1. Developing Immunotherapy for Autoimmune Diseases
Premkumar Christadoss, M.B.B.S.
Department of Microbiology and Immunology
University of Texas Medical Branch
301 University Blvd.
Galveston, Texas

2. Generalized Myasthenia Gravis

3. MG

4. Neuromuscular Junction (NMJ)
Conti-Fine, B.M. et al. J. Clin. Invest. (116) , 2006

5. NMJ in MG
Normal MG

6. Electronmicroscopy Study of NMJ of an MG Patient
Engel et al.
Mayo clinic proc. 52:267, 1977

7. AChR is a transmembrane glycoprotein formed by five homologous subunits in the stoichiometry a2bgd or a2bed. The molecular weights of the subunits range between 45 and 55 kDa.
The a subunit is Considered to be the highly immunogenic region.

8. Primary immunization: 20 microgram AChR/CFA
EAMG induction
AChR Source
Primary immunization: 20 microgram AChR/CFA
28-30 days
Boost: 20 microgram AChR/CFA
28-30 days
Monitor for clinical EAMG
Immunopathological evaluation

9. MG in Mice
Normal MG

10. Molecular Mechanisms of EAMG
Damage to the neuromuscular junction
Complement activation
AChR
APC
IL-1, IL-12
CD4
Class II
Peptide
(a )
CD4
TCR B7
CD 28
CD40L/CD40
AChR-specific memory T cell
Proliferation and Differentiation
IL-10, TNF-a, IL-6,
IL-12, IL-18
AChR-specific memory B cells
AChR-Ab
Plasma cells NK
IFN-g
IL-18 B

11. COMPLEMENT PATHWAYS C1 C4b C2a MASP1 MASP2 MBL C3 C5 MAC (C5bC9) C3b
CLASSICAL PATHWAY C1
C4b
C2a
MASP1
MASP2
MBL C3 C5
MAC
(C5bC9)
MBL PATHWAY
C3b Bb
C3bBb
ALTERNATIVE PATHWAY

12. AChR-immunized C3-/- and C4-/- mice are resistant to clinical EAMG
Tuzun -Christadoss.
J. Immunol. 171:3847, 2003

13. Serum anti-AChR antibody levels of
AChR-immunized mice
Tuzun- Christadoss.
J. Immunol. 171:3847, 2003

14. RED  -bungarotoxin binding (NMJ) GREEN  C3, MAC or IgG deposits
IF Studies Reveal IgG Deposits But Not C3 or MAC Deposits at the NMJs of Mice with C3 or C4 Deficiency
C4+/+
C4-/-
C3-/- C3
MAC
IgG
RED  -bungarotoxin binding (NMJ)
GREEN  C3, MAC or IgG deposits
Tuzun –Christadoss
J. Immunol. 171:3847, 2003

15. Immune Intervention Disease specific Antigen/organ specific
I. AChR T cell epitope tolerance
II. AChR B cell epitope tolerance
Disease specific
I. Anti-Proinflammatory Cytokine
a. Soluble TNFR (etanercept)
b. IL1-Ra
c. Anti-IL-6
II. Blocking classical complement pathway
a. Anti C1q/C2/C4

16. Targeting Classical Complement Pathway

17. Anti-C1q Administration Suppresses EAMG
Tuzun-Christadoss, J.Neuroimmunol.174: , 2006

18. Dual Effect of Anti-C1q
Tuzun-Christadoss, J.Neuroimmunol.174: , 2006

19. Anti-C1q Ab Treats EAMG B6 RIII
Tuzun-Christadoss, J.Neuroimmunol, 182: , 2007

20. Anti-C1q Ab Treatment Suppresses AChR and Dominant Peptide Specific
IL-6 Production

21. Effect of Cytokine Deficiencies in
Clinical EAMG
Normal
IL-4
IL-10
Gene Depletion
IFN-g
Anti-AChR Ab
Disease
IL-12
IL-18
IL-6
TNF-a p55p75 20 40 60 80
100
120
% clinical disease and anti-AChR antibodies

22. IL-6 and TNF in EAMG Th Th B B TNF TNF AChR specific IL6 IL-6
GC formation
Activation of B cells and generation of effector B cells.
Potentiates production of IgG anti-AChR antibodies (pathogenic) B B
Activates C3
Promotes EAMG pathology

23. Targeting ProinflammatorCytokines
A. Soluble Recombinant HumanTNFR (Etanercept)

24. Soluble TNFR (Etanercept) Treats EAMG
Christadoss and Goluszko
J. Neuroimmunol, 122:186, 2002

25. Etanercept Treatment Fails to Suppress Serum Anti-AChR Ab
Christadoss and Goluszko, J. Neuro. Immunol. 122:186, 2002.

27. A pilot Trial of Etanercept in the Treatment of Steroid-Dependent MG *+
Mean change in QMG score from basline at 6 months was (p=0.041).
Mean change in MMT at 6 months was (p=0.020).
Mean decrease in prednisone dose from baseline to end of study was 17.5 mg/48 hr dose (p=0.0084).
Etanercept was well tolerated, and no severe adverse reaction observed
+ 11 patients enrolled; 8 completed, and 2 patients withdrawn due to disease worsening.

28. Immunological Effect of Etanercept
No reduction in plasma anti-AChR antibody.
Peripheral blood CD4 and B cell (CD19+) counts rose steadily during the 24 week study.
Patients who had higher increases in their cytokine levels had a worse outcome.

29. Targeting ProinflammatorCytokines
b. Recombinant Human IL1-Ra

30. Activation of the Adaptive Immune System with IL-1

31. IL-1Ra Treatment Prevents Clinical EAMG
stopped
Yang –Christadoss, J. Immunol.
175:2018, 2005

32. IL-1Ra Treats EAMG Yang –Christadoss, J. Immunol. 175:2018, 2005
P<0.05
P<0.05
P<0.05

33. Possible Consequence of Down Regulating IL-1 by IL-1Ra in Mice with Clinical EAMG
CD40L, OX40 Expression on T cells
Inflammatory cytokines
IFN-g, IL-2, IL-1, IL-6, TNF-a
Anti-AChR IgG , IgG1 and C3
Anti-AChR antibodies and complement
mediated NMJ pathology

34. IL-6 in MG: Multiple Hit AChR Specific NMJ CD4 IL-6 B IgG2b -C1q
C4-C3-
C5-9 C3

35. IL-6 – A Danger Molecule in EAMG !
1. IL-6 deficient mice are resistant to EAMG and produce less C3
2. C3 and FCγRIII deficient mice are resistant to EAMG and produce less IL-6
3. Amelioration of EAMG following anti-C1q treatment is associated with reduced IL-6

36. Anti-IL-6 Ab Treatment Reduces the Incidence of EAMG
Days after second immunization

37. Anti-IL-6 Ab Treatment Suppresses Serum Anti-AChR IgG
and IgG2b Ab

38. Anti-IL-6 Ab Treatment Suppresses AChR Specific
Cytokine Production

39. Classical Complement Pathway and IL-6 in EAMG Pathogenesis
anti-AChR antibody production
T helper
B cell
Antigen presenting cell
AChR
IL-6 C3
Immune complex formation
AChR
C1q
C1r
C1s
C1q
FcγRIII activation
Classical complement pathway activation
Stimulation of
IL-6, C1q and C3 production
Membrane attack complex formation

40. Balancing the Immune System to Treat Autoimmune Disease (MG)
IL-6, TNF
C3-C5b-C9
Anti-AChR IgG
IL-6, TNF-normal level
Healthy
Suppress anti-AChR
and C5b-C9

41. Targeting IL-6 and Classical Complement Pathway
Suppressed anti-AChR antibody production
T helper
B cell
Antigen presenting cell
AChR
IL-6 C3
Immune complex formation
AChR
C1q
C1r
C1s
C1q
Classical complement pathway activation
FcγRIII activation
Stimulation of
IL-6, C1q and C3 production
Membrane attack complex formation

42. MG lab in Galveston

43. MG Lab - Galveston Collaborators Huan Yang Bo Wu Stephen Higgs
Erdem Tuzun1,2
Shamsher Saini
Andrey Bednov
Ben Scott 3
Jing Li1
Iris Wingrow3
Huibin Qi
Xiarong Wu
Collaborators
Huan Yang
Bo Wu
Stephen Higgs
Tian Lin Xio
Galen Kaufmann
Mat Merigioli
Juli Rowin
1MG foundation Osserman/Sosin/McClure Post doctoral Fellows
1,2MDA Research Career Award Recipients
3 MG Foundation Henry Viets Fellow
Supported by NIH,MDA, AFM,and MG Foundation