Therapeutic approaches for MG studied in animal models Miriam Souroujon Open University of Israel Weizmann Institute of Science International Conference on Myasthenia Gravis Paris, December 2009
Dec 18, 2015
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
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
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
evCD4+CD25+ nCD4+CD25-Rel
ati
ve
ex
pre
ss
ion
le
vel
s
TGF- b
0
0.5
1
1.5
evCD4+CD25+ nCD4+CD25-Rel
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
Control Anti-IL-60.00
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
Control Anti-IL-60.0
0.5
1.0
1.5
2.0
IL-23R
Control Anti-IL-6
0
1
2
3
4
TGF-
Control Anti-IL-60.00
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