1. Name three different types of transplant rejection and list the time frame following transplantation at which each of these occur. The three different types of transplant rejection are hyperacute, acute, and chronic rejection. Hyperacute rejection is mediated by pre-formed antibodies and complement fixation and occurs within minutes to hours of transplantation. Hyperacute rejection is directly comparable to type III hypersensitivity reactions. No reliable method of treating or reversing hyperacute rejection is yet known. Investigation into plasmaphoresis to remove the plasma of blood and replace with saline, which doesn't have these antibodies in it. Acute rejection is mediated by T cell activation which attack the organ graft and destroy it. Because activation and clonal selection must occur, acute rejection takes 5-7 days to occur. Because memory cells are formed from this activation, further transplantation from the same source would elicit acute rejection in less time. Acute rejection functions with the same underlying mechanisms as type IV hypersensitivity reactions. Currently, patients are monitored for acute rejection and treated with immunosuppressive drugs and anti-T-cell antibodies. Chronic rejection occurs months or years after transplantation and is characterized by reactions in the vasculature of the graft that cause thickening of the vessel walls and narrowing of the lumina, resulting in ischemia, loss of function, and death of the graft. The cause and treatment of chronic rejections are not yet elucidated and may be related to antibody-mediated mechanisms or long-term use of immunosuppressive drugs. 2. Describe what happens in graft-versus-host disease. In GVHD, the bone marrow transplantation replaces the host immune system. If not all allograft T cells in the graft are depleted, these donor T cells will attack the transplantation recipient in a graft vs. host manner. 3. Define the following terms: autograft, allograft, isograft, allogeneic, syngeneic, and xenogeneic. Autografts are transplantations within the same individual. Allograft or allogeneic transplantation is between two individuals of the same species. Isograft or syngeneic transplantation is between identical twins. Xenograft or Xenogeneic transplantations are between two different species. 12-10. In blood transfusion, donors and recipients are matched for the A, B, O system of blood group antigens. 4. Draw O antigen, A antigen, B antigen and an example of Galactose alpha-1,3 galactose bearing glycolipid as might be found on the surface of a porcine erythrocyte. [Figure 12.12] O antigen is a Fucose-Galactose-R where R is an oligosaccharide consisting of N-acetyl galactosaminegalactose-glucose-ceramide with ceramide attached to the erythrocyte surface. Fuc-Gal | R A antigen is the same as O antigen except there is an additional N-acetyl galactosamine (GalNAc) attached to the galactose (gal): GalNAc | Fuc-Gal | R B antigen is the same as O antigen except there is an additional galactose attached to the galactose: Gal | Fuc-Gal | R Pigs have a galactose-alpha 1,3 galactose bearing glycolipid on the surface of their erythrocytes which all humans have antibodies pre-made against. Thus, transplantation of porcine erythrocytes would cause a hyperacute rejection. Gal-Gal 5. Explain how a cross-match is performed prior to blood transfusion and prior to organ transplantation. Human erythrocytes do not express HLA class I or II molecules but do have structural polymorphisms in the carbohydrates on the glycolipids of the erythrocyte cell surface, forming the basis for the ABO system of blood group antigens. Except for Rh blood group antigens, other polymorphic systems of blood groups exist but are less important for matching in blood transfusions. To eliminate unexpected transfusion reaction, the recipient's serum is tested directly for its reactivity with donor RBCs to avoid hyperacute rejection -- basically, recipient serum is mixed with donor RBCs to see if the antibodies and complement in the serum will attack the RBCs and cause hemolysis. Cross-match tests are not performed between the recipient's RBCs and serum from the donor because the antibodies in the transfused blood that will bind to recipient RBCs is insufficient to trigger hemolysis or produce a detrimental effect. For organ transplantation, testing recipient serum with cells from the donor organ directly is not practical. Instead, donor lymphocytes are tested as a surrogate because they express HLA class I or II, which are the basis of tissue compatibility. Remember that B cells express both HLA class I and II but T cells typically only express HLA class I. Because human complement isn't so good at lysing antibody coated lymphocytes, rabbit complement is used instead. The cross-test for organ transplantation tests reactivity with B cells and T cells separately to elucidate whether or not the recipient has antibodies against donor HLA class I or II: If T cells are killed, then there are recipient antibodies against HLA class I (and B cells should also be killed when tested). If B cells are killed but T cells are not, then there are recipient antibodies against HLA class II. If B cells are killed and no test is done for T cells, it cannot be elucidated whether the recipient has antibodies against HLA class I or II. 12-11. Antibodies against A, B, O or HLA antigens cause hyperacute rejection of transplanted organs. 6. Explain the mechanism of action of hyperacute rejection. Hyperacute rejection is mediated by pre-formed antibodies and complement. It is directly comparable to type III hypersensitivity reactions in which immune-complex deposition causes complement activation within blood vessel walls. 7. Know the types of cells on which HLA class I antigens are expressed. HLA class I antigens are expressed in nearly all cells in the body. Exceptions are non-nucleated cells (such as RBCs), sperm cells, and most cells in the brain. Human erythrocytes do not express HLA class I or II antigens. 8. Know the types of cells on which HLA class II antigens are expressed. HLA class II antigens are expressed on professional antigen-presenting cells only, such as B cells, dendritic cells, and macrophages. Human erythrocytes do not express HLA class I or II antigens. 12-12. Anti-HLA antibodies can arise from pregnancy, blood transfusion, or previous transplants. 9. Explain why mothers are more likely than fathers to have a positive cross-match with one of their children. Mothers are more likely than fathers to have a positive cross-match with one of their children because mothers can getexposed to their kid's antigens during labor and develop antibodies. These antibodies are actually directed against the antigens inherited from the father. Therefore, if the mother requires a transplant many years later, the father cannot be the donor because the mother has developed antibodies against the child and the father. The conundrum is why mothers, when exposed to the antigens of their first child, do not reject their second child with the same father. 10. Know that blood transfusions are matched for A, B, O type but NOT HLA type. Blood transfusions are matched for ABO type and Rh blood group but not HLA. This is because erythrocytes don't express HLA class I or II but do express polymorphic systems of blood groups. 11. Define panel reactive antibody (PRA). The PRA is a method to quantify how difficult it is to transplant an organ into somebody based on the number of HLA antigens in a given donor population the recipient has formed antibodies against. Basically, the PRA test is an ELISA. A 96 well plate is coated with 96 antigens common in a given donor population (eg. 30 HLA-A, 50 HLA-B, 20 HLA-DR). Recipient serum is added to tests against each HLA antigen and washed off. Any antibodies against any HLA antigens will remain attached. A secondary antibody conjugated with a substrate-reactive enzyme is added which binds to any bound recipient antibody; any excess is washed off. Finally, substrate is added which changes color if the well contains the secondary antibody. If 10 wells turn color, the PRA is 10. If 96 wells turn color, the PRA is 96. Basically, the lower the PRA, the easier it is to transplant to somebody in terms of avoiding hyperacute rejection. Disparities in expression of certain HLA antigens in a given population are probably due to past survival of a disease by an ancestor with a certain HLA that was particularly good at presenting antigens from that disease. This led to selection of certain HLA antigens, making some more common in a given population than another. 12. Explain why blood transfusion increases PRA. Blood transfusions increase a recipient's PRA because that person is exposed to more HLA antigens on nucleated cells in donor blood, such as lymphocytes, etc. This leads to development of more antibodies against more HLA antigens by the recipient. However, current practices deplete blood for transplantation of nucleated cells to prevent spread of viruses, making transplanted blood less likely to increase PRA. 13. Explain why previous organ transplantation increases PRA. Similar to blood transfusions, previous organ transplantation exposes the recipient to more HLA antigens, causing the development of more antibodies against those HLA antigens, and increasing the recipient's PRA. 12-13. Organ transplantation involves procedures that inflame the donated organ and the transplant recipient. 14. Define ischemia. Ischemia is the cessation of blood flow to an organ/tissue resulting in anoxia, lack of substrate, and lack of tissue perfusion, leading to accumulation of metabolic products. Ischemia will result in cell death. This is different than hypoxia which means only a deficiency of oxygen. 15. Know that ischemia causes activation of endothelium and complement leading to leukocyte infiltration and cytokine production. Ischemia causes activation of endothelium and complement leading to leukocyte infiltration and cytokine production. 12-14. Acute rejection is caused by effector T cells responding to HLA differences between donor and recipient. 16. Know the predominant effector cells of transplant rejection: CD4 TH1 helper cells directed against the grafts’s MHC class II molecules and CD8 killer cells directed against the graft’s MHC class I molecules. CD4+ T helper cells are directed against the graph's MHC-II molecules. Because acute rejection is primarily T cell mediated, Th1 cells are primarily involved in acute rejection. Th1 cells release IFN-g and IL-2 (and TNF-b) which help activate a cellular T cell mediated response. CD8+ CTLs are directed against MHC-I molecules. To avoid acute rejection, it is important to prevent Th1 cell activation; i.e., avoid differences in MHC-II. This is why it is more important to match MHC-II between donors and recipients than MHC-I, though both are important! 17. Know the difference between a primary and secondary response to transplant antigens. The primary acute rejection response to transplant antigens occurs 5-7 days after transplantation, as T cells are activated and proliferate. Because the immune system becomes primed and has memory cells after the primary response, if another transplantation from the same donor is done, a secondary acute rejection response will occur, but more rapidly than the primary response -- probably 3 days or so. 18. Explain the mechanism of action of acute rejection Acute rejection is mediated by an alloreactive T cell response that produces effector CD4+ and CD8+ T cells that attack the organ graft and destroy it. Because T cell activation and proliferation must occur, acute rejection takes longer than hyperacute rejection to occur, typically 5-7 days rather than minutes to hours. The effector mechanisms underlying acute rejection are the same as those causing type IV hypersensitivity reactions. 19. Explain how the mechanism of thymic selection generates a T-cell repertoire with a high proportion of T cells capable of recognizing allogeneic HLA molecules presenting self peptides. Because different HLA allotypes bind different sets of self-peptides, each allotype selects a different repertoire of TCRs during thymic selection. Consequently, the T cell repertoire selected by the recipient's HLA type contains numerous T cell clones that can respond to the HLA:self-peptide complexes presented by donor cells of different HLA type. Because of this, alloreactive T cell responses stimulated by HLA differences are stronger than T cell responses to a vaccine or a pathogen. 20. Understand the difference between direct and indirect antigen presentation pathways. The direct pathway involves recipient T cells being stimulated by direct interaction of their TCRs with the allogeneic HLA molecules expressed by donor APCs. It produces effector T cells to migrate to the transplanted organ where Th1 cells activate macrophages and CD8+ CTLs to systematically kill cells of the transplanted organ and inflame the tissue. The indirect pathway is too complicated to teach. 12-15. Chronic rejection of organ transplants is due to the indirect pathway of allorecognition. 21. Define chronic rejection. Chronic rejection is poorly understood. See #1. 22. Explain the phenomenon of chronic rejection and know that its mechanisms are poorly understood. Chronic rejection occurs months or years after transplantation and is characterized by reactions in the vasculature of the graft that result in thickening of the vessel walls and narrowing of the lumina, causing eventual ischemia, loss of function, and death of the graft. Chronic rejections is correlated with the presence of antibodies specific for the HLA class I molecules of the graft, suggesting a B cell response. However, the mechanisms are poorly understood. 12-16. Matching donor and recipient for HLA class I and class II allotypes improves the outcome of transplantation. 24. Define histocompatibility. Histocompatibility means tissue compatibility or immunologic similarity that permits successful transplantation (no antibodies against the graft). 25. Know that the important HLA loci for clinical transplantation are: HLA-A, -B, and –DR. The important HLA loci for clinical transplantation are HLA-A, HLA-B, HLA-C and HLA-DR. 12-17. Allogeneic transplantation is made possible by the use of immunosuppressive drugs. 26. Know that most patients receive HLA-mismatched transplants and thus require immunosuppression. The majority of patients receive organs that are mismatched at one or more HLA loci, requiring use of immunosuppressive drugs. 27. Know three different categories of immunsuppressants: steriods with anti-inflammatory properties, cytotoxic drugs that interfere with DNA replication and thus prohibit replication, and microbial products that interfere with T-cell activation. Steroids block the IL-1 signal from the APC that activates the T helper cell to secrete IL-2 and generally suppress the inflammatory / immune response. Steroids are bad - they cause diabetes, buffalo humps in women, thinning skin, alopecia (hair loss), HTN, avascular necrosis, osteoporosis, etc. You don't want to be on them if possible! Newer drugs are helping us eliminate them from transplantation immunosuppression regimens. Cyclosporin and Tacrolimus (Prograf) block the IL-2 production from the T helper cell to prevent activation of CTL. They do this by binding intracellular proteins cyclophilin and FKBP to form complexes that bind calcineurin, preventing its activation by Ca2+ and blocking activation of NFAT which normally forms a transcription factor that activates IL-2 secretion (Figure 12.27). Unfortunately, these drugs cause nephrotoxicity. Rapamycin, Azathioprine and Mycophenolate block CTLs from proliferating in response to IL-2. Mycophenolate prevents cell proliferation by acting as a purine antagonist. Cytotoxic drugs are used to prevent proliferation of activated immune cells by killing all rapidly proliferating cells. This can cause side effects such as anemia, leukopenia, diarrhea, etc. It can also be used to treat certain cancers by killing all the rapidly proliferating cancer cells and replacing the destroyed hematopoietic cells with a bone marrow transplant. Other immunosuppressive agents exist such as IL-2 receptor and TCR antagonists, interfering with T cell activation. Also, OKT3 is an anti-CD3 antibody that prevents TCR signaling and activates complement. Antibodies specific to the IL-2 receptor have been produced because Anti-IL2 Receptor antibodies targets only activated T cells, whereas anti-CD3 antibodies target all T cells. 12-18. Corticosteroids change patterns of gene expression. 28. Know that prednisone is a synthetic derivative of hydrocortisone. Only pharmacologists will ask this question. Prednisone is a synthetic derivative of hydrocortisone or cortisol, the principle steroid made by the adrenal cortex. Prednisone is often given to transplant patients because it is 4x more potent at reducing inflammation. 29. Know that the biologically active metabolite of prednisone is prednisolone. Only pharmacologists will ask this question. Prednisone is a pro-drug with no biological activity until it is converted in vivo to prednisolone. 30. Understand the mechanism of action of corticosteroids: they diffuse across the plasma membrane, bind to receptors in the cytoplasm and enter the nucleus to alter gene transcription. Corticosteroids act by diffusing across the plasma membrane and binding to specific receptors in the cytoplasm. Steroid receptors are often associated with Hsp90 which dissociates after the steroid binds to the receptor, allowing the steroid receptor (with bound steroid) to enter the nucleus and alter gene transcription. 31. Know that steroids upregulates IkB which bind to NFkB preventing NFkB from entering the nucleus and upregulating proinflammatory genes such as IL-1. In context of anti-inflammatory action, the important effect of corticosteroids is to inhibit the function NFkB, a transcription factor responsible for cellular activation and cytokine production in the immune response. In quiescent cells, NFkB is held in the cytoplasm in association with IkBa. On cellular activation, IkBa becomes phosphorylated, allowing NFkB to initiate gene transcription. Corticosteroids increase IkBa production, preventing NFkB from gaining access to the nucleus, preventing the upregulation of proinflammatory genes such as IL-1. 32. Know side effects of steroids: fluid retention, weight gain, diabetes, loss of bone mineral, and thinning of the skin. Corticosteroid drugs have many adverse effects including fluid retention, weight gain, diabetes, loss of bone mineral, and thinning of the skin. 33. Know that steriods are the drug of first choice for treating acute rejection. Corticosteroids are the most effective immunosuppressive drugs when they are first administered before transplantation. This allows the patterns of cytokine gene expression to have already been altered by the time of alloantigenic challenge (i.e. transplantation). This allows corticosteroids to be used for treating acute rejection. 12-19. Cytotoxic drugs kill proliferating cells. 34. Know that a commonly used immunosuppressant is azathioprine. A commonly used cytotoxic drug used in solid organ transplantation is azathioprine. 35. Be able to explain the mechanism of action of azathioprine (it is metabolized into an inhibitor of purine metabolism and thus inhibits DNA replication). Azathioprine is a pro-drug that is converted in vivo to 6-meracaptopurine and then to 6-thioinosinic acid. 6-thioinosinic acid inhibits production of inosinic acid, an intermediate in the synthesis of adenine and guanine nucleotides, effectively inhibiting DNA replication. Azathioprine has no effect on cells until they attempt to replicate their DNA, whereupon they will die. Horribly. 36. Know the side effects of azathioprine: damages replicating tissues of the body such as bone marrow, intestinal epithelium and hair follicles leading to anemia, leukopenia, thrombocytopenia, diarrhea and loss of hair. Although azathioprine is helpful in inhibiting proliferation of alloantigen-activated lymphocytes, it damages all tissues of the body that are normally actively dividing. These tissues include the bone marrow, intestinal epithelium, and hair follicles, leading to anemia, leukopenia, thrombocytopenia, intestinal damange, and hair loss. If pregnant women take cytotoxic drugs, fetal development can be adversely affected. Because azathioprine cannot act until the patient's immune system has been stimulated by alloantigen, it needs only to be administered after transplantation. 12-20. Cyclosporin A, tacrolimus and rapamycin selectively inhibit T-cell activation. 37. Describe the mechanism of action of cyclosporin noting intraellular calcium, calcineurin, NFAT, AP-1 and IL-2 transcription. Cyclosporin A inhibits activation of T cells by antigens by disrupting the transduction of signals from the TCR. Normally TCR signaling leads to generation of IP3 and Ca2+ release from intracellular stores. This activates calcineurin which turns on NFAT by dephosphorylating it. NFAT then enters the nucleus to bind transcription factor AP-1 and turn on gene transcription of IL-2. Cyclosporin interferes with calcineurin activity by binding with peptidyl-prolyl isomerase enzymes called cyclophilins. The cyclosporin-cyclophilin complex binds calcineurin and inhibits its phosphatase activity, preventing NFAT activation. 38. Describe the mechanism of action of tacrolimus (FK-506) noting its similarity and differences from cyclosporin (in particular noting FK-binding proteins). Tacrolimus or FK-506 is a macrolide that is structurally distinct from cyclosporin (which is a cyclic decapeptide). However, it also suppresses T cell activation by inhibiting calcineurin by a similar mechanism. 39. Define immunophillins. While cyclosporin binds cyclophins, tacrolimus binds different peptidyl-prolyl isomerases called FKbinding proteins or immunophilins. 40. Know side effects of cyclosporin and tacrolimus include nephrotoxicity. Although the principle effect of cyclosporin A and tacrolimus is to inhibit T cell activation, they also suppress B cell and granulocyte activation. They do not target proliferating cells so intestinal damage and reduction in hematopoiesis does not occur. However, continued use of cyclosporin A or tacrolimus causes nephrotoxicity. 41. Describe the mechanism of action of rapamycin (sirolimus) noting binding to FK-binding protein but no blockade of calcineurin. Rather rapamycin appears to prevent signal transduction from the IL-2 receptor to the nucleus thereby preventing the T cell from entering the cell cycle and undergoing replication. Rapamycin is another macrolide that also binds FK-binding proteins. However, it does not interfere with calcineurin like cyclosporin A or tacrolimus. Instead, it blocks T cell activation at a later stage by preventing signal transduction from the IL-2 receptor. Rapamycin is more toxic that cyclosporin A and tacrolimus. 12-21. Antibodies specific for T-cells are used to control acute rejection. 42. Know that clinically used anti-T-cell antibodies are produced in sheep, goats, or mice. Ployclonal anti-T cell antibodies are made from sheep and goats that have been immunized with human thymocytes or lymphocytes. This produces antibody-containing fractions called antithymocyte globulin (ATG) or antilymphocyte globulin (ALG). Anti-T cell antibodies are also made from hybidoma cell lines that make mouse mAbs specific to proteins only on the T cell surface like CD3. 43. Understand that because these antibodies are produced from animals, humans can generate an antibody response to these xenogeneic antibodies that can reduce their immunosuppressant activity with subsequent, repeat use. Immunosuppressive antibodies work in 2 ways. ALG and ATG destroy lymphocytes they bind to through complement fixation and phagocytosis. Monoclonal Anti-CD3 interferes with the function of T cells to which it binds, reducing expression of CD3:TCR complexes on the cell surface and depleting these cells from circulation. Because these immunosuppressive antibodies are xenogeneic, they tend to stimulate an antibody response against them. This reduces their immunosuppressive activity when used on subsequent occasions because the patients antibodies form immune complexes with the immunosuppressive antibodies and clear them from circulation before they can bind to T cells. 44. Understand the result of patients making an antibody response against these immunosuppressive antibodies, is that physicians generally use each immunosuppressive antibody to treat just one episode of rejection per patient. Because the patient will make antibody response against the xenogeneic immunosuppressive antibodies, multiple administrations of immunosuppressive antibodies can cause immune complexes to form, resulting in serum sickness. For this reason, physicians generally use each xenogeneic immunosuppressive antibody to counter just one episode of acute rejection per patient. Fortunately, anti-CD3 antibodies and other immunosuppressive antibodies have been humanized by incorporating minimal amounts of mouse antigen-binding site into the framework of a human antibody. This allows them to be used on multiple occasions in the same patient and can possibly used to prophylactically prevent acute rejection. 12-22. Patients needing a transplant outnumber the available organs. 45. Know that the waiting lists for transplantation are growing at a faster rate than the number of transplants performed each year. The waiting list for transplantation grow faster than the number of transplants performed each year. For example, there are 75,000 people waiting for kidney transplants each year, of which only 17,000 transplants are performed. 12 people die every day because there are not enough organs. 46. Define xenotransplantation. Xenotransplantation is a transplantation where the donor and the recipient are of different species. The graft tissue in a xenotransplantation is a xenograft. The most suitable donor species for humans are pigs. However, humans have circulating antibodies called xenoantibodies that would bind to pig antigens called xenoantigens and cause hyperacute rejection. 47. (Understand that complement regulation does not work efficiently across species barriers and that pigs transgenic for human decay accelerating factor may improve the survival of xenotransplanted organs. After a porcine xenograft transplant, human blood will perfuse through the pig organ. Animals have their own species-specific anti-complement barrier lining their blood vessels, so the pig vessels cannot stop binding and activation of human complement unless they are genetically modified to do so by including human decay accelerating factor to improve survival of the xenograft organ.) 12-23. Bone marrow transplantation is treatment for genetic diseases of blood cells. 48. Understand pluripotent stem cell engraftment makes bone marrow transplantation possible. Pluripotent stem cell engraftment makes bone marrow transplantation possible because it permanently replaces an individual's entire hematopoeitic system, including the immune system. The pluripotent stem cells reconstitute the recipient's immune system, RBCs, and platelets by undergoing engraftment and colonization of the recipient's bones. With time, the transplant replaces the defective hematopoietic system. 49. Know two indications for bone marrow transplantation: genetic diseases of hematopoiesis such as thalassemia, sickle cell anemia, Fanconi’s anemia and SCID and treatment of malignancies such as leukemia and lymphoma. Bone marrow transplantation can be used to treat genetic diseases of hematopoisesis such as thalassemia, sickle cell anemia, Fanconi's anemia, and SCID. It can also be used to treat malignancies such as leukemia and lymphoma because the cytotoxic drugs to treat cancers often kill the recipient's bone marrow and require replacement. 12-24. The alloreactions in bone marrow transplantation attack the patient, not the transplant. 50. Know that graft-versus-host-reaction (GVHR) is the major cause of morbidity and mortality after bone marrow transplantation. GVHR is the major cause of morbidity and mortality after bone marrow transplantation. 51. Know that the condition caused by GVHR is called graft-versus-host-disease (GVHD), that it can attack almost every tissue of the body, but principally involves the skin, liver, and intestines. GVHD primarily affects the skin, liver, and intestines. 52. Understand that a graft-versus-host-response can sometimes be beneficial as when directed against leukemia. GVHD response can sometimes be beneficial when directed against leukemia. The bone marrow replaces the entire hematopoietic system, including the immune system; the new immune cells that develop may undergo a GVHD response which can be beneficial if it attacks leftover leukemia cells from the old immune system. 12-25. The impact of alloreactions on the type of tissue or organ transplanted. 53. Know that cornea transplantation is not vascularized and does not require immunosuppression. Cornea transplantation is not vascularized and does not require immunosuppression. 54. Know that the outcome of liver transplantation is not improved by matching the donor and recipient of for HLA class I or II antigens. The outcome of liver transplantation is not improved by matching the donor and recipient for HLA class I or II antigens.