1. 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

2. Immunogenesis of MG Anti-AChR Abs AChR T T T T T T T T B B Plasma cell APC / DC Complement 1. 2. 3. 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.

4. Immature DCs ClassII/B7 low IL12, Il 6, IL10 neg Semimature DCs ClassII/B7 hi IL12, Il 6, TNFα neg IL10 +/- Mature DCs ClassII/B7 hi IL12, 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 TGFβIL-10IFNγIL-2

5. Von Hearrath et al., Nature Reviews Immunology 3, 223-232 (March 2003) A Homeostatic Balance Exists Between Teff & Tregs

6. GM-CSF in experimental autoimmunity Disease ModelResultAssociated 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.

7. treatment boosttreatment Therapeutic effects of GM-CSF in EAMG Sheng JR, et al., Clin Immunol 2008;128:172-180 Muscle AChR Content Loss

8. Control 22.4 6.9GM-CSF B cell proliferation w/ AChR stim A.A. B. Autoantibody and B cell Responses

9. DC expression of MHC II, CD8α, and cytokines Sheng JR, et al., Clin Immunol 2008;128:172-180

10. GM-CSF treatment mobilizes Tregs (FOXP3 + GM-CSF treatment mobilizes Tregs (FOXP3 + ) and shifts the cytokine response Sheng JR, et al., Clin Immunol 2008;128:172-180

11. 13.4 4.8 PBSGM-CSF 4.262.27 IFN-r IL-10 IL-17 10.2 4.9 611 IL-6 GM-CSF modulation of cytokine milieu GM-CSF Modulation of Cytokine Milieu

12. 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

13. Tregs from GM-CSF-treated mice are more potent suppressors of AChR-stimulated T cell proliferation

14. GM-CSF 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 OVAAChRmTg Co-culture 1:1 AChR-primed OVA-primed

15. 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

16. Tregs (CD25 + ) EAMG A. B. Adoptive Transfer of Tregs Treg Control

17. What is the mechanism of GM-CSF’s effects? How are antigen-specific Tregs expanded ??

18. Control 8a+GM-CSF 8a+Control 8a-GM-CSF 8a- Isotype control CD25 Foxp3 4.1 10.24.7 0.1 4.3 5.7 5.04.2 TAChR primed T cells Naive T cells 6.0 GM-CSF 8a-Control 8a-GM-CSF 8a+Control 8a+ %CD25+Foxp3+ cells 0 2 4 6 8 10 12 with TAChR No Ag ** Antigen presentation by GM-CSF (CD8a-) DCs induce expansion of Tregs

19. Proliferation of Foxp3+ CD4+CD25+ T cells expanded by CD8a+ or CD8a- DCs from GM-CSF and control mice 2.60.2 3 0.4 4.6 0.45.3 0.4 11 29 37 16 23 39 7 18 40 26 14 34 CD4+25- CFSE dilution Foxp3 CD4+25+ GM-CSF 8a-Control 8a-GM-CSF 8a+Control 8a+

20. 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

21. Regulatory Immune cells - Interactions B B T T DC IgG Th1, Th17 Tregs B reg IL-2, IL-4 BAFF APRIL IL-10, IL12 Activated DC

22. 11.2 5.84 PBSGM-CSF Regulatory Cells FoxP3 CD4 15 CD1d CD5 1.46 T cells B cells

23. 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

24. Acknowledgements Dr. Jianrong Sheng Dr. Liangcheng Li Dr. Bellur S. Prabhakar Support: NIH/NINDS K08NS058800-03 Myasthenia Gravis Foundation of America Muscular Dystrophy Association

25. AChR-specific Tregs DCs Culture Dendritic cell Treat ε α α δ β AChR GM-CSF, other cytokines Ex Vivo expansion of Tregs T T T T T T BM DCs Peripheral Monocytes

26. 7.143.95 16.10 CD4 Foxp3 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 BMDC GM-CSF

27. T reg T eff AChR GM-CSF In vivo expansion of T reg cell population In vivo expansion of T reg cell population T reg T eff AChR T reg + AChR Ex vivo expansion of AChR-specific T regs Ex vivo expansion of AChR-specific T regs T reg T eff Suppression Clinical translation?