1. Rozen Le Panse CNRS UMR 8162 Centre Chirurgical Marie lannelongue Le Plessis Robinson - France Rozen Le Panse CNRS UMR 8162 Centre Chirurgical Marie lannelongue Le Plessis Robinson - France Thymic changes in Myasthenia Gravis: Involvement of chemokines in thymic hyperplasia associated with an inflammatory environment International conference on Myasthenia December 1-2, 2009 - Paris - France

2. Implication of the thymus in MG AChR + Myasthenia Gravis (MG) AChR expression (Wakkach et al., 1996) Anti-AChR autoreactive T cells (Melms et al., 1988) B cells producing anti-AChR antibodies (Leprince et al., 1990 AChR B B B B B B Thymus Muscle B B B B

3. Méraouna et al., Blood 2006 Ectopic germinal centers (GCs) Germinal centers (CD21 labeling) Epithelial cells (keratin labeling) Adult controlMG + corticoïdsMG Thymic hyperplasia in AChR + MG % of CD21 + areas 0.01 0.1 1 10 p<0.003p<0.001 MG + CorticoMGControls

4. Neoangiogenesis is associated with thymic hyperplasia MG HEVs GCs Ectopic high endothelial venules (HEVs) Berrih-Aknin et al., Ann. of Neurol. 2009 Lymphangiogenesis VEGFR3 mRNA (AU) PROX1 mRNA (AU) Adult control HEVs Stroma GCs

5. Role of chemokine in thymic hyperplasia Expression of chemokines involved in GC formation Thymic transcriptome analysis revealed a chemotactic signature in the hyperplastic thymus of MG patients. Chemotactic assays Le Panse et al., J. Immunol 2006 CXCL13CCL21 ELISA

6. Analysis of CXCL13 overexpression in MG Méraouna et al., Blood 2006 CXCL13 overexpression by thymic epithelial cells CXCL13 mRNA (AU) Q-PCR on thymic epithelial cells Laser-capture microdissection Q-PCR on CXCL13

7. Generation of transgenic mice for CXCL13 Increased expression of CXCL13 and B-cell recruitment Weiss et al., in progress  Heterozygous… B220 Keratin Q-PCR on CXCL13 Immunofluorescence

8. Analysis of CCL21 overexpression in MG VEGFR3 CCL21 % of cell migration CCL21 overexpression by lymphatic vessels HEVs CCL21 Berrih-Aknin et al., Ann. of Neurol. 2009 Immunofluorescence Transwell assaysQ-PCR for PROX1 and D6

9. Julia Weiss : Poster 16 Expression of SDF1 on thymic HEVs Search for chemokines on HEVs HEVs SDF1 MG Transcytosis ? Immunofluorescence

10. Roles of chemokines in thymic hyperplasia Signals triggering these thymic dysregulations ? Thymus Epithelial cells CXCL13 Lymphatic endothelial cells CCL21 GC T Tn DC B B B B B B B B B B B B B FDC B T T T B T B Tn T T T T B B B B B B B B B B B B FDC B B B B B B B B B B BB BB BB B BB BB BB BB BB BB BB BB B BB T T B TT B Tn HEV SDF1-α CXCL10 (Feferman et al., 2003) CXCL9-CXCL11 (Transcriptomic analysis) RANTES (Colombarra et al., 2005) Peripheral cell recruitment

11. Microarray analysis revealed a role for IFN-I in MG Increased expression of IFN-  in MG thymuses Cufi et al., in progress  -IFN  -IFN Microarray data Q-PCR and ELISA

12. Roles of IFN-I on chemokine expression IFN-IIFN-II U/ml CCL21 Lymphatic endothelial cells (Dermal LECs) Perrine Cufi : Poster 14 IFN-I induces the expression of : CXCL13 and CCL21  -AChR Thymic epithelial cells IFN-IIFN-II U/ml CXCL13 IFN-I  -AChR

13. Potential involvement of viral infection in MG Cufi et al., in progress Specific effect of Poly(I:C) Selective effect on the  -AChR subunit Poly(I:C) (µg/ml) TLR1/2 TLR3 TLR4TLR6/2TLR7TLR7/8TLR9TLR5

14. In vivo confirmation of Poly(I:C) effects on  -AChR Poly(I:C)Control  -AChR  -AChR Cufi et al., in progress Thymic upregulation of  -AChR expression by Poly(I:C) Q-PCR on AChR subunits

15. Could a viral infection, under particular conditions, trigger thymic changes involved in MG? Conclusion Thymic Hyperplasia

16. Julia Weiss Perrine Cufi Nathalie Ruhlman Géraldine Cizeron-Clairac Jacky Bismuth Frédérique Truffault Amel Méraouna Sonia Berrih-Aknin Nicole Kerlero de Rosbo WEIZMANN INSTITUTE Idit Shachar Frida Lantner UMR 8162 Acknowledgements