1. Immune regulation defect in human Myasthenia Gravis Sonia Berrih-Aknin CNRS UMR-8162, IPSC International Conference on Myasthenia Gravis, December 1-2, Paris

2. ControlMG Cortico+MG Cortico- Female 26 yrsFemale 22 yrsFemale 24 yrs In Green: follicular dendritic and B cells stained with anti-CD21 antibodies; in Red; staining with anti-keratin antibodies Scanner sections of human thymus (Meraouna et al, Blood, 2006)

3. Anti-acetylcholine receptor autoimmune response occurs in the thymus of MG patients Cytokines Th2 B IL-4 T cells V  5.1 Fas resting T activated T MHC B7 X APC Anti-AChR Antibodies Regulatory T - AChR Inflammatory environment Why a chronic inflammation? Are regulatory cells present and efficient? Corticoids -

4. Percentage of CD4+CD25+ thymocytes is unchanged in MG patients % CD25 + among CD4 + cells 0 10 20 30 MG patients adults newborns Controls N.S. % CD25 hi among CD4 + cells 0 2 4 6 N.S. MG patients adults newborns Controls What about the function?

5. Ratio of CD4 + CD25 + / CD25 - cells MG Adult Newborn 0 20 40 60 80 100 120 00.20.40.60.811.2 % of proliferative response The suppressive function of CD4+CD25+ cells in MG patient thymus is severely impaired (Balandina et al, Blood, 2005, 1% top citation) Are peripheral CD4+CD25+ cells also defective?

6. Treg cells are also deficient in PBMC, but at a lesser extent % Proliferation 0 60 80 20 40 100 ControlsMG % Proliferation 0 60 80 20 40 100 ControlsMG THYMUSPBMC Is the phenotype of thymic and peripheral CD4+CD25+ cells different?

7. Expression of CD127neg/low in CD4+CD25+ cells THYMUS PBMC In the thymus or in PBMC of MG patients, there is no difference in the expression of IL-7R in the CD4+CD8+ population. What about FAS?

8. Compared phenotype of Treg cells in the thymus and PBMC *** ** * In MG, CD95 (Fas) is highly increased in thymic, but not peripheral lymphocytes CD95 in CD4+ thymocytes CD95 in CD4+ PBL

9. Compared phenotype of Treg cells in the thymus and PBMC In MG, both CXCR3 and CXCR5 are increased in thymic, but not peripheral CD4Treg cells

10. These results suggest that the thymic environment influences locally the thymic CD4 and Treg cells Role of Thymic epithelial cells?  Major role in the differentiation of lymphocytes  Overproduce pro-inflammatory cytokines in MG

11. D6 Control thymic epithelial cells (TEC) maintain Treg phenotype of control CD4+CD25+ thymocytes CD4 cells alone CET+CD4 D6

12. TEC protect the Treg phenotype CD25 D3/D0 x 100 - TEC + TEC ** - TEC + TEC FoxP3 D3/D0 x 100 ** CD25 FoxP3 Total CD4+ cells

13. The effet is TEC specific FoxP3 CD25 FoxP3 CD25 FoxP3 CD25 + CET + CACO2 + MITC Cell number (Standardized %) Cell number (Standardized %) Cell number (Standardized %) Cell number (Standardized %) CD25 FoxP3 + CET + CACO2 + MITC % Control + TEC + MITC FGM 42 80 + TEC + MITC FGM 28 40 + TEC + CACO2 FGM 31 67 + TEC + CACO2 FGM 31 67

14. In contact with MG TEC, normal CD4+ cells has a low suppressive ability Teff Treg Teff Treg Co-culture (3 days) Control CD4 + Cont TEC MG TEC % proliferation Suppression assay *

15. In contact with normal TEC, MG CD4+ cells have an increased suppressive ability compared to cells before culture

16. Conclusions  MG Treg are defective both in the thymus and at the periphery (at a lesser extent). This is not due to higher number of Teff cells  TEC involved in Treg phenotype and function  MG TEC induce defective Treg  Normal TEC reverse partially the defect of MG Treg Defects of Treg from MG patients probably linked to the inflammatory thymic environment, potentially reversible: Cell therapy

17. Anti-acetylcholine receptor autoimmune response occurs in the thymus of MG patients Cytokines Th2 B IL-4 T cells V  5.1 Fas resting T activated T MHC B7 X APC Anti-AChR Antibodies Regulatory T - AChR Inflammatory environment Why a chronic inflammation? Are regulatory cells present and efficient?

18. Collaborators and financial support D. Nazzal F. Truffault J. Bismuth N. Kerlero de Rosbo

19. Thank you for your attention

20. M1 Normal cells MG cells CD4+CD25neg cells from MG patients present a defect of FoxP3 regulation

21. FoxP3 on human thymus sections Newborn thymus Adult thymus MG thymus Keratin FoxP3 CD21 FoxP3

22.  Number of CD4+CD25+ is normal  Phenotype of CD4+CD25+ cells  IL-7R normal  mRNA FoxP3 decrease  Fas increase  Function of CD4+ CD25+ cells  Suppressive function defective FoxP3  Cell number not decreased  Located in the medulla and GC  Regulation defective CD4+25+ cells in the thymus of MG patients present phenotypic and functional abnormalities What are the consequences of Treg defects?

23. Overproduction of Il-1 and IL-6

24. CD4+CD25+ Treg cells play a major role in prevention of autoimmunity, namely through the transcription factor, FoxP3, whose expression is highly correlated with the suppressive function. Numbers of thymic CD4+CD25+ Treg cells do not differ in MG patients and control individuals, while these cells present a severe functional defect in MG patients. A similar analysis in peripheral blood showed a similar defect in Treg function, although at a lower extent. In parallel, we investigated the phenotype of MG and control Treg. In the periphery, Treg cells from MG patients are very similar to control cells, while in the thymus Treg cells form MG patients express higher level of DR, Fas and CXCR3 compared to controls. These thymic-specific features suggest that thymic Treg cells may be influenced by unique interactions within the thymus. This leads us to analyze in more detail, the functional cross-talks between Treg and thymic epithelial cells (TEC) that participate to the development of self-tolerant T cells. We showed that TEC (from normal or MG thymus) are able to support the expression of CD25 and FoxP3 on CD4+ cells. Interestingly, the suppression activity of CD4+CD25+ issued from co-culture with TEC from MG patients, and not with non-MG TEC, was impaired as attested by functional assays. Altogether, these results suggest that functional Treg defects in MG patients are at least partially issued from defective interactions between developing T cells and TEC. Since we previously showed that TEC from MG patients overproduce IL-1 and IL-6, they could shift Treg cells towards a pro- inflammatory phenotype.

25. Control thymic epithelial cells (TECs) maintain Treg phenotype of control CD4+CD25+ thymocytes CD25FoxP3 No. of cells (Standardized %) Treg No. of cells (Standardized %) Treg + TEC Treg + TEC

26. Regulatory CD4+CD25+ T cells The CD4 + CD25 + Treg provide protection from T cell-mediated autoimmune disorders CD25 + cells are naturally anergic in vitro and they inhibit the proliferation of co- cultured CD4 + CD25 -

27. Organigramme Chercheurs Sonia Berrih-Aknin, DR INSERM Rozen Le Panse-Ruskoné, CR CNRS Nicole Kerlero de Rosbo, CDD EU Nadine DRAGIN-MAMAVI, CDD EU Angeline Gradolatto, CDD EU Dani Nazzal, CDD EU Sylvain Bougoin, CDD EU ITA Frederique Truffault, CCML Jacky Bismuth, CCML Perrine Cuffi, CDD ANR Margot Schmolinsky, CDD EU Etudiants: Julia Weiss (PhD, ANR)

28. Angeline Margot Treg IFN, virus et chemokine

29. Defective suppression of CD4+CD25+ cells Defective suppression of CD4+CD25+ cells 2 hypothesis 1)FoxP3 = transcription factor required for the suppressive function 2 ) Imbalance between Treg and activated effector cells IL-7R FAS

30. IL-7 R = CD127 IL-7 R = CD127 J. Exp. Med, 2006

31. Fas++ thymocytes accumulate in MG patients with anti-AChR antibodies (Moulian et al., Blood, 1997) 1,3% MG1, 15 yrs, 0 nM 1,3% Control, 17 yrs 1,2% Fas MG2, 36 yrs, 3 nM 4,1% 14,7 % MG3, 19 yrs, 37 nM Controls <1 1-10>10 nM 0 5 10 15 MG patients N.S. % of Fas++ thymocytes p<0.006p<0.001

32. Fas expression in CD4+CD25+ cells % Fas hi % Fas lo MG adults newborns Controls p<0.0001 0 20 60 40 10 30 50 70 p<0.0001 N.S. 20 60 40 0 80 MG adults newborns Controls Fas lo = Treg? Fas hi = effector?

33. CD25 + CD25 - + ++ + Fas hi Fas lo +++ + Fas hi Fas lo ControlsMG patients Both CD4+CD25+ Faslo and Fashi are functionally suppressive in controls but defective in MG patients

34. Fas phenotype of CD4+CD25+ cells (Balandina et al, Blood, 2005) Thymocytes CD4 + CD25 + thymocytes from MG patients  Increase of Fas hi  Decrease of Fas lo CD4 + CD25 - CD4 + CD25 + FAS 59 % MFI=263 7 % Fas hi MFI=51 MFI=5 20 % MFI=117 MFI=11 Fas hi MFI=9 MG Adult Newb