1

Job’s Syndrome:Establishing Mouse Models

for a Complex Human Disease

John P Manis M.D.Joint Program in Transfusion Medicine

Children’s Hospital BostonDepartment of PathologyHarvard Medical School

HyperHyper IgEIgE SyndromeSyndrome(Job(Job ’’ss SyndromeSyndrome ))

First Patients were described in 1966Classical Triad- Eosinophilia

EczemaRecurrent Skin & Pulmonary Infections

1972: IgE was recognized as elevated in serum from patients

Classification expanded and in 1979Recognition of skeletal and connective tissue abnor malities

HyperHyper IgEIgE SyndromeSyndrome(Job(Job ’’ss SyndromeSyndrome ))

Two types of disease now recognized:

Type 1Sporadic- more than 90% of cases

Skeletal and connective tissue abnormalitiesBone fractures, scoliosisRetention of deciduous teethHyperextensibilityPneumatocele

Type 2Familial with autosomal recessive inheritance

Lack developmental abnormalitiesViral infections (HSV, molluscum contagiosum)absence of pulmonary cysts

Hyper IgE SyndromeHyper IgE SyndromeAutosomal RecessiveAutosomal Recessive

�� DescribedDescribed in 2004 in a in 2004 in a groupgroup ofof patientspatients thatthat lackedlacked skeletalskeletal defectsdefects

�� IntactIntact TCR TCR signalingsignaling , , butbut withwith recurrentrecurrent viralviral infectionsinfections and the and the presencepresence ofof infectionsinfections withwith intracellularintracellular pathogenspathogens ((mycobacterialmycobacterial ) )

suggestedsuggested cytokinecytokine signalingsignaling defectsdefects : : defectivedefective interferon interferon alphaalpha --mediatedmediated signalingsignalingdefectivedefective ILIL--12 12 signalingsignaling

��TYK TYK 22 mutationmutation foundfound in a single in a single patientpatient in 2006in 2006��MemberMember ofof the JAK family the JAK family kinaseskinases

��DefectiveDefective typetype 11 IFN, ILIFN, IL--12, IL12, IL--23, IL23, IL--10, IL10, IL--66

Clinical inflammatory changes and susceptibility to viral infections led work to study IFN signaling pathways uncovering mutations in Tyk2 for Autosomal recessive patients

Minegishi 2008

Hyper IgE SyndromeHyper IgE SyndromeAutosomal DominantAutosomal Dominant

�� MultisystemMultisystem disorderdisorder :: immune system, skeletal/dental system, immune system, skeletal/dental system, soft soft tissuetissue //connectiveconnective tissuetissue changeschanges ((hyperhyper extensibilityextensibility ))

�� AAbnormalitiesbnormalities ofof head and facehead and face -- distinctivedistinctive facies facies withwith broadbroad nosenose andandforeheadforehead , , deepdeep set set eyeseyes

��BoneBone fracturesfractures , , nonnon --traumatictraumatic , , hyperhyper extensiveextensive jointsjoints , , scoliosisscoliosis , , retainedretained deciduousdeciduous teetteet

��RRecurrentecurrent skinskin abscessesabscesses and and cystcyst formingforming pneumoniapneumonia

��InfectionsInfections and and inflammationinflammation : : usuallyusually StaphylococcalStaphylococcal��DysregulatedDysregulated locallocal inflammatoryinflammatory changeschanges

��CellCell signalingsignaling: : IntactIntact ILIL--12, 12, IFNIFN--alphaalpha, , defectivedefective forfor ILIL--6, IL6, IL--10 10 suggestedsuggested StatStat 33 signalingsignaling defectsdefects

2

Clinical Features of Patients with AD-HIESClinical Features of Patients with AD-HIESMineguishi and coMineguishi and co --workers found STAT3 mutations affecting the workers found STAT3 mutations affecting the DNADNA--binding domain, binding domain, associated w/ clinical manifestations of HIES associated w/ clinical manifestations of HIES (Mineguchi et al., Nature 2007)(Mineguchi et al., Nature 2007)

STAT3STAT3

Stat 3 has a central signaling roles for multiple p athways including:

IL-6gp 130 family (leptin, LIF)MAPKSHP pathways in T cellsmalignant transformation of cells (Ras)mutations associated with Crohn’s diseasemutations associated with lymphoma

Holland 2009

Mutant Stat 3: Mechanism

TH17 T cell differentiation is defective in patien tsWith AD HIES

Absent TH17 T cells in patients with AD-HIESReported by multiple groups- accounts for defective immunity

Infections however are primarily lung and skinKeratinocytes and bronchial epithelial cells are mor e dependent

on TH17-mediated cytokines for function

Other T cell subsets involved?Defective antigen specific antibody production:

encapsulated pathogens

TH17 T cell defect does not explain the multiple immune defects

Woellner et al 2010

Characteristics of patients with Stat 3 mutations i n AD HIES

1/3 of AD HIES patients have no Stat 3 mutation

Spectrum of mutations in Stat3

Woellner et al 2010

3

Generate a mouse model of Job’s Syndrome

Challenges:

-Stat 3 is important in maintaining ES cell pluripotency, thus mutant

Stat3 cannot be expressed in targeted ES cells for germline expression

-Job’s Syndrome affects many tissues, it would be useful to express the

mutation in a tissue specific fashion

-Stat3 null mutant mice are embryonic lethal, heterozygous mice have

no phenotype c/w Job’s Syndrome

STAT3 cDNA

R382Q

HA

AscIAscI

loxploxp

Selectable markers

•Neomycin•Diptheriatoxin A

R382QR382Q--STAT3 in ROSA26 STAT3 in ROSA26

STOP

Eco RI Eco RI15.4 Kb

probe A

Ex1 Ex2 Ex3

SA NEO STAT3 R328Q IRES-EGFP pA

Eco RI Eco RI5.1 Kb

loxP loxP frt frt

Rosa26 locus

SA NEO STOP STAT3 R328Q IRES-EGFP pA DT

Targeted allele

Targeting construct

Eco RI Eco RI

TARGETING ES CELLS

R382 Stat 3 is targeted into the Rosa 26 locus, and driven off the endogenous promoter

0 102 103 104 105

FITC-A

0

100

200

300#

Cel

ls

99.9 0.042

0 10 2 103 104 10 5

FITC-A

0

100

200

300

# C

ells

1.6 98.4

GFP

0 102 103 104 105

FITC-A

0

20

40

60

80

100

% o

f Max

GFP expression by FACS- gate on ES population define d by FSC vs SSC

AC deleted cloneundeleted clone

Overlay:

undeleted clone

AC deleted clone

R382Q Stat 3 is overexpressed in a 1:1 ratio with wildtype Stat 3

RQ

70

100

130

kDa

WT

clon

e (T

C1)

AC del

eted

clo

neW

T cl

one

(TC

1)

AC del

eted

clo

ne #

1

AC del

eted

clo

ne #

2

WT

clon

e (T

C1)

AC del

eted

clo

ne #

1

AC del

eted

clo

ne #

2

total cell extracts ααααHA IPs ααααHA eluate

IPs

αααα STAT3

αααα tubulin

Confirmation of R382Q protein expression of Stat3 i n Gene-targeted ES cells

4

Nuclear fraction of Stat 3 is maintained at wildtype levels in the

R382Q over expressing ES cells

Nuclear fraction

WT

AC

-DE

L #

1

AC

-DE

L #

2

UD

EL

70

100

130

α α α α STAT3

Cytoplasmic fraction

70

100

130

WT

AC

-DE

L #

1

AC

-DE

L #

2

UD

EL

α α α α STAT3

α α α α tubulin

Role of Stat3 R382Q in ES cell

differentiation

Does DNStat3 R382Q alter differentiating patterns of ES

cells, account for the variable phenotypes seen in patients?

What lineages are affected most during differentiation of

R382Q ES cells

35

55

70100

130

WTS

tat3

+ R

A

WT

α α α α OCT3/4

αααα tubulin

Oct4 is downregulated at early passages in R382Q ES cells

WTS

tat3

DN

Sta

t3. 1

DN

Sta

t3. 2

KDa

Oct4 Nanog Sox2 Fgf5 Nestin

Rel

ativ

e Q

uant

ifica

tion WT ES

DNStat3.1

DNStat3.2

Mesenchymal fate differentiation program is prefere ntially affected

+LIF +MEFS

p1 p5 p10

No LIF no MEFS

OCT4

p2 p6 p8

wt

DN1

DN2

wt

DN1

DN2

Fgf5

+LIF +MEFS

No LIF no MEFS

p1 p5 p10

p2 p6 p8

wt DN1DN2

5

WT R382Q Stat3

Alkaline phosphatase staining of ES cell colonies (p assage 10)

ES Cell Conclusions:

R382Q ES cells differentiate faster than WT ES cell s, via impaired LIF signaling

LIF independent pathways exist to maintain pluripot ency

Lineage that is most affected is mesenchymal (microa rray data)

Neural progenitor differentiation is maintained

Implications for clinical syndrome:Chimerism seen inpatientsImplications for ability of HSC cells to maintain pluripotency

Generation of B cell Mouse Model

Deletion of Stat3 in mice is embryonic lethal:conditional deletion- completely lack Stat 3 (unlike AD-HIES)

“Knock-in” of mutation in mouse ES cells- marked chim erismand selection against mutation

Expression of R382Q is induced in a Cre specific fas hion

Mb-1 (Ig-alpha)-Cre mice, express only in B cells, a t the pro-B cell stage of development

All B cells in this mouse will express Dominant Neg ative Stat3

Exploit the ability to exploit the DN Stat3 in a ti ssue specificfashion

BM IgM B220- GFP expression

0 102 103 104 105

0

102

103

104

105

0 102 103 104 1050

20

40

60

80

100

0 102 103 104 105

0

102

103

104

105

B220

IgM

WT Rosa STAT3-Mb1

0 102 103 104 1050

20

40

60

80

100

GFP

B220 negB220 pos

0 102 103 104 105

0102

103

104

105

59.3

0 102 103 104 105

0102

103

104

105

59

SPLEEN IgM B220- GFP expression

B220

IgM

WT Rosa STAT3-Mb1

GFP

0 102 103 104 1050

20

40

60

80

100

0 102 103 104 1050

20

40

60

80

100

B220 negB220 pos

0 102 103 104 105

<PerCP-Cy5-5-A>: IgM

0102

103

104

105

<P

E-A

>: I

gD 38.2

15.9

WT

IgM IgD spleen

0 102 103 104 105

<PerCP-Cy5-5-A>: IgM

0102

103

104

105

<PE

-A>:

IgD 40.1

14.2

Rosa Mb1

T1T2

Mature B cell compartment appears intact in 8 week old mice

6

CD21CD23 on B220 pos -spleen

WT

0 102 103 104 105

<APC-Cy7-A>: CD23

0102

103

104

105<P

E-A

>: C

D21

64.8

5.97

Rosa Mb1

0 102 103 104 105

<APC-Cy7-A>: CD23

0

102

103

104

105

<PE

-A>:

CD

21

63

4.31

Fo

MZ

Marginal zone and Follicular cell B cell compartmen ts appear intact

0 102 103 104 105

<PE-A>: g1

0

102

103

104

105

<A

PC

-A>

: B22

0

94.1 5.32

0.0830.51

0 102

103

104

105

<PE-A>: g1

0102

103

104

105

<A

PC

-A>

: B22

0

95.5 3.8

0.140.53

0 102 103 104 105

<PE-A>: g1

0

102

103

104

105

<AP

C-A

>: B

220

93.8 5.28

0.130.8

0 102 103 104 105

<PE-A>: g1

0

102

103

104

105

<AP

C-A

>: B

220

14.3 82.1

2.441.14

0 102 103 104 105

<PE-A>: g1

0102

103

104

105

<A

PC

-A>

: B

220

19.8 75.3

3.081.91

0 102 103 104 105

<PE-A>: g1

0102

103

104

105

<A

PC

-A>

: B

220

14 81.2

3.171.58

αCD40

αCD40 +IL4

WT Rosa Mb1#1 Rosa Mb1#2

Class switch assay day4

Serum levels of Immunoglobulin in R382Q MB-1 Mice are normal Conclusions from B cell Mouse Model

B cell numbers are unaffected- in resting unimmunize d mice

DN Stat 3B cells do not intrinsically undergo incre ased class switching to IgE or other Isotypes (need immunization studies)

Fewer plasma cells are found in older mb-1 mice (4 months), based on immunohistochemical staining of the bone ma rrow

Future work:CD4 T cell specific expression Bone specific expressionSkin specific expression

Summary:

AD-HIES is a heterogeneous disease- genetically and clinicallynot all patients have Stat 3 mutations

Most patients with Stat 3 mutations are sporadic in natureindicating importance in embryonic development

Developmental selection is severely influenced by D N-Stat 3murine ES cells severely affected

Preliminary studies suggest no intrinsic B cell def ect for class switchingin context of normal T cells

Mouse models may elucidate therapeutic targets for non-immune defectsBone and skin manifestations

• Joyce Chang Mark Geyer

• Nicole Walsh Alice Chang

• Shilpee Dutt Kate Graves

• Matt Lynch

• Lianne Kaylor

Children’s Hospital Boston

Luigi Notarangelo

Francesca Rucci

UT Houston Dana Farber Cancer Center

• Phil Carpenter Margaret Shipp

• Julio Morales Kuni Takeyama

Harvard Medical School MIT/Broad Institute

• Sean Rooney Todd Golub

• Shan Zha Stefano Monti

• Klaus Rajewsky

Acknowledgements