Therapeutic approaches for MG studied in animal models Miriam Souroujon Open University of Israel...

Therapeutic approaches for MG studied in animal models

Miriam Souroujon

Open University of IsraelWeizmann Institute of Science

International Conference on Myasthenia GravisParis, December 2009

Experimental animal models for Myasthenia Gravis

Active immunizationTorpedo AChR

EAMG

Passive transferAnti-AChR Abs

Animal models for MuSK+ MG

Humanized SCID/NOD mouse model for MG

Treatment modalities tested in EAMG

Targeting AChR-specific T and B cell responses•Apheresis of pathogenic anti-AChR antibodies - immunoadsorption on

ECDs• Anti-TCR (Vb5.1) antibodies • Targeting the antigen-binding site of HLA-restricting alleles• Anti-idiotypes (IVIG)

Antigen (AChR)- specific

Immunization by AChR recombinant fragments or synthetic peptides

Denatured AChRSynthetic AChR peptides: Altered peptides, Dominant T cell epitopesAChR recombinant fragments (mucosal tolerance)

Xenogeneic, SyngeneicPathogenic B-cell epitope free

Cell based• Dendritic cells• Mesenchymal stem cells• T regulatory (Treg) cells

Non-cell based targets• Cytokine networks (IFN-g, IL-18, TNF-a, IL-1) • Costimulatory signaling (CD40L) • Complement pathways• Fc receptor• Cholinergic balance at the neuromuscular junction• AChR-associated anchor protein, rapsyn• Chemokines and chemokine receptors (IP-10/CXCR3)• Phosphodiesterases

Treatment modalities tested in EAMGImmunomodulatory approaches

Following PTX treatment

PDE 4

TNF-a, IL-18, IL-12

IL-10

Foxp3

T cell proliferation

Anti-AChR Ab

PDE 1, 4, 7

TNF-a

Cathepsin-l

PDE 2,3,4,7

Cathepsin-l In EAMG PDE 1,3,4,7

Immune systemMuscle

PTX acts as a steroid-sparing agent

PDE4B

Control MG0.0

0.5

1.0

1.5

PDE4D

Control MG0.0

0.5

1.0

1.5

PDE2

Control MG0.0

0.5

1.0

1.5

2.0

2.5

PDE7

Control MG0.0

0.5

1.0

1.5

2.0

2.5

PDE1

Control MG0.0

0.5

1.0

1.5

2.0

2.5

3.0

PDE3

Control MG0.0

0.5

1.0

1.5

2.0

2.5

PDE expression in thymus of MG patients

PDE4B

Control MG0.0

0.5

1.0

1.5

PDE4D

Control MG0.0

0.5

1.0

1.5

2.0

PDE1

Control MG0.0

0.5

1.0

1.5

2.0

2.5

PDE3

Control MG0.0

0.5

1.0

1.5

PDE7

Control MG0.0

0.5

1.0

PDE2

Control MG0.0

0.5

1.0

1.5

2.0

PDE expression in PBL of MG patients

Are phosphodiesterase (PDE) levels altered

in other autoimmune diseases?

• Experimental models ?

• Human diseases ?

*** *** *** **

LNC

*** *** ***

* *

*

0

50

100

150

200

250

PDE1 PDE2 PDE3 PDE4 PDE7 TNF-a

Gen

e/A

ctin

(%

)

CFA

EAE

0

50

100

150

200

250

PDE1 PDE2 PDE3 PDE4 PDE7 TNF-a

Gen

e/A

ctin

(%

)

CFA

EAE

CD4+ cells

*

*

*

******

***

PDE expression in PBL of Multiple Sclerosis patients

PDE1

CONTROL MS

0.0

0.2

0.4

0.6

0.8

1.0

1.2

PDE3

CONTROL MS

0

10

20

30

40

50

PDE7

CONTROL MS

0

1

2

3

4

5

6

PDE4B

CONTROL MS

0.0

0.5

1.0

1.5

2.0

PDE4D

CONTROL MS

0

1

2

3

• The expression levels of selective PDE subtypes are upregulated in EAMG and MG.

• The general PDE inhibitor PTX acts as a steroid sparing agent in EAMG.

• The expression levels of selective PDE subtypes are upregulated also in multiple sclerosis (MS) and its animal model, EAE.

• PDEs may be potential therapeutic targets in various autoimmune diseases.

Summary IPDE

Cell based• Dendritic cells• Mesenchymal stem cells• T regulatory (Treg) cells

Non-cell based targets• Cytokine networks (IFN-g, IL-18, TNF-a, IL-1) • Costimulatory signaling (CD40L) • Complement pathways• Fc receptor• Cholinergic balance at the neuromuscular junction• AChR-associated anchor protein, rapsyn• Chemokines and chemokine receptors (IP-10/CXCR3)• Phosphodiesterases

Treatment modalities tested in EAMGImmunomodulatory approaches

Treg abnormalities are observed in MG

• Functional impairments were found in thymic Treg cells of MG patients

• Decreased CD4+CD25high cell numbers were found in PBL of MG patients

• Successful treatments or thymectomy result in elevated numbers of CD4+CD25high cells

• CD4+CD25+ cells are involved in the suppressive action of various effective therapies in EAMG

EAMG rats have reduced levels of Treg cells

AChR CFA Naive

123456789

p<0.05

AChR CFA Naive

1

2

3

4

5

6p<0.05

AChR CFA Naive

1.00

1.25

1.50

1.75

2.00

p<0.01

Treg-based treatments

CancerInfectious disease Autoimmune

Inflammatory disease

• Administration of Exogenous Treg

• Modulation of endogenous Treg

Treg

Ex-vivo generation of CD4+CD25+ regulatory T cells

Spleen from healthy or EAMG donor rat

Negative selection of CD4+ cells using magnetic beads

Y Y

Y Y

Y

Y

Y Y

YYY

Y

Y

YYY

Y YY

90%

CD

4

+ TGF- ,b IL-2

culture cells on anti-CD3 and

anti-CD28 coated plates

Characterization of ex vivo generated CD4+CD25+ Treg cells

5%

CD

4

CD25

EAMG Splenocytes + + + + Co-culture with - evCD4+CD25+

nCD4+CD25+ nCD4+CD25-

0

1000

2000

3000

4000

5000

6000

CP

M

* *

96%

DAY 0 DAY 3

Foxp3

0

0.1

0.2

0.3

0.4

0.5

evCD4+CD25+ nCD4+CD25-Rel

ativ

e ex

pre

ssio

n l

evel

s

CTLA-4

0

0.5

1

1.5

2

2.5

3

3.5


ati

ve

ex

pre

ss

ion

le

vel

s

TGF- b

0

0.5

1

1.5


ativ

e ex

pre

ssio

n l

evel

s

Treg generated ex vivo from healthy donors suppress EAMG

0 1 2 3 4 5 6 7 80.0

0.5

1.0

1.5

2.0

2.5 PBS

evCD4+CD25+

nCD4+CD25-

Weeks

0 1 2 3 4 5 6 7 80

20

40

60

80

100

Weeks

CD25

Healthy

EAMG

0

0.5

1

1.5

2

2.5

3

3.5

4

0 1 2 3 4 5 6 7 8 9 10 11 12

Weeks

Clin

ical score

evCD4+CD25+ from EAMG donors

CONTROL (PBS)

evCD4+CD25+ from healthy donors

Treg from myasthenic rats exacerbate EAMG

Revisiting the Th1-Th2 Paradigm

Naive T cell

Dendritic cell

TGF-

IFN IL-4 + IL-6 - IL-6

Th1Tbet

Th2Gata 3

Th17RORT

TregFoxp3

IL-12R

IL-12

IL-23R

IL-23

Adapted from Reiner et al., Cell, 2007

Autoimmunity Inflammation Cancer

Extracellular bacteria

Allergy and asthma

Systemic pathology

Harmful role

Counter regulation

Parasitic worms

Intracellular pathogens

Protective role

Naive CFA EAMG

0

1

2

3

Naive CFA EAMG

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

3 Weeks 7 Weeks

Expression of Foxp3

IL-23

Naive CFA EAMG

0.0

0.3

0.6

0.9

1.2

1.5

1.8 *IL-23

Naive CFA EAMG

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

IL-23R

Naive CFA EAMG

0

1

2

3

IL-23R

Naive CFA EAMG

0.0

0.5

1.0

1.5

2.0 *

IL-17

Naive CFA EAMG

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5*

IL-17

Naive CFA EAMG

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

IL-17R

Naive CFA EAMG

0.00.10.20.30.40.50.60.70.80.91.01.1

IL-17R

Naive CFA EAMG

0.00

0.25

0.50

0.75 *

3 Weeks 7 Weeks

Expressionof Th17-relatedgenes

TGF-

Naive CFA EAMG

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

*

TGF-

Naive CFA EAMG

0.3

0.4

0.5

0.6

0.7

0.8

0.9

IL-6

Naive CFA EAMG

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

IL-6

Naive CFA EAMG

0.0

0.1

0.2

0.3 *

IL-6R

Naive CFA EAMG

0.6

0.8

1.0

1.2

1.4

1.6

IL-6R

Naive CFA EAMG

0.45

0.65

0.85

1.05

*

3 Weeks 7 Weeks

Expressionof regulatory

cytokines

Treatment by anti-IL-6 Abs starting at the acute phase of EAMG

0

0.5

1

1.5

2

2.5

3

3.5

4

0 1 2 3 4 5 6 7 8 9 10

weeks

clin

ical score

PBSanti IL-6

0

0.5

1

1.5

2

2.5

3

3.5

4

0 1 2 3 4 5 6 7 8 9 10 11

weeks

clin

ical score

anti IgG

anti IL-6

Treatment by anti-IL-6 Abs starting at the chronic phase of EAMG

*

Total anti-AChR IgG

control anti IL-6

0.0

0.1

0.2

0.3

control anti IL-6

0

10

20

30

40

% of splenic B cells

*

Expression following anti-IL-6 treatment

Foxp3

Control Anti-IL-60

1

2

3

IL-6

Control Anti-IL-60

1

2

3

IL-6R

Control Anti-IL-60.0

0.5

1.0

1.5

2.0

2.5

Cytokines expression following anti IL-6 treatment

IL-17A

Control Anti-IL-60

1

2

3

4

5

6

7

8

9

IL-17R


0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

IL-21

Control Anti-IL-60

1

2

3

4

IL-23


0.5

1.0

1.5

2.0

IL-23R

Control Anti-IL-6

0

1

2

3

4

TGF-


0.25

0.50

0.75

1.00

1.25

1.50

1.75

Serum IL-17 in anti-IL-6 treated rats

control Anti-IL-6

0

1

2

3

4

5

6

Revisiting the Th1-Th2 Paradigm

Naive T cell

Dendritic cell

TGF-

IFN IL-4 + IL-6 - IL-6

Th1Tbet

Th2Gata 3

Th17RORT

TregFoxp3

IL-12R

IL-12

IL-23R

IL-23

Adapted from Reiner et al., Cell, 2007

Autoimmunity Inflammation Cancer

Extracellular bacteria

Allergy and asthma

Systemic pathology

Harmful role

Counter regulation

Parasitic worms

Intracellular pathogens

Protective role

Summary II

Treg

Suppression of EAMG can be achieved by:

¨ Administration of exogenous Treg from healthy donors

¨ Shifting the balance between endogenous Treg and Th17 in favor of Treg

Hopefully, the vast repertoire of therapeutic approaches studied in experimental models of MG will pave the way to clinical studies

that will eventually improve the management of MG

Thanks !!Thanks !!

• Sara Fuchs• Revital Aricha• Tali Feferman• Keren Mizrachi

• Sonia Berrih-Aknin

• Ariel Miller (MS)• Avi Ben-Nun (EAE)

• Sara Fuchs• Revital Aricha• Tali Feferman• Keren Mizrachi

• Sonia Berrih-Aknin

• Ariel Miller (MS)• Avi Ben-Nun (EAE)

Recipients of Treg have elevated CD4+CD25+FoxP3+ cells

FoxP3+

PBS treated

31%

FoxP3+

54%

evCD4+CD25+ treated

0

1

2

3

4

PBS nCD4+CD25- evCD4+CD25+

% o

f C

D25

+am

on

g C

D4+

cel

ls

*

0

1

2

3

PBS nCD4+CD25- evCD4+CD25+% o

f C

D25

+F

oxP

3+am

on

g C

D4+

ce

lls

% F

oxP

3+ am

on

g C

D4+

CD

25+ cells o

f recipien

ts

Therapeutic approaches for MG studied in animal models Miriam Souroujon Open University of Israel...

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