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Immune regulation defect in human Myasthenia Gravis

Immune regulation defect in human Myasthenia Gravis. Sonia Berrih-Aknin CNRS UMR-8162, IPSC. International Conference on Myasthenia Gravis, December 1-2, Paris. Scanner sections of human thymus. ( Meraouna et al, Blood, 2006). Fe male 22 yrs. Fem ale 24 yrs. Fem ale 26 yrs. Control.

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Immune regulation defect in human Myasthenia Gravis

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  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. Scanner sections of human thymus (Meraouna et al, Blood, 2006) Female 22 yrs Female 24 yrs Female26yrs Control MG Cortico- MG Cortico+ In Green: follicular dendritic and B cells stained with anti-CD21 antibodies; in Red; staining with anti-keratin antibodies

  3. Regulatory T X - activated T Anti-acetylcholine receptor autoimmune response occurs in the thymus of MG patients - Corticoids Inflammatory environment Why a chronic inflammation? Are regulatory cells present and efficient? Cytokines APC IL-4 MHC resting T B Th2 B7 T cells Vb5.1 Fas AChR Anti-AChR Antibodies

  4. Percentage of CD4+CD25+ thymocytes isunchanged in MG patients % CD25+ among CD4+ cells % CD25hi among CD4+ cells N.S. N.S. 6 N.S. N.S. 30 4 20 2 10 0 0 newborns adults newborns adults MG patients MG patients Controls Controls What about the function?

  5. The suppressive function of CD4+CD25+ cells in MG patient thymus is severely impaired (Balandina et al, Blood, 2005, 1% top citation) 0 0.2 0.4 0.6 0.8 1 1.2 120 100 MG 80 60 % of proliferative response 40 Adult Newborn 20 0 Ratio of CD4+CD25+/ CD25- cells Are peripheral CD4+CD25+ cells also defective?

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

  7. Expression of CD127neg/low in CD4+CD25+ cells PBMC THYMUS 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 CD95 in CD4+ PBL CD95 in CD4+ thymocytes * *** ** In MG, CD95 (Fas) is highly increased in thymic, but not peripheral lymphocytes

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

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

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

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

  15. In contact with normal TEC, MG CD4+ cells have an increased suppressive abilitycompared to cellsbefore 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. Regulatory T X - activated T Anti-acetylcholine receptor autoimmune response occurs in the thymus of MG patients Inflammatory environment Why a chronic inflammation? Are regulatory cells present and efficient? Cytokines APC IL-4 MHC resting T B Th2 B7 T cells Vb5.1 Fas AChR Anti-AChR Antibodies

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

  19. Thank you for your attention

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

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

  22. CD4+25+ cells in the thymus of MG patients present phenotypic and functional abnormalities • 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 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 FoxP3 CD25 Treg + TEC Treg Treg + TEC Treg No. of cells (Standardized %) No. of cells (Standardized %)

  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 Etudiants: Julia Weiss (PhD, ANR) • ITA • Frederique Truffault, CCML • Jacky Bismuth, CCML • Perrine Cuffi, CDD ANR • Margot Schmolinsky, CDD EU

  28. Margot Treg IFN, virus et chemokine Angeline

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

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

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

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

  33. + + + + + + CD25- Faslo CD25+ Fashi Faslo Fashi + + Both CD4+CD25+ Faslo and Fashi are functionally suppressive in controls but defective in MG patients Controls MG patients

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

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