Scurfy Mouse (outside) X-linked recessive inheritance Lethality at 21-25 days Wasting syndrome...
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Transcript of Scurfy Mouse (outside) X-linked recessive inheritance Lethality at 21-25 days Wasting syndrome...
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
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?
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
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