Immunosuppression and Transplantation

1. Immunosuppression and Transplantation Yufang Shi, D.V.M, Ph.D. E-mail: shiyu@umdnj.edu

2. Transplantation in Clinic Body parts: Facial, Limbs, Organs: Heart, kidney, liver Tissue: Islets, hair follicles, bone, bone marrow Cells: Stem cells, lymphocytes

3. Transplantation Hurdles Most surgical procedures are well established **Immunosuppression Organ variation: liver is easier to tolerize Cornea very little rejection Skin is most difficult

4. The dream of transplants The 3rd Century, Saints Cosmas and Damian

6. Relationships between Donor and Recipient Syngeneic - between genetically identical individuals, usually the same individual, identical twins or isogenic strains Allogeneic - from one individual to another of the same species Xenogeneic - between individuals of different species.

7. Immunological Rejection Major Histocompatibility Complex (MHC) is the major concern. Rejections: Antibody mediated T cells mediated Hyperacute rejection e.g., Blood type mismatch Acute Graft Rejection Direct recognition of allogeinic MHC; rejection about 10 days Chronic rejection Take many months to years. Due to failure of immunosuppressants

8. Milestones In Organ Transplantation (1) Source: National Kidney Foundation, Inc. 1906 The first kidney transplantations were done without anti-rejection drugs. Kidneys from sheep, pigs, goats and primates are used. 1936 Dr. Voronoy, a Russian, reports the first human-to-human kidney transplant, when a kidney from a cadaver is transplanted to a recipient with a different blood type. **1944 A British scientist, Sir Peter Medawar, reports that rejection of a transplant is based on immunologic factors. This discovery eventually transforms transplant surgery from a largely unsuccessful experiment to an accepted form of treatment. 1954 Surgeons Joseph E. Murray and John Hartwell Harrison, in collaboration with nephrologist John P. Merrill, perform the first successful kidney transplant -- between identical twins -- at the Peter Bent Brigham Hospital in Boston. 1963 Dr. Thomas E. Starzl performs the first human liver transplant at the University of Colorado Medical School; however, lack of effective immunosuppressives limits the success. Four years later, the availability of more effective immunosuppressives enables Dr. Starzl to perform the first successful liver transplant. 1963 Dr. James D. Hardy performs the first lung transplant at the University of Mississippi at Jackson; however, the patient survives only a few days because of the lack of effective immunosuppression drugs. Twenty years later, with improved immunosuppressives, Dr. Joel Cooper performs the first successful lung transplant at Toronto General Hospital. 1967 Dr. Christiaan Barnard performs the first heart transplant at Groote Shuur in Cape Town, South Africa.

9. 1968 Dr. Norman Shumway performs the first U.S. heart transplant at Stanford University. 1968 Drs. Richard Lillehei and William Kelly perform the first pancreas transplant at the University of Minnesota Hospital. **1979 U.S. trials of Sandimmune (cyclosporine) in cadaver kidney transplants begin at the Peter Bent Brigham Hospital in Boston and at the University of Colorado. The results show that Sandimmune (cyclosporine), combined with steroids, controls rejection better than any drug therapy in the past. 1983 The Federal Drug Administration releases Sandimmune (cyclosporine) for general use in the U.S., heralding a new era for kidney, liver and heart transplantation. 1986 Dr. A. Benedict Cosimi and his associates at Massachusetts General Hospital introduce monoclonal antibodies into clinical medicine in the form of OKT3 antibodies, which have a selective effect on the immune system and are intended primarily for reversing kidney transplant rejection. 1989 Clinical investigators begin using an experimental drug called FK 506 for kidney, liver, heart and lung recipients. Results suggest that this drug is effective, but clinical trials continue to assess its safety and efficacy. 1993 Continuing shortages in organ donation lead to renewed interest in transplanting organs from animals such as baboons (often referred to as xenografting). Baboon-to-human liver and heart transplants have been attempted, with limited success. A new research strategy involves developing a line of pigs with the appropriate human genes to help prevent rejection of organs such as hearts, livers and kidneys transplanted from these animals. Milestones In Organ Transplantation (2) Source: National Kidney Foundation, Inc.

10. Milestones In Organ Transplantation (3) Source: National Kidney Foundation, Inc. 1994 The FDA approves a new medication for use in transplant recipients: Prograf (formerly known as FK506) marks a significant advance in the understanding and suppression of the human rejection response and in the lessening of unwanted side effects. 1995 A new study by Dr. Paul Terasaki and colleagues at UCLA shows that spouses are an important source of living-donor kidney transplants. According to the Terasaki study, the 3-year graft survival rate for spouse-to-spouse transplants (85%) is comparable to that seen in parent-to-child transplants (82%) and better than that seen in transplants from cadaver donors (70%). Living donation is becoming an increasingly important source of kidney and other transplants because of continuing shortages of cadaver donors. 1995 Two more new medicines are approved by the FDA for use in transplant recipients. These are: CellCept (mycophenolate mofetil), and Neoral, a new formulation of cyclosporine. These drugs hold promise for providing even better control of rejection with fewer side effects. 1995 At Johns Hopkins Bayview Medical Center, Lloyd Ratner, M.D. and Louis Kavoussi, M.D., perform the world's first laparoscopic live-donor nephrectomy in which a patient's kidney is removed through a hole slightly larger than a silver dollar. Laparoscopic live-donor nephrectomies mean fewer post-op days in the hospital, speedier recovery, less scarring and decreased post-operative pain. 1996 The number of kidney transplants using living donors (both related and unrelated) continues to grow. A total of 11,099 kidney transplants were performed in 1996 -- 3,389 of which involved kidneys recovered from living donors. 1997 The Department of the Navy Bureau of Medicine and Surgery announces a research breakthrough that they are now able to prevent kidney transplant rejection in primates with different histocompatibility factors through the use of a combination of a specific fusion protein and a specific monoclonal antibody. Further trials are necessary to determine future applicability of the technique to humans.

11. Anti-inflammatory and Immunosuppressive Drugs Nonsteroid anti-inflammatory drugs: Aspirin, Vioxxx (no longer used), and Celebrex. Work through COX1/2 (cylooxygeneases, which are involved in the synthesis of prostaglandins) Antihistamines: Blockers of histamine receptors: Allegra, Claritin, Clarinex, Benadryl *Steroid hormones: Glucocorticoid derivatives: prednisone, dexamethasone, and hydrocortisone *Lymphocyte specific immunosuppressants: Cyclosoprine, FK506, rapamycin, FTY720, specific antibodies and receptors (bioactive). Cytotoxic agents:cyclophosphamide

12. Simplified Schematic Representation of an Immune Response proliferation & differentiation CD8+ T cells CD8+ cytolytic T cells Class I IFNg, IL-2 APC proliferation & differentiation CD4+ immune cells (delayed hypersensitivity) Class II CD4+ T cells Cytokines Costim. Mol. IL-4,-5,-6 B cells Protein antigens Plasma cells proliferation & differentiation antibody production MHC class II/peptides APCs

13. TCR Costimulation © 2003 by LIPPINCOTT WILLIAMS & WILKINSFundamental Immunology

14. Families of Costimulation Molecules B7:CD28 superfamily: B7.1/B7.2:CD28/CTLA4; ICOSL:ICOS; PD-L1/PD-L2:PD-1; B7-H3:? TNF:TNFR superfamily: CD134 (OX40):CD134L; CD27:CD70; 4-1BB:4-1BBL; CD30:CD30L; RANK (OPG):RANKL; LIGHT:HVEM, and GITR:GITRL CD2 superfamily: CD2:CD58/CD48; CD48:CD244 CD150, CD84, CD299, Ly-108, and BLAM Some integrins: CD44, CD43, and heat-stable antigen (HSA).

15. TCR Activation

16. T-cell Activation Blockers: Cyclosporine, Tacrolimus (FK506), and Sirolimus (Rapamycin) • CsA and FK506 act on T-cells to inhibit T-cell receptor activation and induction of cytokines • CsA may also inhibit IgE-stimulated mast cell degranulation and stimulate TGF- expression • Rapamycin acts to inhibit lymphocyte response to cytokines • Rapamycin and analogues are also used to sensitize cancer cells to chemotherapeutic reagents

17. Targets of Immunosuppressants Cyclophilin FKBP Rapamycin Cyclosporine FK506 mTor Calcineurin NFAT Translocation Cytokine Signaling Genes lead to T cell Activation

18. Target of Rapamycin

19. Mechanism of Action of Helper T-cell blockers X X From Hardman and Limbird, The Pharmacological Basis of Therapeutics

20. Immunophilins Cyclophilinis a peptidyl-prolyl cis-trans-isomerase which catalyzes the cis-trans isomerization of proline imidic peptide bonds. Helps protein folding. FKBPs are also known to participate in many cellular processes such as cell signaling, protein transport (such as Notch) and transcription.

21. Biology of Glucocorticoids Newton, Thorax 2000;55:603-613

22. Mechanisms of Glucocorticoid Action • Inhibit the production of • proinflammatory cytokines • Promote the production of • inflammatory cytokines • Induce apoptosis in • inflammatory cells • 4. Interfere with cytokine signals Newton, Thorax 2000;55:603-613

23. Glucocorticoid-sensitive sites of the immune response proliferation & differentiation X MHC Class I/peptides APCs CD8 T-cell CD8 cytolytic T-cells X GC IL-2 GC MHC Class II/peptides APCs proliferation X CD4 immune cell (delayed hypersensitivity) CD4 T-cell (helper T-cells) X IL-1 IL-1, -4,-5,-6 B-cell Protein antigen Plasma cell proliferation & differentiation antibody production

24. Use of Glucocorticoid as Immunosuppressants • Most widely used effective anti-inflammatory drugs • Used with other immunophilin inhibitors to prevent transplant rejection and GVHD • natural glucocorticoids not used due to mineralocorticoid activity • Prednisone and prednisolone are used orally at moderate to high doses; Very high doses of methylprednisolone used i.v. during acute organ rejection • Used before and after anti-thymocyte Abs to inhibit allergic reactions

25. General Principles of Immunosuppression • Primary immune responses are more easily suppressed than secondary (memory) • Different immunosuppressants have different effects on different immune reactions • Suppression is more likely achieved if therapy begins before exposure to the immunogen

26. Uses of Calcineurin inhibitors (TCR activation blockers) • Cyclosporine commonly used with prednisone and other immunosuppressants to prevent allograft rejections in renal, hepatic and cardiac transplants, and in RA and psoriasis • use is delayed posttransplantation due to neurotoxicity concerns • FK506 (Tacrolimus) is approved for prevention of solid-organ allograft rejection, and eczema (topical) • Treatment begins prior to surgery, and is maintained well afterwards

27. Glucocorticoid effects related to immunosuppression • Reduced immune cell content of lymph nodes, spleen and blood • lymphopenia, monocytopenia, eosinopenia, but neutrophilia • Interference with APC, T-cell and macrophage functions

28. Sirolimus (Rapamycin, Rapamune): a new T-cell blocker • different mechanism of action • blocks mTOR kinase • similar poor bioavailability as cyclosporine and tacrolimus, much longer half-life; 62 h v. 18 and 12 h • same metabolism (CYP3A) and potential drug interactions • used for prophylaxis of organ transplant rejection in combination with a calcineurin inhibitor and glucocorticoids • toxicities include: • hyperlipidemia, lymphocoele, anemia, leukopenia, thrombocytopenia, fever, GI effects, hyper- or hypokalemia

29. Toxicity of Glucocorticoids • Major side effects are common due to high doses necessary for suppression • Cushings syndrome • glucose intolerance • infections • bone dissolution • muscle wasting

30. Cytotoxic Agents as immunosuppressants • Antineoplastic drugs will also prevent clonal expansion of T- and B-cells • azathioprine (prodrug of nucleotide anti-metabolite) • mycophenolate mofetil • becomes MPA; inhibits IMP dehydrogenase • cyclophosphamide (DNA alkylating agent) • methotrexate (inhibits dihydrofolate reductase)

31. Uses of cytotoxic agents • Azathioprine; with cyclosporine and/or prednisone for organ transplant rejection and severe RA • Mycophenolate mofetil; with cyclosporine and prednisone for renal transplants • Cyclophosphamide; for BMT • Methotrexate; GVHD prophylaxis

32. Bioactive Immunosuppressants • Anti-thymocyte antibodies • 3 types available • all derived from non-human sources • Rh(D) immune globulin • OKT3, OKT4, Anti-CD20, anti-TNF, anti-ICAMs, and CTLA4-Ig • Repeated blood transfusion; transfusion of apoptotic cells

33. History of Immunosuppressants (1) Time author Experiment or Clinical application Result 1950 Billingham Corticosteroids Prolongation of Skin Graft of Rabbit and Mice 1950 Hume Cadaveric Kidney Tx+ TLC Recipient Died of Systemic Infection 1952 Baker cortisteroid+Splenectomy Prolongation of canine Kidney Graft Survival 1952 Loutit X-irradiation+BMT Prolongation of Mouse Skin Graft Survival 1959 Schwartz,Dameshek 6-MP Inhibition of Antibody Production, Prolongation of Skin Graft Survival 1959 Hamburger TLI+Autogeneic BMT Prolongation of Cadaveric Kidney Graft Survival 1960 Calne,Zckoski Azathioprin(Aza) Significant Prolongation of canine Kidney Graft Survival 1960 Kuss TLI+6-MP Prolongation of Cadaveric Kidney Graft Survival 1963 Starzl Aza+Corticosteroid Became a Clinical Routine

34. History of Immunosuppressants (2) Time Author Experiment or Clinical Application Results 1963 Srarzl Preoperative Splenectomy Not so Good, Seldom Used 1964 McGregor,Gowans Thoracic Duct Drainage Transient Effect，Technical Difficulty 1967 Russell Monaco ALS Prolongation of Graft Survival of Mouse Skin and Canine Kidney 1967 Starzl ALS Clinical Use of ALS, Still in Use Clinically 1969 Wolf Irradiation of Cadaveric Kidney No Obvious Effect 1970 Starzl CHX To Replace Aza When Severely Toxic 1973 Opelz Prooperative Blood Transfusion Enhanced Survival Rate of Cadaveric Kidney Graft by 20% 1976 Borel The Finding of CsA Strong Immunosuppresion and Less Infection

35. Currently Used Immunosuppressants Category Drugs Alkyl Agent Cyclophosphamide Antimetabolic Agent Azathioprin (Aza) Steroids Predenisone,Prednisolone, Dexamethasone, etc ALG (anti-lymphocyte globulins), ATG (anti-thymocyte globulins), OKT3 Biological Agents Fungus Products CsA FK506Rapamicin，Cellcept (mycophenolate mofetil ) Chinese Medicine

36. Ideal Immunosuppressant • Strongly Immunosuppressive • Specific, No Overall Immunosuppression • Anti-infection ability • Low Toxicity for Vital Organs • Low cost • Long in vivo bioactivity • Easy to use

37. Clinical Tolerance Established • Absence of Pathogenic Immune Response Against Graft Tissue • With little or no Immune Suppressant • With the Retention of Immune Responses Against Other Infectious Antigens

38. Help on the Way Adult bone-marrow-derived mesenchymal stem cells are immunosuppressive and prolong the rejection of mismatched skin grafts in animals. We transplanted haploidentical mesenchymal stem cells in a patient with severe treatment-resistant grade IV acute graft-versus-host disease of the gut and liver. Clinical response was striking. The patient is now well after 1 year. We postulate that mesenchymal stem cells have a potent immunosuppressive effect in vivo. Lancet. 2004 May 1;363:1439-41.