Date post: | 01-Jan-2016 |
Category: |
Documents |
Upload: | oswald-lambert |
View: | 221 times |
Download: | 0 times |
Mobilization of Regulatory Immune Cells Utilizing GM-CSF in Experimental Myasthenia Gravis
Matthew N. Meriggioli, M.D.
Neuromuscular Disorders ProgramDepartment of Neurology and Rehabilitation
Immunogenesis of MGImmunogenesis of MG
Anti-AChRAbs
AChR
TT
TT
TT
TT
BB
Plasma cellPlasma cell
APC / DC
ComplementComplement
1.
2.
3.
NT NT
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.
Immature DCsClassII/B7 lowIL12, Il 6, IL10 neg
Semimature DCsClassII/B7 hiIL12, Il 6, TNFα negIL10 +/-
Mature DCsClassII/B7 hiIL12, Il 6, TNFα pos
Modulation of DC maturation can affect the fate of a T cell response
Functional unresponsiveness
Regulatory T cells(FoxP3, CD25+)
Effector T cells
TGFβ IL-10 IFNγIL-2
Von Hearrath et al., Nature Reviews Immunology 3, 223-232 (March 2003)
A Homeostatic Balance Exists Between Teff & Tregs
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.
0
0.5
1
1.5
2
2.5
0 5 10 15 20 25 30 35 40 45 50 55 60
Days After First Treatment
Mean
Cli
nic
al
Sco
reGM-CSF
Control
treatment boost treatment
Therapeutic effects of GM-CSF in EAMGTherapeutic effects of GM-CSF in EAMG
Sheng JR, et al., Clin Immunol 2008;128:172-180
Muscle content loss
0%
20%
40%
60%
80%
100%
Control GM-CSF PBS
Muscle AChR Content LossMuscle AChR Content Loss
Control 22.4
6.9GM-CSF
B cell proliferationw/ AChR stim
A.
B.
Autoantibody and B cell Responses
DC expression of MHC II, CD8α, and cytokines
Sheng JR, et al., Clin Immunol 2008;128:172-180
GM-CSF treatment mobilizes Tregs GM-CSF treatment mobilizes Tregs (FOXP3(FOXP3++) and shifts the cytokine
response
Sheng JR, et al., Clin Immunol 2008;128:172-180
13.4 4.8
PBS GM-CSF
4.262.27
IFN-r
IL-10
IL-17
10.2 4.9
611
IL-6
GM-CSF modulation of cytokine milieuGM-CSF Modulation of Cytokine Milieu
CD25+ cells from GM-CSF-treated mice suppress T cell proliferation (AChR-induced) and are IL-10 dependent
BUT, do not suppress non-specific proliferation to mitogenic stimuli
Tregs from GM-CSF-treated mice Tregs from GM-CSF-treated mice are more potent suppressors of are more potent suppressors of
AChR-stimulated T cell proliferationAChR-stimulated T cell proliferation
GM-CSF
EAMG EAMG
PBS
Isolate CD25+ nTregs
mTg primed
Splenic CD4+ T cells
CD25-
Co-culture 1:1
Do GM-CSF-induced Tregs have an antigen-specific suppressive effect?
Splenic CD4+ T cells
Isolate CD25+ nTregs
CD25-
Co-culture 1:1
CD25-
? AChR-specific Tregs
Polyclonal Tregs
OVA AChR mTg
Co-culture 1:1
AChR-primedOVA-primed
Baseline
Add Tregs (CD25+) from untreated Mice (polyclonal Tregs)
Add Tregs (CD25+) from GM-CSF-treated mice
Tregs mobilized by GM-CSF are potent and relatively specific suppressors of AChR-induced lymphocyte proliferation
0
10
20
30
40
50
60
70
80
OVA TAChR Tg
Baseline
Treg untreated
Treg GM-CSF
Tregs(CD25+)
EAMG
Clinical score after the adoptive transfer
0
0.5
1
1.5
2
2.5
Days after the adoptive transfer
Mea
n c
linic
al s
core
CD25+
CD25-
Adoptive transfter CD25+/CD25- cells to EAMG mice
00.5
11.5
22.5
3
CD25+ CD25-
Mea
n cl
inic
al S
ocre
Before
After
A.
B.
Adoptive Transfer of Tregs
Treg
Control
What is the mechanism of GM-CSF’s effects?
How are antigen-specific Tregs expanded ??
Control 8a+GM-CSF 8a+Control 8a-GM-CSF 8a-
Isotype control
CD25
Foxp3
4.110.2 4.7
0.1
4.3
5.7
5.0 4.2
TAChR primed T cells
Naive T cells
6.0
GM-CSF 8a- Control 8a- GM-CSF 8a+ Control 8a+
%C
D25
+F
oxp3
+ c
ells
0
2
4
6
8
1012
with TAChRNo Ag
**
Antigen presentation by GM-CSF (CD8a-) DCs induce expansion of Tregs
Proliferation of Foxp3+ CD4+CD25+ T cells expanded by CD8a+ or CD8a- DCs
from GM-CSF and control mice
2.6 0.2 3 0.44.6 0.45.3 0.4
11
29
37 16
23
39 7
18
4026
14
34
CD4+25-
CFSE dilution
Foxp3
CD4+25+
GM-CSF 8a- Control 8a- GM-CSF 8a+ Control 8a+
Proposed Mechanism of GM-CSF induced EAMG suppression
Controlled AChR specific responses ameliorate disease
GM-CSF
Tregs and IL-10 suppress AChR specific responses
Semi-mature DCs (low pro-inflammatroy cytokines)
Induction of Tregs
Regulatory Immune cells - Interactions
BB
TTDC
IgG
Th1, Th17
Tregs
B reg
IL-2, IL-4
BAFF APRIL
IL-10, IL12
ActivatedDC
11.2 5.84
PBSGM-CSF
Regulatory Cells
Fo
xP3
CD4
15
CD
1d
CD5
1.46
T cells
B cells
Summary GM-CSF can prevent and treat EAMG (and T1D and
EAT)
GM-CSF selectively expands & “tolerizes” CD8a-
DCs 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
AcknowledgementsDr. Jianrong Sheng
Dr. Liangcheng Li
Dr. Bellur S. Prabhakar
Support:• NIH/NINDS K08NS058800-03• Myasthenia Gravis Foundation of
America• Muscular Dystrophy Association
AChR-specific Tregs
DCs
Culture
Dendritic cell
Treat
εα α
δ βAChR
GM-CSF, other cytokines
Ex Vivo expansion of Tregs
TT TT TT
BM DCsPeripheral Monocytes
7.143.95 16.10
CD4
Fo
xp
3
CD4+ cells from AChR- primed mice
(+ AChR)
GM-CSF derived BMDCs are potent inducers of Foxp3+ Tregs in DC/T-cell co-cultures
.
spDC control
spDC GM-CSF
BMDCGM-CSF
TregTreg
TeffTeff
AChRAChR
GM-CSFGM-CSF
In vivo expansion of Treg cell populationIn vivo expansion of Treg cell population
TregTreg
TeffTeff
AChRAChRTregTreg
+ AChR+ AChR
Ex vivo expansion of AChR-specific Tregs
Ex vivo expansion of AChR-specific Tregs
TregTreg
TeffTeff
SuppressionSuppression
Clinical translation?