Treatment of Myasthenia gravis by in vitropreconditioned human MSCs

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1 Treatment of Myasthenia gravis by in vitropreconditioned human MSCs ECell France 4th Annual Meeting Grenoble Institute of Neuroscience - 22 May 2017 Sonia Berrih-Akin Rozen Le Panse Jean-Thomas Vilquin

2 Myasthenia gravis: pathologies affecting the Neuromuscular Junction Shigemoto et al. Geriatr Gerontol Int 2010

3 Myasthenia Gravis: an heterogeneous disease q Congenital Myasthenia Gravis q Autoimmune Myasthenia Gravis

4 Congenital myasthenic syndromes n Very rare diseases: less than 1/ n Genetic diseases (autosomal recessive or dominant diseases) n Different forms: ü Presynaptic: deficiency in acetylcholine transferase (ChAT) ü Synaptic: deficiency in acetylcholinesterase (AChE) ü Post-synaptic: mutations on AChR subunits, on proteins of the neuromuscular junction (MuSK, Dok7, rapsyn ) Dr. Sonia Berrih-Aknin

5 Dr. Rozen Le Panse Physiopathology of Myasthenia Gravis Diagnosis for autoimmune MG Symptoms Autoantibodies Physiopathology Pathogenic mechanisms of autoantibodies Role of the thymus Etiology : a multifactorial disease Therapies Conventional treatments New animal model Treatment using conditioned MSCs Perspectives

6 Autoimmune myasthenia gravis n Rare disease ü prevalence: 1/20.000; incidence: 2-5 cases/year/million n n Acquired disease due to autoantibodies ü Against postsynaptic components (Myasthenia gravis - MG) ü Lambert-Eaton Myasthenic Syndrome (LEMS) with abs against a presynap-c component Consequence: defective transmission between nerve and muscle n Symptoms: Fatigable muscle weakness of skeletal muscles: ocular - respiratory muscles n Different forms Specificity of autoantibodies Affected muscles Age of onset < > Thymic abnormalities Dr. Rozen Le Panse

Pr. Bruno Eymard Myasthenia diagnosis: clinical signs n Muscle weakness and fatigability increased by effort and ameliorated by rest n Several muscle groups affected Ocular muscles: diplopia, ptosis

7 Pr. Bruno Eymard Myasthenia diagnosis: clinical signs n Muscle weakness and fatigability increased by effort and ameliorated by rest n Several muscle groups affected Ocular muscles: diplopia, ptosis Arms and legs muscles Bulbar muscles: chewing and swallowing difficulties, nasal voice, cervical muscles, facial paresia n The worst myasthenic crisis: paralysis of respiratory muscles n Chronic and fluctuating disease

8 Osserman Classification

Physiopathology of Myasthenia Gravis Diagnosis for autoimmune MG Symptoms Autoantibodies Physiopathology Pathogenic mechanisms of autoantibodies

9 Physiopathology of Myasthenia Gravis Diagnosis for autoimmune MG Symptoms Autoantibodies Physiopathology Pathogenic mechanisms of autoantibodies Role of the thymus Etiology : a multifactorial disease Therapies Conventional treatments New animal model Treatment using conditioned MSCs Perspectives

Myasthenia diagnosis: NMJ targets for autoantibodies Motor neuron q AChR for acetylcholine receptor AChE ColQ LRP4 Agrin Acetylcholine q MuSK for Muscle Specific Kinase q LRP4 for Low- density

10 Myasthenia diagnosis: NMJ targets for autoantibodies Motor neuron q AChR for acetylcholine receptor AChE ColQ LRP4 Agrin Acetylcholine q MuSK for Muscle Specific Kinase q LRP4 for Low- density lipoprotein Receptor- related Protein 4 q Agrin, Collagen Q, cortac-n Muscle end- plate MuSK Rapsin AChR ErbB Simplified scheme of the neuromuscular junction Gilhus & Vershuuren, Lancet Neurology 2015

11 Physiopathology of Myasthenia Gravis Diagnosis for autoimmune MG Symptoms Autoantibodies Physiopathology Pathogenic mechanisms of autoantibodies Role of the thymus Etiology : a multifactorial disease Therapies Conventional treatments New animal model Treatment using conditioned MSCs Perspectives

12 Pathogenic effects of anti-achr antibodies Gilhus et al., Nature Rev. Immunol. 2016

13 Physiopathology of Myasthenia Gravis Diagnosis for autoimmune MG Symptoms Autoantibodies Physiopathology Pathogenic mechanisms of autoantibodies Role of the thymus Etiology : a multifactorial disease Therapies Conventional treatments New animal model Treatment using conditioned MSCs Perspectives

Implication of the thymus in Myasthenia Gravis Cellules épithéliales corticales Thymocytes Cellules épithéliales médullaires Normal Thymus ü Matura-on and educa-on of T cells (thymocytes) ü Central

14 Implication of the thymus in Myasthenia Gravis Cellules épithéliales corticales Thymocytes Cellules épithéliales médullaires Normal Thymus ü Matura-on and educa-on of T cells (thymocytes) ü Central role of thymic epithelial cells (TECs) ü TECs and myoid cells express AChR subunits Thymus in MG pa8ents ü Hyperplasia with germinal centers are more frequent in young pa-ents ü Thymoma are more frequent with age Dr. Rozen Le Panse

Thymic hyperplasia in Myasthenia Gravis n n n

Mainly observed in early-onset MG patients

control MG MG + corticoïds Epithelial cells

labeling) % of CD21 + areas 10 1 0.1 0.01 p<0.

15 Thymic hyperplasia in Myasthenia Gravis n n n Specific of patients with AChR autoantibodies Mainly observed in early-onset MG patients Abnormal development of germinal centers Adult control MG MG + corticoïds Epithelial cells (keratin labeling) Germinal centers (CD21 labeling) % of CD21 + areas p<0.001 p<0.003 Méraouna et al., Blood 2006 Controls MG MG + Cortico

16 Implication of the thymus in Myasthenia Gravis B B B B B B B B B Cellules épithéliales corticales Thymocytes Cellules épithéliales médullaires Thymus in early onset AChR + MG ü Neoangiogenic processes (HEVs) ü Chemokine overexpression (CXCL13, CCL21,...) ü Germinal center development ü An-- AChR autoreac-ve T cells ü B cells producing an-- AChR an-bodies ü Thymectomy of MG pa-ents AChR Dr. Rozen Le Panse

17 Physiopathology of Myasthenia Gravis Diagnosis for autoimmune MG Symptoms Autoantibodies Physiopathology Pathogenic mechanisms of autoantibodies Role of the thymus Etiology : a multifactorial disease Therapies Conventional treatments New animal model Treatment using conditioned MSCs Perspectives

18 Myasthenia Gravis, a multifactorial disease Polymorphisms on HLA and other genes Female predisposition in EOMG Genetic predisposition Environmental factors Gender bias Immune dysregulations Pathogens Regulatory cells

19 Female predisposition to autoimmunity Whitacre, Nat Immunol McCarthy M. Lancet % of patients with an autoimmune disease are women

20 Dragin et al., JCI 2016 Why are women more susceptible to autoimmunity? Thymic Epithelial Cell (mtec) AIRE AIRE-dependent TSAs Woman ** Man Estrogen Thymic Epithelial Cell Methylation AIRE Depletion of Autoreactive T cells Central tolerance AIRE mrna expression Man Woman ** Child Pubescent Adult *

21 Central role of IFNs in anti-achr response Viral infection Early- onset MG IFN- β IFN- I Thymoma- associated MG IFN- α2, - α8, - ω, - β DC T DC T T T T BB T B B B B B B B B α- AChR IFN-α2 mrna (Log) *** ** Germinal center 1 Ad MGT T AChR attack Some viral infections may be involved in MG Dr. Rozen Le Panse

Suppressive function of T cells is defective in MG Treg FoxP3 TGF- β IL- 10 Regulatory (CD4 + CD25 + ) Th1 T- bet IFN- γ TNF- α Conventional (CD4 + CD25 - ) Th2 GATA3 IL- 4 IL- 5 IL- 13 Th17 RORγt

22 Suppressive function of T cells is defective in MG Treg FoxP3 TGF- β IL- 10 Regulatory (CD4 + CD25 + ) Th1 T- bet IFN- γ TNF- α Conventional (CD4 + CD25 - ) Th2 GATA3 IL- 4 IL- 5 IL- 13 Th17 RORγt IL- 17A IL- 17F IL- 21 IL- 22 Tfh Bcl6 IL- 21 IL- 4 IL- 10 Is the default due to regulatory or conventional T cells? Treg do not suppress proliferation Tconv are resistant to suppression Gradolatto et al., J Autoimmunity 2014

23 Involvement of BREG cells in MG Modulate the immune response (tumors, infectious diseases, autoimmunity). Contribute to the maintenance of tolerance (immune-regulatory cytokines). All mechanisms rely on IL-10. Autocrine IL-35 to expand or induce BREG. Decreased numbers of BREG in MG patients, especially the most severe Sun et al, Muscle & Nerve 2014

24 Physiopathology of Myasthenia Gravis Diagnosis for autoimmune MG Symptoms Autoantibodies Physiopathology Pathogenic mechanisms of autoantibodies Role of the thymus Etiology : a multifactorial disease Therapies Conventional treatments New animal model Treatment using conditioned MSCs Perspectives

25 Therapeutic strategies Pr. Bruno Eymard Ø Anticholinesterases drugs (to increase presence of ACh at NMJ) Ø If thymoma : thymectomy Ø Generalized seropositive, until 40 years-old : thymectomy Ø Severe forms (swallowing, respiratory, CV < 60) Ø Intensive care unit if myasthenic crisis suspected Ø Short term : intravenous immunoglobulins / plasma exchanges Ø Long term : Ø Corticoids (anti-inflammatory, modulation of gene expression) Ø Imuran (inhibition of DNA synthesis and cell proliferation) Ø Mycophenolate mofetil (inhibition of synthesis of purine) Ø Other therapies: Ø Cyclosporine, Tacrolimus (Immunosuppressants) Ø Cyclophosphamide Methotrexate Ø Monoclonal antibodies (ongoing assays)

26 Physiopathology of Myasthenia Gravis Diagnosis for autoimmune MG Symptoms Autoantibodies Physiopathology Pathogenic mechanisms of autoantibodies Role of the thymus Etiology : a multifactorial disease Therapies Conventional treatments New animal model Treatment using conditioned MSCs Perspectives

Set up of a new animal model of MG (MG- NSG) Human thymic

cells, germinal centers and T cells can be detected Anti-AChR

27 Set up of a new animal model of MG (MG- NSG) Human thymic fragments are grafted under the skin of SCID mice CD21 B cells, germinal centers and T cells can be detected Anti-AChR antibodies (nm) Vasculariza-on of the grax 45 days later MG 1.0 Control Days An-bodies to AChR appear from 2 weeks in mice receiving MG fragments CD4+CD8 Sudres et al., 2017

Sicsic (Hadassa Hospital), 2017 An-- AChR Ab in the sera of the mice AChR content in the diaphragm 5 0.

28 An-- human AChR an-bodies and AChR loss are generated by MG thymic transplants M. Sudres, Collabora;on T. Brenner and C. Sicsic (Hadassa Hospital), 2017 An-- AChR Ab in the sera of the mice AChR content in the diaphragm < pmol/ml serum % of the CTRL mean < CTRL MG seropos low MG seropos high 60 CTRL MG low MG high The result reflects the pathology of the donor of the thymus

29 The anti-human AChR antibodies generated by MG thymic transplants mediate MG symptoms sick animal percentage 100 MG seropos high 80 MG seropos low CTRL days post graft % of animals with at least one criteria of MG Number of MG-positive mice /52 4% Control 44/49 90% MG Most animals display at least one sign of the disease Sudres et al., 2017

Human cells home to mouse lymphoid organs A B h C D 4 5 + p e rc e n t o f to ta l c e lls 6 0 4 0 2 0 0 C C T R L M G S p B l B

e n t o f to ta l c e lls in s p le e n 6 0 ** 4 0 2 0 0 C T R L M G *** **** u n g ra fte d C T R L M G Upon sacrifice of

30 Human cells home to mouse lymphoid organs A B h C D p e rc e n t o f to ta l c e lls C C T R L M G S p B l B M S p B l B M D CTRL MG LaminA/C (human nucleus) s p le e n w e ig h t/m o u s e w e ig h t E h C D p e rc e n t o f to ta l c e lls in s p le e n 6 0 ** C T R L M G *** **** u n g ra fte d C T R L M G Upon sacrifice of grafted animals, the presence of human cells (control, MG) was assessed. Cells circulated and homed to the spleen. Sudres et al., 2017

31 The Thymus of MG patients produce antibodies to AChR when grafted in NSG mice Why Mesenchymal Stem cells?

32 Physiopathology of Myasthenia Gravis Diagnosis for autoimmune MG Symptoms Autoantibodies Physiopathology Pathogenic mechanisms of autoantibodies Role of the thymus Etiology : a multifactorial disease Therapies Conventional treatments New animal model Treatment using conditioned MSCs Perspectives

33 MSCs inhibit T cell prolifera-on Proliferating cells: 76.9% 80 *** *** - MSC Proliferating cells: 4.1% Proliferating cells (%) *** +MSC (MSC/PBMC:1/5) 0 -MSC +MSC -MSC +MSC Controls (n = 27) MG patients (n = 10) PBMC from Healthy Controls do not proliferate when grown with adipose-derived human MSC PBMC from MG patients also respond to MSC inhibition (to a lesser extent) Ben Ami et al, Autoim Rev, 2014

34 Conditioned MSC are more efficient in vivo (MG-NSG) Some adipose-derived MSC have been previously conditioned by coculture with human PBMC Anti AChR Ab (nm) MG rmsc cmsc before treatment 2w post treatment % of the CTRL mean MG rmsc cmsc The titer Ab is decreased in animals receiving conditioned MSCs The percentage of free NMJ is increased in animals receiving conditioned MSCs Sudres et al., 2017

35 Conditioned MSCs reduce the symptoms in NSG-MG mice Score Incidence score (AU) % of sick mice MG rmsc cmsc < MG rmsc cmsc days post graft What are the mechanisms? Sudres et al., 2017

100 50 0 Ki67 in the thymus mki67 in thymus 150 0.0092 0.

36 Sudres et al., 2017 Condtioned MSC inhibit proliferation of cells in MG-NSG mice mki67 mrna expression (%MG 2 -ΔCt ) Ki67 in the thymus mki67 in thymus MG rmsc cmsc - MSC Spleen histology Dapi LaminA/C KI-67 mrna expression (%MG 2 -ΔCt ) Ki67 in the spleen mki67 in spleen cmsc Less Ki67+ cells in the cmsc-treated mice compared to untreated animals 0 MG rmsc cmsc

37 Condtioned MSC modify the expression of several genes CD55 (% MG 2delta Ct) MG rmsc cmsc TNF-α (%MG 2 - ΔCt ) MG rmsc cmsc cmscs increase the level of CD55 in the grafted thymus, a regulator of complement pathway (RT-PCR) cmscs inhibit the expression of TNF-alpha in the grafted thymus (RT-PCR) Sudres et al., 2017

38 Hypothetical mechanistic scheme in the thymus Sonia Berrih-Aknin, Muriel Sudres, 2017

39 Conclusions u NSG-MG: a new robust MG model to test therapies targeting human molecules or cells u MSCs reduce levels of anti-achr Ab, improve the AChR at the NMJ and decrease clinical symptoms u Conditioned MSCs >> resting MSCs u Mechanisms involved: u Decreased proliferation of human cells in mice u Reduced expression of pro-inflammatory cytokines and co-stimulatory molecules u Increased expression of CD55, a regulator of complement pathway u A preconditioning step promotes the therapeutic effects of MSCs u cmscs : a promising strategy for treating MG (and other autoimmune diseases?) Sonia Berrih-Aknin, Muriel Sudres, 2017

40 Physiopathology of Myasthenia Gravis Diagnosis for autoimmune MG Symptoms Autoantibodies Physiopathology Pathogenic mechanisms of autoantibodies Role of the thymus Etiology : a multifactorial disease Therapies Conventional treatments New animal model Treatment using conditioned MSCs Perspectives

Collaborators, Acknowledgments Laboratory Members Muriel Sudres Marieke Robinet Marie Maurer Jacky Bismuth Nadine Dragin Clinicians B. Eymard (Institut de Myologie) F.

41 Collaborators, Acknowledgments Laboratory Members Muriel Sudres Marieke Robinet Marie Maurer Jacky Bismuth Nadine Dragin Clinicians B. Eymard (Institut de Myologie) F. Bolgert (La Pitié Salpêtrière) T. Sharshar (Hôpital de Garches) E. Fadel (Hôpital Marie Lannelongue) D. Gossot (Institut Mutualiste Montsouris) N. Santelmo (Hôpital Civile de Strasbourg) Scientific collaborators T. Brenner, C. Sicsic (Hadassa Hospital) E. Ben Ami and A. Miller (Technion, Haifa) Thank you for your attention!

42 Collaborators, Acknowledgments Equipe Myasthénie - Centre de recherche en Myologie UMRS 974 UPMC - INSERM - CNRS - AIM Sonia Berrih-Aknin Rozen Le Panse FP5

BALANCE BETWEEN ESTROGENS AND PROINFLAMMATORY CYTOKINES REGULATES CHEMOKINE PRODUCTION INVOLVED IN THYMIC GERMINAL CENTER FORMATION

BALANCE BETWEEN ESTROGENS AND PROINFLAMMATORY CYTOKINES REGULATES CHEMOKINE PRODUCTION INVOLVED IN THYMIC GERMINAL CENTER FORMATION BALANCE BETWEEN ESTROGE AND PROINFLAMMATORY CYTOKINES REGULATES CHEMOKINE PRODUCTION INVOLVED IN THYMIC GERMINAL CENTER FORMATION Nadine Dragin 1, 2, 3,, Patrice Nancy 4, José Villegas 2, 3, 5, Régine