Control of autoimmune diabetes in NOD mice by GAD expression or suppression in β Cells

1. Control of autoimmune diabetes in NOD mice by GAD expression or suppression in β Cells Presented by Johann Campbell Yoon, J., C. Yoon, H. Lim, Q. Huang, Y. Kang, K. Pyun, K. Hirasawa, R. Sherwin, and H. Jun. 1999. Control of autoimmune diabetes in NOD mice by GAD Expression or suppression in β cells. Science. 284: 1183-1186.

2. GAD and Autoimmune Diabetes • GAD is found to be an important autoantigen in Type 1 Diabetes • Type 1 Diabetes results from auto immune insulin-producing cell destruction

3. Suppressing GAD expression • Isoforms of GAD are GAD 65 and GAD 67 • Antisense GAD65.67 suppresses expression of normal GAD65.67 • Antisense GAD65.67 produced using antisense transgene for GAD65.67 • Antisense genes prevent translation of mRNA

4. Suppressing GAD expression • Rats were selected based on expression of antisense GAD65.67 • PCR revealed three types of expression • High expression of antisense GAD65.76 or H-AS-GAD-NOD/Hk-AS-GAD-NOD • Medium expression of antisense GAD65.67 or M-AS-GAD-NOD/Mk-AS-GAD-NOD • Low expression of antisense GAD65.67 or L-AS-GAD-NOD/Lk-AS-GAD-NOD

5. Suppressing GAD expression • Northern Blot shows different levels of antisense mRNA Western Blot shows different levels of expression of normal GAD in pancreatic cells • GAD expression normal in brain cells

6. Suppressing GAD expression • Anti-GAD staining showed varying levels of GAD in transgene (-), L-AS-GAD-NOD and M-AS-GAD-NOD mice • GAD was suppressed H-AS-GAD-NOD mice

7. Disease development in NOD mice • 0% of H-AS-GAD-NOD mice developed diabetes after 40 weeks • 67% M-AS-GAD-NOD mice developed diabetes after 40 weeks • 75% L-AS-GAD-NOD mice developed diabetes after 40 weeks • 81% transgene-negative mice after 40 weeks

8. Assessing effects of GAD (normal) absence and diabetes progression • L-AS-GAD-NOD and M-AS-GAD-NOD developed diabetes after 40 weeks • Transgene-negative (normal GAD) also developed diabetes after 40 weeks • H-AS-GAD-NOD did not develop diabetes after 40 weeks • Results were similar for second line of transgenic mice

9. Pancreatic and salivary status of NOD mice • 80% of H-AS-GAD-NOD mice had intact pancreatic cells after 20 weeks • Less than 20% of those pancreatic cells (H-AS-GAD-NOD) showed inflammation • The other groups (M and L) showed significant damage to pancreatic cells • The second line of transgenic mice (Hk, etc) showed similar results • Hk-AS-GAD-NOD – 2.8% developed diabetes in the same time frame • Mk-AS-GAD-NOD – 83.3% developed diabetes in the same time frame • Lk-AS-GAD-NOD – 80.8% developed diabetes in the same time frame

10. Further investigation into GAD’s role in autoimmune diabetes • Mice were developed with incorporated transgene for murine leukemia proviral env protein • 79% of transgenic for env protein mice developed diabetes • 82% transgenic-negative for env protein developed diabetes • Diabetes prevention is not achieved with transgenic expression of other autoantigens

11. Mechanism initiated by GAD expression in autoimmunity • Cells from H-AS-GAD-NOD mice were transplanted to NOD.scid mice • Cells from transgene-negative mice were transfused to NOD.scid mice • None of the H-AS-GAD-NOD recipients developed diabetes • Almost 90% of transgene-negative recipients developed diabetes • T-cells in the autoimmune reaction of the diabetes seem to be stimulated by the presence of normal GAD

12. Other mechanisms involved in T-cell production • H-AS-GAD-NOD mice treated with GAD did not develop T-cells towards GAD • Treatment with HSP60 and insulin did produce insignificant amounts of T-cell towards either antigen • Also transplanting with splenocytes from Hk-AS-GAD-NOD mice did not induce T-cell production towards autoantigens

13. Investigating response to GAD-suppressed cells • GAD-suppressed cells and transegene-negative (GAD-expressing) were transplanted to young diabetic NOD mice • GAD-suppressed cells helped prevent the recurrence of diabetes, as seen in steady gloucose levels. • All recipients of GAD-expressing cells had a recurrence of diabetes and destruction of grafted cells • About 80% of grafted GAD-suppressed cells remained intact while the ltter 20% showed peri-insulitis • Transplanting with Hk-AS-GAD-NOD displayed similar results • Cells from H-AS-GAD-NOD mice were transplanted to transgene-negative mice with no cell destruction • Transgene negative recipients displayed marked pancreatic tissue damage

14. Conclusion • GAD has to be present on β-cell in order to initiate an immune response • The initiated immune response causes destruction of β-cells