesis)synthesis)yesyes

neutropenineutropeniaa

nono nono yesyes nono (yes)(yes)

endocrineendocrine nono nono nono nono yesyes

GI-GI-problemsproblems (yes)(yes) nono nono nono yesyes

FOXP3 - function (1)FOXP3 - function (1)

• Forkhead (FKH) proteins regulate Forkhead (FKH) proteins regulate lineage commitment, developmental lineage commitment, developmental differentiationdifferentiation

• Scurfin localizes to the nucleus Scurfin localizes to the nucleus FKH domain requiredFKH domain required

• FKH binding cites adjacent to NFAT sites FKH binding cites adjacent to NFAT sites were identified in cytokine Promotorswere identified in cytokine Promotors

FOXP3 - function (2)FOXP3 - function (2)

• FOXP3 transgenic mice overexpress scurfin FOXP3 transgenic mice overexpress scurfin decrease of Tcell number (ly nodes, spleen) decrease of Tcell number (ly nodes, spleen) poor proliferation poor proliferation decreased IL-2. IFN gammadecreased IL-2. IFN gammadecreased expression of CD40L decreased expression of CD40L reduced Ab responses to T dependent Agreduced Ab responses to T dependent Ag

• Lack of T helpLack of T help

APECEDAPECED

•AutoimmuneAutoimmune•PolyendocrinopathyPolyendocrinopathy

•CandidiasisCandidiasis•Ectodermal DysplasiaEctodermal Dysplasia

APECED - Clinical APECED - Clinical phenotypephenotype

• Chronic mucocutaneous candidiasisChronic mucocutaneous candidiasis• HypoparathyroidismHypoparathyroidism• Addison DiseaseAddison Disease• Diabetes, type 1, gonadal atrophy, Diabetes, type 1, gonadal atrophy,

pernicious anemia, hypothyroidismpernicious anemia, hypothyroidism• Autoimmune hepatitisAutoimmune hepatitis• Ectodermal dysplasia: enamel, nails, Ectodermal dysplasia: enamel, nails,

alopecia, vitiligo, calcification of TMalopecia, vitiligo, calcification of TM

APECED -Founder EffectAPECED -Founder Effect

Finnish

1:25K

67

100%

79%

72%

52%

R257X

Exon 6

Sardinia

1:14K

11

83

93

73

not rep.

R139X

Exon 3

Iran Jews

1:9K

16

18

96

22

most

Y85C

Exon 2

N-America

16

75

100

93

not rep.

C322

de113

bp>stop

Incidence

N

MCC

Hypopara

Adrenal

Nails

Predomin.AIRE

mutation

APECED - genetics (1)APECED - genetics (1)

• Mapped to 21 822.3Mapped to 21 822.3• Gene: AutoIimmune REgulator (AIRE)Gene: AutoIimmune REgulator (AIRE)• Function: transcriptional regulator, Function: transcriptional regulator,

has characteristic domains for has characteristic domains for Induction and maintenance of Induction and maintenance of immune functionimmune function

• 14 exons, encoding a 545 aa protein14 exons, encoding a 545 aa protein• Expressed in Thymus epithelium, Expressed in Thymus epithelium,

lymphnodes, spleen, fetal liverlymphnodes, spleen, fetal liver

APECED genetics (3)APECED genetics (3)

• 45 known mutations of AIRE45 known mutations of AIRE

• Distributed throughout the AIRE geneDistributed throughout the AIRE gene

• Variable clinical phenotypeVariable clinical phenotype

type of mutation type of mutation environmental environmental HLA-DRHLA-DR (B 103 - Addison (B 103 - Addison disease)disease)

(B 104 - alopecia)(B 104 - alopecia)

APECED Aire-knockout APECED Aire-knockout micemice

• APECED featuresAPECED features• Lymphocytic infiltrates (ovary, liver)Lymphocytic infiltrates (ovary, liver)• Atrophy of thymus, adrenals, ovaries Atrophy of thymus, adrenals, ovaries

(42%)(42%)• Autoimmune hepatitis (50%)Autoimmune hepatitis (50%)• Autoantibodies (73%), testes (8/15), Autoantibodies (73%), testes (8/15),

liver (3/15), pancreas (7/15), liver (3/15), pancreas (7/15), adrenal gland (3/15)adrenal gland (3/15)

• In vitro hyperproliferation of sensitized In vitro hyperproliferation of sensitized LyLy

ALPS - geneticsALPS - genetics• Autosomal dominant (mostly), or Autosomal dominant (mostly), or

recessiverecessive

• Mutations of Mutations of APT1APT1 encoding encoding Fas/APO-1/CD95 is present in most ALPSFas/APO-1/CD95 is present in most ALPS

• Mutation of FasL or Caspase 10 is rareMutation of FasL or Caspase 10 is rare

• Animal models:Animal models: lprlpr mutation (Fas) mutation (Fas) gldgld mutation (FasL) mutation (FasL)

phenotype is strain phenotype is strain dependentdependent

malabsorption

IPEX (FOXP3)

Autoantibody

Lymphocyticinfiltrates

Tx: immuno-suppressive

Immunedysregulation

Diarrhea

Skinrashes

Apoptosis

Double negative T cells

lymphoma

Hyper

IgG, M, A

Alopecia

Endocrino-pathies

T cell defect

Hepatitis

Ectodermaldysplasia

APECED(Aire)

ALPS(Fas/FasL/

Caspase10)