Why transplant? “Cure for organ specific disease”

1. Why transplant? “Cure for organ specific disease” (or islets since 2000) Adapted from “Immunology” by Goldsby, Kindt, Osborne, Kuby

2. ISOGRAFT AUTOGRAFT XENOGRAFT (Experimental) ALLOGRAFT

3. ISOGRAFT AUTOGRAFT XENOGRAFT (Experimental) ALLOGRAFT POTENT IMMUNE RESPONSE

4. Immune rejection of organ transplants is a major barrier to success • 3 manifestations defined by when and how occur • Hyperacute • Acute • Chronic

5. Hyperacute rejection - within hours - Ab mediated Adapted from “Immunology” by Goldsby, Kindt, Osborne, Kuby

6. Problematic pre-existing antibodies and how to deal with these • Allotransplants • -natural antibodies to ABO blood group antigens • -anti-HLA antibodies raised during previous transfusion, transplant or pregnancy • Solution: test recipient serum for ABO compatibility and negative crossmatch • Xenotransplants • natural antibodies to Gala1-3Gal epitope present in lower mammals • Solution: agalactosyl transferase knockout pig

7. Acute rejection - within weeks Adapted from “Immunology” by Goldsby, Kindt, Osborne, Kuby

8. T cells play a central role in acute rejection Adapted from “Immunology” by Goldsby, Kindt, Osborne, Kuby

9. Induction in draining lymph nodeT cells are activated by 2 pathways Indirect (classical antigen presentation by host DC) Transplant-derived peptide on host MHC/HLA interacts with host T cell receptor Response is weak for allografts but strong for xenografts Host T cell Host T cell Host DC Transplant Donor DC Direct (antigen presentation by passenger donor DC) Transplant-derived peptide on donor MHC/HLA or donor MHC/HLA alone interacts with host T cell receptor Response is strong for allografts but weak for xenografts

10. allo xeno Destruction by various effectors at graft site DC Adapted from “Immunology” by Goldsby et al

11. Solutions to acute rejection • MHC match • Serologic methods • Cellular assays • Molecular biological methods • Critical for bone marrow and helpful for kidney, but limited organ availability and highly polymorphic MHC means often not practical. • No MHC match for xenografts • Immunosuppress recipient • Cocktail of drugs with 3 distinct modes of action

12. Actions of immunosuppressive drugs 1. Inhibit T cell signaling e.g. CyclosporinA or FK-506 block calcineurin activity and thereby IL2 synthesis. 2. Anti-proliferative e.g.Azathioprine or mycophenolic acid inhibit synthesis of purines required for cell division. Inhibit B and T cell proliferation. 3. Anti-inflammatory e.g. Corticosteroids bind to intracellular steroid receptors and thereby regulate transcription of a number of genes including cytokines, adhesion molecules and class II molecules.

13. Chronic rejection - months or years after grafting Poorly understood combination of immunological + other factors Damage at time of grafting + acute rejection episodes + ongoing indirect response result in low grade damage to graft vascular endothelium, smooth muscle proliferation and migration leading to vascular occlusion Pascual et al, N. Engl. J. Med. 346:580 (2002) Solution: No effective immunosuppressive regimen to date, but predict better control of the immune response will decrease incidence of chronic rejection.

14. Organ transplantation is a life saving procedure, but there are major problems……

15. Problem 1 Systemic immunosuppression facilitates graft survival at a price- drug targets are not limited to the immune system resulting in toxicity for other organs-systemic immunosuppression increases susceptibility to cancer and infection-more specific drugs? (several in clinical trials) -induce graft tolerance? (“Holy Grail”)-local immunosuppression? (Our approach) Possible solutions under investigation

16. Transplant girl's blood change a 'miracle' The Sydney Morning Herald. January 25, 2008 Graft tolerance by chance and deduction

17. N ENGL J MED 358: 369-374 (2008).

18. O- O+ Case history • Hepatitis/liver failure • Profound lymphopenia O+ O+ Immunosuppression No immunosuppression 9 Y.O. • Predominantly donor white cells but approx. 2%host B cells • IgG on RBC progressing to active hemolysis • Donor liver survival • Loss of pre-existing immunity/successful reimmunization • TCR excision circles liver graft 12 Y.O.

19. How was tolerance established ?

20. O- O+ Presumed events resulting in graft tolerance • Hepatitis/liver failure • Profound lymphopenia Donor leukocytes/stem cells not rejected + space for expansion O+ O+ Immunosuppression No immunosuppression 9 Y.O. • Donor liver survival • Loss of pre-existing immunity/successful reimmunization • TCR excision circles • Predominantly donor white cells but approx. 2%host B cells • IgG on RBC progressing to active hemolysis liver graft 12 Y.O.

21. O- O+ Presumed events contributing to graft tolerance • Hepatitis/liver failure • Profound lymphopenia Donor leukocytes/stem cells not rejected + space for expansion O+ O+ Immunosuppression No immunosuppression 9 Y.O. • Donor liver survival • Loss of pre-existing immunity/successful reimmunization • TCR excision circles • Predominantly donor white cells but approx. 2%host B cells • IgG on RBC progressing to active hemolysis Donor leukocytes outgrow host leukocytes but residual host B cells make antibody to donor red blood cells liver graft Aggressive host B cells rejected by donor T cells upon withdrawal of immunosuppression 12 Y.O.

22. O- O+ Presumed events contributing to graft tolerance • Hepatitis/liver failure • Profound lymphopenia O+ O+ Immunosuppression No immunosuppression Thymic engraftment Donor cells educated to be tolerant to host and donor but responsive to foreign antigen 9 Y.O. Active thymus • Predominantly donor white cells but approx. 2%host B cells • IgG on RBC progressing to active hemolysis • Donor liver survival • Loss of pre-existing immunity/successful reimmunization • TCR excision circles liver graft Stem cells 12 Y.O.

23. T cell precursor T cell receptor gene rearrangement TCR+ Immature thymocyte TCR+ Positive selection of cells whose T cell receptor bind MHC TCR+ TCR+ Death of cells that do not interact with MHC Epithelial cell TCR+ TCR+ TCR+ TCR+ Negative selection and death of high affinity self reactive cells DC TCR+ Mature T cells tolerant of self into circulation TCR+ Thymic education

24. T cell precursor T cell receptor gene rearrangement TCR+ TCR+ Immature thymocyte TCR+ TCR+ Positive selection of cells whose T cell receptor bind MHC TCR+ Death of cells that do not interact with MHC Epithelial cell TCR+ TCR+ TCR+ TCR+ TCR+ TCR+ TCR+ TCR+ TCR+ TCR+ TCR+ TCR+ DC DC TCR+ Mature T cells Tolerant of host and donor into circulation TCR+ TCR+ Negative selection and death of high affinity host-reactive and donor-reactive cells Thymic education in mixed chimera

25. Tolerance is ideal, but for most patients the risks are too great.What else can we do…..

26. Local immunosuppression Engineer graft to produce its own immunomodulatory molecules thus concentrating immunosuppression in the graft microenvironment “Helping the graft help itself”

27. Type 1 diabetes Autoimmune destruction of insulin producing beta cells Usually strikes in childhood or early adulthood Affects over 140,000 Australians Up to 6 insulin injections each day for rest of life

28. Parenteral insulin is not good enough Adapted from

29. Somatostatin ( cell) Pancreatic polypeptide (PP cell) Insulin  cell Glucagon  cell Pancreatic islet transplantation is a cure for Type 1 Diabetes Adapted from seungkimlab.stanford.edu/ islet.html

30. Islet transplantation www.jdrf-hms-islets.org

31. Westmead Hospital Sydney portal vein infusion of islets into the liver

32. Generating local immunosuppression at the graft site systemic Local

33. Viral • Transient eg. adeno or stable eg. lenti • Reproducible • Control expression level • Allo/xenotransplants • Transgenic • Stable • Reproducible • Select expression level • xenotransplants Methods of expressing immunomodulatory molecules in primary tissues Immunohistolgy from seungkimlab.stanford.edu/ islet.html

34. Problem 2Shortage of donor organs

35. 2002 2003 2004 2005 2006 2007 Number of Deceased Donors Solid Organ Transplantsand Patients on the Waiting List 2002 - 2007 Australia Source:ANZOD Registry (www.anzdata.org.au)

36. Donor organ shortage and Type 1 diabetes • >140,000 Australians with Type 1 diabetes • 198 organ donors in 2007 Xenotransplantation may provide the solution

37. Why pigs? • Reproduce quickly and have large litters • Organs of similar size to humans • Beta cells regulate blood glucose appropriately in humans & pig insulin used for many years in humans • Relatively easy to rear in conditions free of particular pathogens • Can be genetically modified to reduce the risk of immune rejection

38. Why not pigs? • Ethical objections • Religious objections • Risk of xenozoonosis (transmission of infection between species) • Breed free of known pathogens • Breed free of potential pathogens (PERVS) • Long-term monitoring of recipients • Current moratorium in Australia, but experience in approx. 150 people worldwide show no evidence of pig derived infection

39. Add modulators of host pathways eg. complement modulators eg. anticoagulant molecules Remove target epitopes eg. Gala(1,3)Gal Add local immunosuppression eg. costimulation blockade eg. depleting antibodies Add pro-survival molecules Pigs can be modified to remove detrimental and add beneficial molecules