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?