1. Complement in Heart Allograft Biopsies E. Rene Rodriguez W. M. Baldwin, III

4. Variables in Heart Studies Patient Population Percent Sensitized LVADLVAD Blood TransfusionsBlood Transfusions PregnancyPregnancy Previous transplantPrevious transplant Immunosuppression

5. Prospective Study of Cardiac Transplants All biopsies from Jan ‘01 to Dec ‘03 All biopsies from Jan ‘01 to Dec ‘03 665 biopsies from 165 patients 665 biopsies from 165 patients 107 males: 58 females 107 males: 58 females 34 patients followed from time of transplantation 34 patients followed from time of transplantation almost all patients had 0%PRA almost all patients had 0%PRA Rodriguez et al AJT 2005 (in press)

6. ROUTINE DIAGNOSTIC PROTOCOL BIOPSIES FROM CARDIAC TRANSPLANTS 1 mo 2 mo 3 mo 6 mo 1 yr H&E, IP (CD68) FITC (IgG, IgM, IgA, C1q, C4d, C3d)

7. Number of Patients with Positive Biopsies 16 (9.6%) 17 (10.3%)

9. C3d+C3d - C4d+ 13 (8%)*3 (2%) C4d-4 (2%)145 (88%) * One patient 10% PRA C4d and/or C3d Deposits (665 consecutive biopsies from 165 patients)

11. C4d C3d C4d + C3d n = 3 n = 4 n = 13 Incidence of C4d and C3d deposits Dysfunction n = 5

12. Patients with AMR ISHLT grade C4d + C3d CD 68 Dys- function #graft Donor Sp. Ab. HLA (I or II) 1B +++2nd + I + II 1A +++2nd - - 0 +++2nd +I 1B +++ 1st - - 1A +++ 1st +II

13. ANTIBODY ASSOCIATED REJECTION 7 years after transplantation anti-DR7 and DRw53

14. Macrophages

15. Response to Therapy Plasmapheresis and IVIg Graft function improved Graft function improved DSA decreased or eliminated DSA decreased or eliminated (C consumption not measured) (C consumption not measured) C3d deposits cleared first C3d deposits cleared first Low levels of antibodies (as detected Flow beads) can persist beyond C4d Low levels of antibodies (as detected Flow beads) can persist beyond C4d

16. Association of Cardiac Allograft Vasculopathy with C4d C3d and AMR

17. C4d C3d C4d + C3d n = 3 n = 4 n = 13 Incidence of C4d and C3d deposits Dysfunction n = 5

18. 1A 1B No antibodies Class I or II Second Heart Transplant with Repeat HLA Mismatch Strong C4d, Little C3d, No HLA antibodies, Good Function 1st heart HLA-A1 2nd heart HLA-A1 0 12 Baldwin et al 2004 AJT 4:311

19. C1 C4 C3 C2 C5 C6 C7 C8 C9 C4bp DAF MCP* CR1* Factor I Factor H DAF MCP* CR1* Factor I CD59 Regulation of Complement Cascade

20. C1 C4 C3 C2 C5 C6 C7 C8 C9 C4bp DAF MCP* CR1* Factor I Factor H DAF MCP* CR1* Factor I CD59 C4dC3d C4d and C3d are Products Complement Regulation

21. C1 C4 C3 C2 Sensitization vs Regulation

22. C1 C4 C3 C2 C1 C4 C3 C2 Sensitization vs Regulation

23. C1 C4 C3 C2 C1 C4 C3 C2 C1 C4 C3 C2 C1 C4 C3 C2 C1 C4 C3 C2 C5 C6 C7 C8 C9 Sensitization vs Regulation

24. Epilogue

25. Epilogue …and between different eras in the same center eg, JHU Increased patients with >0% PRA 2 patients with episodes of antibody-mediated rejection in the first weeks after transplantation Required longer treatment with PP & IVIg

26. Summary C4d and C3d in Heart Transplants Incidence depends on sensitization Can occur years after transplantation Needs to be correlated with graft function and Donor Specific Antibody (DSA) for a diagnosis of antibody-mediated rejection May be correlated with increased graft vasculopathy

27. Kenji Minami Kazunuri Murata Clinical studies E. Rene Rodriguez Experimental studies