Download M. Meriggioli

Mobilization of Regulatory
Immune Cells Utilizing GM-CSF
in Experimental Myasthenia
Gravis
Matthew N. Meriggioli, M.D.
Neuromuscular Disorders Program
Department of Neurology and Rehabilitation
Immunogenesis of MG
APC / DC
T
T
T
Anti-AChR
Abs
B
AChR
Plasma cell
T
Complement
Complement
1.
2.
NT
NT
3.
Granulocyte-Macrophage Colony
Stimulating Factor (GM-CSF)
• Produced by mesenchymal cells, macrophages
and T cells
• Stimulates cells of the innate immune system
including bone marrow-derived DC precursors
• Expands myeloid DCs (CD8a-) both in vitro and in
vivo.
• Helps maintain CD8a- DCs in a semi-matured
status
• Currently used to stimulate white blood cells such
as neutrophils and macrophages following
chemotherapy.
Modulation of DC maturation can
affect the fate of a T cell response
Immature DCs
ClassII/B7 low
IL12, Il 6, IL10 neg
Semimature DCs
ClassII/B7 hi
IL12, Il 6, TNFα neg
IL10 +/-
TGFβ
Functional
unresponsiveness
IL-10
Regulatory T cells
(FoxP3, CD25+)
Mature DCs
ClassII/B7 hi
IL12, Il 6, TNFα pos
IL-2
IFNγ
Effector T cells
A Homeostatic Balance Exists
Between Teff & Tregs
Von Hearrath et al., Nature Reviews Immunology 3, 223-232 (March 2003)
GM-CSF in experimental
autoimmunity
Disease Model
Result
Associated findings
EAMG (1,2)
-Suppression of disease
induction
-Amelioration of chronic disease
- ↓ Anti-AChR antibodies
- Semi-mature DCs / ↑ Tregs
T1D (NOD) (3)
-Protection against diabetes
- ↓ Pancreatic islet inflammation
- Semi-mature DCs / ↑ Tregs
EAT (4)
-Suppression of disease
induction
-Amelioration of ongoing
disease
- ↓ Thyroid inflammation
- Semi-mature DCs / ↑ Tregs
EAT = experimental autoimmune thyroiditis, T1D (NOD) = Type 1 diabetes (Non-Obese Ddiabetic),
EAMG = experimental autoimmune myasthenia gravis, DC = dendritic cells, Tregs = regulatory T cells
1. Sheng, J.R., L.C. Li, B.B. Ganesh, et. al. 2006. J. Immunol. 177: 5296-306.
2. Sheng JR, Li L, Ganesh BB, Prabhakar BS, Meriggioli MN. Clin Immunol 2008;128:172-180.
3. Gaudreau S, Guindi C, Menard M, Besin G, Dupuis G, Amrani A, J. Immunol. 179; 2007: 3638-3647.
4. Gangi, E., C. Vasu, D. Cheatem D, et al. 2005. J. Immunol. 174: 7006-7013.
Therapeutic effects of GM-CSF in EAMG
Mean Clinical Score
2.5
GM-CSF
Control
2
1.5
1
0.5
treatment
boost treatment
0
0
5
10 15 20 25 30 35 40 45 50 55 60
Days After First Treatment
100%
Muscle
content loss
Muscle
AChR
Content Loss
80%
60%
40%
20%
0%
Control
GM-CSF
PBS
Sheng JR, et al., Clin Immunol 2008;128:172-180
Autoantibody and B cell Responses
A.
B.
Control
22.4
B cell proliferation
w/ AChR stim
GM-CSF
6.9
DC expression of MHC II, CD8α, and cytokines
Sheng JR, et al., Clin Immunol 2008;128:172-180
GM-CSF treatment mobilizes Tregs
(FOXP3+) and shifts the cytokine
response
Sheng JR, et al., Clin Immunol 2008;128:172-180
GM-CSF modulation of cytokine milieu
GM-CSF Modulation of Cytokine Milieu
PBS
IFN-r
GM-CSF
13.4
4.8
2.27
4.26
10.2
4.9
11
6
IL-10
IL-17
IL-6
CD25+ cells from GM-CSFtreated mice suppress T cell
proliferation (AChR-induced)
and are IL-10 dependent
BUT, do not suppress nonspecific proliferation to
mitogenic stimuli
Tregs from GM-CSF-treated mice
are more potent suppressors of
AChR-stimulated T cell
proliferation
Do GM-CSF-induced Tregs have an antigen-specific suppressive effect?
EAMG
GM-CSF
Splenic CD4+ T cells
EAMG
OVA-primed
mTg primed
PBS
Splenic CD4+ T cells
CD25-
Isolate CD25+
nTregs
AChR-primed
CD25-
CD25-
Isolate CD25+
nTregs
Polyclonal
Tregs
OVA
AChR
Co-culture 1:1
Co-culture 1:1
mTg
? AChR-specific Tregs
Co-culture 1:1
Tregs mobilized by GM-CSF are potent
and relatively specific suppressors of
AChR-induced lymphocyte proliferation
80
Baseline
70
60
50
40
30
Baseline
Add Tregs (CD25+) from untreated
Treg untreated
Mice (polyclonal Tregs)
Treg GM-CSF
20
10
0
Add Tregs (CD25+) from GM-CSFtreated
mice
OVA
TAChR
Tg
A.
Clinical score after the adoptive transfer
CD25+
2.5
Mean clinical score
CD25-
Adoptive Transfer
of Tregs
Control
2
1.5
1
Treg
0.5
9
10
D
D
8
D
7
D
6
D
7
D
6
D
5
D
4
D
3
D
D
1
D
2
0
Tregs
(CD25+)
Days after the adoptive transfer
B.
Mean clinical Socre
EAMG
Adoptive transfter CD25+/CD25- cells to EAMG mice
3
2.5
2
1.5
1
0.5
0
Before
After
CD25+
CD25-
What is the mechanism of
GM-CSF’s effects?
How are antigen-specific
Tregs expanded ??
Antigen presentation by GM-CSF (CD8a-) DCs
induce expansion of Tregs
GM-CSF 8aIsotype
control
10.2
Control 8a4.7
GM-CSF 8a+
Control 8a+
5.7
4.1
TAChR
primed T
cells
0.1
6.0
4.3
5.0
Foxp3
CD25
%CD25+Foxp3+ cells
**
12
10
8
with TAChR
No Ag
6
4
2
0
GM-CSF 8a- Control 8a- GM-CSF 8a+ Control 8a+
4.2
Naive T
cells
Proliferation of Foxp3+ CD4+CD25+ T
cells expanded by CD8a+ or CD8a- DCs
from GM-CSF and control mice
GM-CSF 8a5.3
0.4
Control 8a4.6
0.4
GM-CSF 8a+
Control 8a+
2.6
3
0.2
0.4
CD4+25-
26
34
11
37
16
39
7
40
CD4+25+
14
Foxp3
CFSE
dilution
29
23
18
Proposed Mechanism of GM-CSF
induced EAMG suppression
Semi-mature DCs (low proinflammatroy cytokines)
GM-CSF
Induction of Tregs
Tregs and IL-10 suppress
AChR specific responses
Controlled AChR specific
responses ameliorate disease
Regulatory Immune cells Interactions
Th1, Th17
IL-10, IL12
Tregs
Activated
DC
T
DC
IL-2, IL-4
BAFF
APRIL
B
IgG
B reg
Regulatory Cells
GM-CSF
T cells
5.84
FoxP3
11.2
PBS
CD4
CD1d
B cells
15
CD5
1.46
Summary
 GM-CSF can prevent and treat EAMG (and T1D and
EAT)
 GM-CSF selectively expands & “tolerizes” CD8aDCs
 Antigen presentation by CD8a-DCs induces Tregs
 Tregs suppress EAMG (in vitro and in vivo)
Treg induction by GM-CSF could be an effective
strategy to treat MG as well as other autoimmune
diseases
Acknowledgements
Dr. Jianrong Sheng
Dr. Liangcheng Li
Dr. Bellur S. Prabhakar
Support:
• NIH/NINDS K08NS058800-03
• Myasthenia Gravis Foundation of
America
• Muscular Dystrophy Association
Ex Vivo expansion of Tregs
AChR-specific
Tregs
Treat
T
Culture
α
BM DCs
Peripheral Monocytes
DCs
Dendritic cell
T
ε
α
δ β
AChR
T
GM-CSF derived BMDCs are potent inducers
of Foxp3+ Tregs in DC/T-cell co-cultures
.
7.14
16.10
Foxp3
3.95
CD4+ cells
from
AChRprimed
mice
(+ AChR)
CD4
spDC
control
spDC
GM-CSF
BMDC
GM-CSF
Clinical translation?
In vivo expansion of
Treg cell population
GM-CSF
Treg
Teff
AChR
Treg
Suppression
Ex vivo expansion of
AChR-specific Tregs
Treg
Teff
AChR
Treg
+ AChR
Teff