TRANSPLANTATION IMMUNOLOGY

1. TRANSPLANTATION IMMUNOLOGY TALIB HASSAN ALI Ph.D student Experimental immunology and development of innovative therapies

3. Immunologic Basis of Graft Rejection The degree of immune response to a graft varies with the type of graft. The following terms are used to denote different types of transplants: Auto graft: is self-tissue transferred from one body site to another in the same individual. Transferring healthy skin to a burned area in burn patients and use of healthy blood vessels to replace blocked coronary arteries are examples of frequently used auto grafts. Iso graft: is tissue transferred between genetically identical individuals. In inbred strains of mice, an iso graft can be performed from one mouse to another syngeneic mouse. In humans, an iso graft can be performed between genetically identical (monozygotic) twins. Allograft: is tissue transferred between genetically different members of the same species. In mice, an allograft is performed by transferring tissue or an organ from one strain to another. In humans, organ grafts from one individual to another are allografts unless the donor and recipient are identical twins. Xeno graft: is tissue transferred between different species (e.g., the graft of a baboon heart into a human). Because of significant shortages in donated organs, raising animals for the specific purpose of serving as organ donors for humans is under serious consideration.

4. Immunologic Basis of Graft Rejection Schematic diagrams of the process of graft acceptance and rejection. (a) Acceptance of an autograft is completed within 12–14 days. (b) First-set rejection of an allograft begins 7–10 days after grafting, with full rejection occurring by 10–14 days. (c) Second-set rejection of an allograft begins within 3–4 days, with full rejection by 5–6 days. The cellular infiltrate that invades an allograft (b, c) contains lymphocytes, phagocytes, and other inflammatory cells. FULL REJECTION FULL ACCEPTANCE

5. Transplant Rejection • Graft rejection depends on host recognition the grafted tissue as foreign. • The antigens responsible for such rejection are those of the major histocompatibility antigen (HLA) system. • Rejection is a complex process in which both cell-mediated immunity and circulating antibodies play a role. • The relative contributions of these two mechanisms to rejection vary among grafts and are often reflected in the histologic features of the rejected organs.

6. T Cells Play a Key Role in Allograft Rejection For example, nude mice, which lack a thymus and consequently lack functionalT cells, were found to be incapable of allograft rejection; indeed, these mice even accept xenografts

7. T Cells Play a Key Role in Allograft Rejection Experimental demonstration that T cells can transfer allograft rejection. When T cells derived from an allograft-primed mouse are transferred to an unprimed syngeneic mouse, the recipient mounts a second-set rejection to an initial allograft from the original allogeneic strain.

8. T Cells Play a Key Role in Allograft Rejection The role of CD4+ and CD8+ T cells in allograft rejection is demonstrated by the curves showing survival times of skin grafts between mice mismatched at the MHC. Animals in which the CD8+ T cells were removed by treatment with an anti-CD8 monoclonal antibody (red) showed little difference from untreated control mice (black). Treatment with monoclonal anti-CD4 (blue) improved graft survival significantly, and treatment with both anti-CD4 and anti-CD8 antibody prolonged graft survival most dramatically (green). [Adapted from S. P. Cobbold et al., 1986, Nature 323:165.]

9. Similar Antigenic Profiles Foster Allograft Acceptance the major histocompatibility complex (MHC) in the mouse and human. The MHC is referred to as the H-2 complex in mice and as the HLA complex in humans. In both species the MHC is organized into a number of regions encoding class I (pink), class II (blue), and class III (green) gene products. The class I and class II gene products shown in this figure are considered to be the classical MHC molecules. The class III gene products include complement (C) proteins and the tumor necrosis factors (TNFα and TNFβ).

10. Similar Antigenic Profiles Foster Allograft Acceptance Illustration of inheritance of MHC haplotypes in inbred mouse strains.

11. Similar Antigenic Profiles Foster Allograft Acceptance

12. Graft donors and recipients are typed for RBC and MHC antigens (a) White blood cells from potential donors and the recipient are added to separate wells of a microtiter plate. The example depicts the reaction of donor and recipient cells with a single antibody directed against an HLA-A antigen. The reaction sequence shows that if the antigen is present on the lymphocytes, addition of complement will cause them to become porous and unable to exclude the added dye

13. Graft Donors and Recipients Are Typed for RBC and MHC Antigens Typing procedures for HLA antigens. (a) HLA typing by microcytotoxicity. (b) Because cells express numerous HLA antigens, they are tested separately with a battery of antibodies specific for various HLA-A antigens. Here, donor 1 shares HLA-A antigens recognized by antisera in wells 1 and 7 with the recipient, whereas donor 2 has none of HLA-A antigens in common with the recipient

14. Graft donors and recipients are typed for RBC and MHC antigens Typing procedures for HLA antigens. (b) HLA typing by Mixed lymphocyte reaction Mixed lymphocyte reaction to determine identity of class II HLA antigens between a potential donor and recipient.

15. Total lymphoid irradiation eliminates lymphocytes the recipient receives multiple x-ray exposures to the thymus, spleen, and lymph nodes before the transplant surgery. The typical protocol is daily x-irradiation treatments of about 200 rads per day for several weeks until a total of 3400 rads has been administered. The recipient is grafted in this immunosuppressed state. Because the bone marrow is not x-irradiated, lymphoid stem cells proliferate and renew the population of recirculating lymphocytes. These newly formed lymphocytes appear to be more tolerant to the antigens of the graft

16. Graft donors and recipients are typed for RBC and MHC antigens The effect of HLA class I and class II antigen matching on survival of kidney grafts. Mismatching of one or two class I (HLA-A or HLA-B) antigens has little effect on graft survival. A single class II difference (line 4) has the same effect as 3 or 4 differences in class I antigens (line 3). When both class I and class II antigens are mismatched, rejection is accelerated. [Adapted from T. Moen et al., 1980, N. Engl. J. Med. 303:850.]

17. Cell-mediated graft rejection occurs in two stages The process of graft rejection can be divided into two stages: 1- A sensitization phase, in which antigen-reactive lymphocytes of the recipient proliferate in response to alloantigens on the graft, 2- An effector stage, in which immune destruction of the graft takes place.

18. Transplant rejection Mechanism • T Cell-Mediated Reactions: • It involves both delayed type hypersensitivity and T cell mediated cytotoxicity. • The host recognition of donor HLA by two ways: • Indirect recognition: • Host CD 4+ T cells recognize donor HLA after they are processed and presented by the host’s APC. • This recognition activates DTH. • Direct recognition: • Host T cells recognize HLA molecules on the surface of APC of the donor. • Host T cells encounter the donor dendritic cells within the grafted organ or after these cells migrate to the draining lymph nodes. • Both host CD4+ and the CD8+ T cells are involved in this reaction.

19. SENSITIZATION STAGE

20. EFFECTOR STAGE Effector mechanisms (purple blocks) involved in allograft rejection. The generation or activity of various effector cells depends directly or indirectly on cytokines (blue) secreted by activated TH cells. ADCC = antibody-dependent cell-mediated cytotoxicity.

21. EFFECTOR STAGE Antibody-dependent cell-mediated cytotoxicity (ADCC). Nonspecific cytotoxic cells are directed to specific target cells by binding to the Fc region of antibody bound to surface antigens on the target cells. Various substances (e.g., lytic enzymes, TNF, perforin, granzymes) secreted by the nonspecific cytotoxic cells then mediate target Cell destruction.

22. EFFECTOR STAGE Generation of effector CTLs. Upon interaction with antigen–class I MHC complexes on appropriate target cells, CTL-Ps begin to express IL-2 receptors (IL-2R) and lesser amounts of IL-2. Proliferation and differentiation of antigen-activated CTL-Ps generally require additional IL-2 secreted by TH1 cells resulting from antigen activation and proliferation of CD4+ T cells. In the subsequent effector phase, CTLs destroy specific target cells

23. Mechanism of graft rejection Both TH and TC are activated - TC cells destroy graft cells by direct contact TH cells secrete cytokines that attract and activate macrophages, NK cells and polymorphs leading to cellular infiltration and destruction of graft - B cells recognize foreign antigens on the graft and produce antibodies which bind to graft cells and . Activate complement causing cell lysis . Enhance phagocytosis, i.e. opsonization . Lead to ADCC by macrophages, NK,PML - Immune complex deposition on the vessel walls induce platelets aggregation and microthrombin leading to ischemia and necrosis of graft

24. Transplant rejection Mechanism • Antibody-Mediated Reactions. • Ab’s produced against donor Ag can also mediate rejection through two forms: • Hyperacute rejection: • Immediate rejection soon after transplantation. • Occurs when preformed antidonor Ab’s are present in the circulation of the recipient. • Seen in: • Recipient who has already rejected a kidney transplant. • Multiparous women who develop anti-HLA antibodies against paternal antigens shed from her fetus. • Recipient of prior blood transfusions from HLA-nonidentical donors, platelets and white cells are particularly rich in HLA antigens.

25. Transplant rejectionMechanism • Anti-HLA humoral Ab’s • It develops concurrently with T-cell mediated rejection. • Seen in recipients not previously sensitized to transplantation antigens, exposure to the class I and class II HLA antigens of the donor may evoke antibodies. • The initial target of these antibodies in rejection appears to be the graft vasculature.

26. Transplant rejectionClassification & Morphology • On the basis of the morphology and the underlying mechanism, rejection reactions are classified as: • Hyperacute. • Acute. • chronic.

27. Transplant rejectionClassification & Morphology • Hyperacute Rejection: • This form of rejection occurs within minutes or hours after transplantation and can be recognized by the surgeon soon after the graft vasculature is anastomosed to the recipient's. • Hyperacutely rejecting kidney rapidly becomes cyanotic, mottled, and flaccid and may excrete few drops of bloody urine. • This form of rejection is due to the presence of preformed antidonor Ab’s in the host circulation. • This form of rejection is rarely seen in today's practice

28. Pre-existing recipient antibodies mediate hyperacute rejection Hyperacute Rejection Steps in the hyperacute rejection of a kidney graft.

29. Transplant rejectionClassification & Morphology • Acute Rejection: • This may occur within days of transplantation in the untreated recipient or may appear suddenly months or even years later, after immunosuppression has been employed and terminated. • Acute graft rejection is a combined process in which both cellular and humoral tissue injuries play parts. • Histologically,humoral rejection is associated with vasculitis, whereas cellular rejection is marked by an interstitial mononuclear cell infiltrate, edam, and tissue injury as well as mild interstitial hemorrhage.

30. Acute rejection is mediated by T-cell responses Cell-mediated allograft rejection manifests as an acute rejection of the graft beginning about 10 days after transplantation .because of a massive infiltration of macrophages and lymphocytes at the site of tissue destruction, suggestive of TH-cell activation and proliferation.

31. Transplant rejectionClassification & Morphology • Chronic Rejection: • Present late after transplantation (months or years). • Chronic changes are commonly seen in the renal allograft. • Patients with chronic renal transplant rejection present clinically with a progressive rise in serum creatinine level over a period of 4 to 6 months. • It is characterised by vascular changes (dense intimal fibrosis), interstitial fibrosis, and loss of renal parenchyma.

32. Chronic rejection occurs months or years post-transplant • Chronic rejection reactions develop months or years after acute • rejection reactions have subsided. The mechanisms of chronic • rejection include both humoral and cell-mediated responses by • the recipient . While the use of immunosuppressive drugs and • the application of tissue-typing methods to obtain optimum match of donor and recipient have dramatically increased survival of allografts during the first years after engraftment, little progress has been made in long-term survival. • Chronic rejection reactions are difficult to manage with immunosuppressive drugs and may necessitate another transplantation

33. Methods of increasing graft survival • Minimization of the HLA disparity between the donor and the recipient by better HLA matching of the donor and the recipient. • Immunosuppressive therapy: • drugs such as azathioprine, steroids, cyclosporine, antilymphocyte globulins, and monoclonal anti-T cell antibodies (e.g., monoclonal anti-CD3) are used.

34. General Immunosuppressive Therapy Most of the immunosuppressive treatments that have been developed have the disadvantage of being nonspecific; -its slowing the proliferation of activated lymphocytes. And any dividing non-immune cells (e.g., epithelial cells of the gut or bone-marrow hematopoietic stem cells) -are also affected, serious or even life-threatening complications. Patients on long-term immunosuppressive therapy are at increased risk of cancer, hypertension, and metabolic bone disease.

35. some Transplantation rejection drugs Mitotic inhibitor Azathioprine (Imuran),, is often given just before and after transplantation to diminish T-cell proliferation in response to the alloantigens of the graft methotrexate. Cyclophosphamide is an alkylating agent that inserts into the DNA helix and becomes cross-linked, leading to disruption of the DNA chain Methotrexate acts as a folic-acid antagonist to block purine biosynthesis such as prednisone and dexamethasone, are potent anti-inflammatory agents that exert their effects at many levels of the immune response. Cyclosporin A (CsA), FK506 (tacrolimus), and rapamycin these drugs block activation of resting T cells by inhibiting the transcription of genes encoding IL-2 and the high-affinity IL-2 receptor (IL-2R), which are essential for cell activation Corticosteroids Fungal Metabolites

36. Specific Immunosuppressive therapy • Monoclonal antibody to the CD3 molecule of the TCR complex • Diphtheria toxin is coupled with the antibody • Monoclonal antibodies specific for the high-affinity IL-2 receptor (anti-TAC). • Monoclonal-antibody therapy for the cell-surface adhesion molecules. ICAM-1 and LFA-1 • Most of these monoclonal antibodies are mouse origin. Many recipients develop an antibody response to the mouse monoclonal antibody, rapidly clearing it from the body. This limitation has been overcome by the construction of human monoclonal antibodies and mouse-human chimeric antibodies Monoclonal Antibodies

37. Diphtheria toxin is coupled with the antibody

38. Blocking co-stimulatory signals can induce anergy TH-cell activation requires a co-stimulatory signal provided by antigen-presenting cells (APCs). Interaction of B7 family members on APCs with CD28 delivers the co-stimulatory signal. Engagement of the closely related CTLA-4 molecule with B7 produces an inhibitory signal. All of these molecules contain at least one immunoglobulin- liké domain and thus belong to the immunoglobulin superfamily

39. CTLA-4Ig, a chimeric suppressor of co-stimulation. (a) CTLA-4Ig, a genetically engineered molecule in which the Fc portion of human IgG is joined to the B7-binding domain of CTLA-4. (b) CTLA-4Ig blocks costimulation by binding to B7 on antigen presenting cells and preventing the binding of CD28, a major co-stimulatory molecule of T cells

40. Blocking co-stimulatory signals at the time of transplantation can cause anergy instead of activation of the T cells reactive against the graft. T-cell activation requires both the interaction of the TCR with its ligand and the reaction of co-stimulatory receptors with their ligands (a). In (b), contact between one of the co-stimulatory receptors, CD28 on the T cell, and its ligand, B7 on the APC, is blocked by reaction of B7 with the soluble ligand CTLA-4Ig. The CTLA4 is coupled to an Ig H chain, which slows its clearance from the circulation. This process specifically suppresses graft rejection without inhibiting the immune response to other antigens

41. Immune tolerance to allografts There are two general cases in which an allograft may be accepted: Is when cells or tissue are grafted to a so-called privileged site that is sequestered from immune-system surveillance. 2. Is when a state of tolerance has been induced biologically, usually by previous exposure to the antigens of the donor in a manner that causes immune tolerance rather than sensitization in the recipient.

42. Privileged sites accept antigenic mismatches These sites include the anterior chamber of the eye, the cornea, the uterus, the testes ,and the brain (blood-brain barrier prevents the entry or exit of many molecules, including antibodies.) cartilage or heart valves Transplantation of artificial privileged tissue (pancreatic islet cells were encapsulated in semi permeable membranes (fabricated from an acrylic copolymer) The transplanted cells are produce insulin were not rejected, because the recipient’s immune cells could not penetrate the membrane

43. Early exposure to alloantigens can induce specific tolerance Experimental support for the notion that tolerance comes from exposure of the developing organism to alloantigens came from neonates of mouse strain experiments. If strain A are injected with cells from strain C they will accept grafts from C strain as adults. Immunocompetence of the injected A-strain mice and specificity of the tolerance is shown by the fact that they reject grafts from other strains as rapidly as their untreated littermates.

44. cattle of dizygotic twin

45. Transplantations routinely used in clinical practice. For the solid organs, the number of transplants performed in the United States in 2000 is indicated. Estimates are included for other transplants if available

46. Clinical Transplantation Kidney • The most commonly transplanted organ is the kidney • Many common diseases, such as diabetes and various types of nephritis, result in kidney failure that can be alleviated by transplantation • Two major problems are faced by patients waiting for a kidney. • is the short supply of available organs, • is the increasing number of sensitized recipients

47. Transplantation of Hematopoietic Cells (BM transplant) • Use of hematopoietic cell transplants for hematologic malignancies, certain nonhematologic cancers, aplastic anemias, and certain immunodeficiency states. • Hematopoietic stem cells are usually obtained from the donor bone marrow but may also be harvested from peripheral blood after they are mobilized from the bone marrow by administration of hematopoietic growth factors.

48. Transplantation of Hematopoietic Cells (BM transplant) • In most of the conditions in which bone marrow transplantation is indicated, the recipient is irradiated with lethal doses either to destroy the malignant cells (e.g., leukemias) or to create a graft bed (aplastic anemias).

49. Transplantation of Hematopoietic Cells (BM transplant) • Two major problems arise in allogeneic bone marrow transplantation: • Graft-Versus-Host (GVH) disease • Transplant rejection.

50. Transplantation of Hematopoietic Cells (BM transplant) GVH disease • Occurs in any situation in which immunologically competent cells or their precursorsare transplanted into immunologically crippled recipients. • GVH disease occurs most commonly in the setting of allogeneic bone marrowtransplantation but may also follow transplantation of solid organs rich in lymphoid cells (e.g., the liver) or following transfusion of un-irradiated blood.