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TRANSPLANTATION IMMUNOLOGY ARPAD LANYI PhD ORGAN, TISSUE OR CELL TRANSPLANT AUTOLOGOUS autograft SYNGENEIC syngraft ALLOGENEIC allograft Skin, muscle, dendritic cell, cartilage Kidney, cornea, liver, heart, lung Bone marrow-derived haematopoietic cells (HSC) Bone marrow-derived haematopoietic cells (HSC) A FEW MILESTONES IN TRANSPLANTATION First Human-To-Human Blood Transfusion 1818 James Blundell, a British obstetrician transfuses four ounces of blood from a man to his wife, replacing the blood she just lost during childbirth--the first well-documented case of person-to-person blood transfusion. Ten other women suffering from similar blood loss receive transfusions, which help half of them. Photo: Blundell's Gravitator Source: NIH/ Pennsylvania State University Libraries A FEW MILESTONES IN TRANSPLANTATION 18th century: succesful transplant experiments is animals (skin grafts) First succesful fresh skin allograft 1869 Jacques Louis Reverdin First Successful Human-To-Human Bone Transplant 1878 This operation, which used bone from a cadaver, remained unusual because there was no way to process and preserve human tissues. Thyroid transplantation 1883 Theodor Kocher Thyroid transplantation became the model for a whole new therapeutic strategy: organ transplantation. After the example of the thyroid, other organs were transplanted in the decades around 1900. First Attempts At Bone Marrow Transplant 1896 First attempts to use bone marrow as treatment for leukemia; patients receive the marrow orally after meals, but it has no effect. In the next few years, intravenous injections of bone marrow to treat aplastic anemia have some success. Discovery of ABO blood groups 1902 Karl Landsteiner First human xenotransplant 1906 A French surgeon inserts slices of rabbit kidney into a child suffering from kidney failure. The immediate results are good, but the child dies after two weeks. Four years later, a monkey kidney is transplanted into a young girl suffering from mercury poisoning; it produces a small amount of urine, but the girl lives for only five days. A FEW MILESTONES IN TRANSPLANTATION Discovery of MHC 1940’ George Snell Human HLA: 1958 Jean Dausset First successful human kidney transplant (syngraft) 1954 Joseph E. Murray First Successful Human Heart Transplant 1967 "It's going to work!" Dr. Christiaan Barnard shouts as the heart of Denise Darvall, a 23-year-old woman who died in an automobile accident, begins to beat in the chest of 54-year old Louis Washkansky. The transplant was performed at Groote Schuur Hospital in Cape Town, South Africa. The heart functioned until Washkansky died of pneumonia eighteen days later because of his suppressed immune system. First Successful Bone Marrow Transplant 1973 Doctors at the Memorial Sloan-Kettering Cancer Center in New York City perform the first successful bone marrow transplant from an unrelated donor. They transplant marrow from a person in Denmark into a five-year-old with severe combined immunodeficiency disease known as bubble boy syndrome. A few successful previous bone marrow transplants had all involved related donors. PEOPLE IN THE US LIVING WITH FUNCTIONING ORGAN GRAFTS TISSUE/SOLID ORGAN TRANSPLANTATION The success of transplantation and maintenance of the donor tissue depends majorly on histocompatibility GRAFTS WITH MATCHING MHC ARE TOLERATED WHILE ALLOGENEIC GRAFTS ARE REJECTED BOTH IN MICE AND HUMANS ALLORECOGNITION DIRECT AND INDIRECT ALLOANTIGEN RECOGNITION MOLECULAR BASIS OF DIRECT RECOGNITION OF ALLOGENEIC MHC MOLECULES T-cell responses to directly presented allogeneic MHC molecules are very strong because there is a high frequency of T-cells (1-2%) that can directly recognize any single allogeneic MHC. 100-1000X times greater than that of during infection. • Many different peptides derived from donor cellular proteins may combine with a single allogeneic MHC molecule: a different clone of recipient T- cells can be activated. • Every APC expresses thousands of copies of different MHC molecules: if these are foreign MHC molecules, many or all of them can be recognized by alloreactive T-cells. (infection: 0.1-0.9%) • Many of the T-cells that respond to an allogeneic MHC molecule, even on first exposure, are memory T-cells (cross-reaction). DIRECT AND INDIRECT ALLOANTIGEN RECOGNITION It is essentially the same as the recognition of any foreign (e.g., microbial) protein antigen ACTIVATION OF ALLOREACTIVE T-CELLS Antibodies against graft antigens (most frequently HLA) also contribute to rejection. Antibody production follows the same sequence of events as any helper T-cell-dependent antibody response (an example of indirect presentation of alloantigens). TRANSPLANT REJECTION BLOOD TRANSFUSION IS THE MOST COMMON FORM OF TISSUE TRANSPLANTATION Incompatibility of blood group antigens causes type II hypersensitivity reactions Incompatibility is tested by the cross-match test. The patient’s serum is tested against the red cells of the potential donors. The patient’s red cells are NOT tested against the serum of the donor!!! (no problem). Organ transplantation requires compatibility of the main blood group antigens HYPERACUTE TRANSPLANT REJECTION ANTIBODY-MEDIATED TYPE II HYPERSENSITIVITY REACTION • The most dramatic form of tissue incompatibility, begins within minutes to hours • BLOOD GROUP ANTIGENS are expressed by endothelial cells of blood vessels (solid vascularized organs) • ANTIBODIES (IgM) – COMPLEMENT FIXATION – NEUTROPHIL, PLATELET ACTIVATION – INTRAVASCULAR COAGULATION - IRREVERSIBLE ISCHEMIC NECROSIS • Cannot be reversed, should be avoided!!!!! • Today, hyperacute rejection by anti-ABO antibodies is extremely rare BUT • HLA I is expressed on vascular endothelium cells, preexisting antibodies (IgG) against HLA class I variants can also cause hyperacute rejection!! Where do anti-HLA antibodies come from? THE SOURCES OF ANTI-HLA ANTIBODIES Pregnancy – during childbirth With successive pregnancies, increasing levels of anti-HLA antibodies can develop Blood transfusions (platelets, leukocytes) - HLA type is not assessed, the matching being restricted to just the ABO and Rhesus D types Previous organ transplant Cross-match!!! CROSSMATCH TESTING Complement-dependent cytotoxicity • Lymphocytes from the donor are isolated. • Serum from the recipient is mixed with the lymphocytes in a multi-well plate. • Complement is then added (usually derived from rabbit serum). • If donor-specific antibody is present and binds to donor cells, the complement cascade will be activated via the classical pathway resulting in lysis of the lymphocytes. DOI: 10.1111/j.1440-1797.2010.01414.x CROSSMATCH TESTING Flow cytometry • More sensitive • T- and B-cells can be separated to detect MHC class I and MHC class II specific antibodies • Anti – MHC I react with both B and T lymphocytes • Anti – MHC II react with B lymphocytes only DOI: 10.1111/j.1440-1797.2010.01414.x CROSSMATCH TESTING Panel reactive antibody (PRA) Beads: Bb • The patient’s serum is tested for reactivity with leukocyte antigens representing the population. • The number of positive reactions is expressed as a percentage PRA. DOI: 10.1111/j.1440-1797.2010.01414.x ACUTE TRANSPLANT REJECTION T-CELL-MEDIATED TYPE IV HYPERSENSITIVITY REACTION Th1, Th17, CD8+ T-cells, macrophages Antibodies, complement, neutrophils, platelets takes days to develop The rejected graft is swollen and has deep-red areas of hemorrhage and gray areas of necrotic tissue. ACUTE TRANSPLANT REJECTION T-CELL-MEDIATED TYPE IV HYPERSENSITIVITY REACTION KIDNEY TRANSPLANTATION HEART TRANSPLANTATION T-CELLS Plasma cells Lymphocytes around renal tubules T lymphocytes in the myocardium Labeled with anti-CD3 antibody Lymphocytes around an arteriole (A) Lymphocytes surrounding the renal tubules (T) Staining of T lymphocytes with anti-CD3 (brown staining) in the same section CHRONIC TRANSPLANT REJECTION TYPE III HYPERSENSITIVITY REACTION Thickening of the vessel wall and narrowing of the lumina Chronic rejection in a kidney allograft with graft arteriosclerosis E: endothel G: granulocyte T: alloreactive T M: macrophage EL: elastic lamina SMC: smooth muscle cell DIFFERENCE IN GRAFT SURVIVAL OF RECIPIENTS RECEIVING KIDNEYS FROM LIVE OR CADAVERIC DONORS 10.5812/numonthly.12182 HLA MATCHING IMPROVES THE SURVIVAL OF TRANSPLANTED ORGANS (for example kidneys) Blue Orange Red Dark blue Green Black Brown HLA-DR, B and A are the most important to match. HLA TYPING MIXED LYMPHOCYTE REACTION The response of alloreactive T-cells to foreign MHC molecules can be analyzed in vitro HLA TYPING DNA-BASED TYPING METHODS PCR-SSOPH: Sequence-specific oligonucleotide probe hybridization Specimen 1 (Type A*0203) Specimen 2 (Type A*0501) TAG C GAT ATC G CTA TAG A GAT ATC TCTA Amplify, denature, and spot onto membranes • Intermediate resolution • Sreening test to identify potential donors or individuals who may later require higher resolution testing Specimen 1 Specimen 2 • High volume, relatively low cost Probe with allele-specific probes ...TAGCGAT..(A*02) Specimen 1 Specimen 2 ...TAGAGAT…(A*05) Specimen 1 Specimen 2 HLA TYPING DNA-BASED TYPING METHODS SSP-PCR: Sequence-specific PCR (allele-specific primers) Reagent blank SSP: Sequence-specific primer SSP matches allele SSP SSP Amplification controls Allele-specific product Primers recognizing different alleles are supplied in a 96-well plate format Amplification Amplification control No amplification SSP does not match allele Allele-specific product Agarose gel Very rapid test that can be performed in 3-4 hours from the time a sample is received. PCR-SSP is used for typing deceased organ donors when speed is an important consideration. HLA TYPING DNA-BASED TYPING METHODS SBT: Sequence-based typing Polymorphic regions are amplified by PCR and then sequenced Isolate DNA The highest resolution HLA typing Reverse PCR primer PCR Forward PCR primer Exon 2 clean amplicons Exon 3 Sequencing primers sequence amplicon Sequences are compared to reference sequences for previously assigned alleles EVEN COMPLETE MATCHING AT THE MHC DOES NOT ENSURE GRAFT SURVIVAL MINOR HISTOCOMPATIBILTY ANTIGENS Peptides derived from polymorphic cellular proteins bound to MHC class I molecules The response to minor H antigens is in many ways analogous to that of viral infection MINOR HISTOCOMPATIBILITY ANTIGENS IMMUNOSUPPRESSION DEPLETING ANTIBODIES Antibodies that broadly react with white blood cells are given to patients before and after transplantation to deplete these cells and generally weaken the immune system. • Rabbit antithymocyte globulin (rATG) • Polyclonal mixture of high-affinity antibodies • Binds to T-, B-, NK, dendritic and endothelial cells • Fixes human complement well, delivers the cells to be killed by phagocytes • Serum sickness • Alemtuzumab: anti-CD52 • CD52-specific humanized rat monoclonal IgG • CD52 is expressed on almost all lymphocytes, monocytes and macrophages • Alemtuzumab induces a profound, long-lasting lymphopenia CORTICOSTEROIDS • Effective immunosuppressive drugs • Used to treat patients before the transplantation and during episodes of rejection • A key immunosuppressive effect: inhibition of NFkB activity (central transcription factor of the inflammatory response) by enhancing IKB • Alters lymphocyte homing, lymphocytes are barred from entering secondary lymphoid tissues • Many adverse side-effects: fluid retention, weight gain, diabetes, loss of bone mineral, thinning of the skin • Prolonged use is avoided wherever possible • Need novel, milder drugs T-CELL ACTIVATION CAN BE TARGETED BY IMMUNOSUPPRESSIVE DRUGS Targeting immunophilins: cyclosporine, tacrolymus Calcineurin inhibitors Side effects: Neurotoxicity, Nephrotoxicity, Diabetes INFLUENCE OF CYCLOSPORINE ON GRAFT SURVIVAL INHIBITION OF T-CELL CO-STIMULATION BY BELATACEPT, A SOLUBLE FORM OF CTLA-4 Belatacept works as well as the best of the older, established drugs and is better than them in preserving kidney function. On the downside, belatacept is associated with increased incidence of episodes of acute rejection. BLOCKING CYTOKINE SIGNALING CAN PREVENT ALLOREACTIVE T-CELL ACTIVATION • Chimeric basiliximab, humanized daclizumab: monoclonal IgG1 antibodies specific for CD25 • First given just before the transplant is performed, subsequent infusions given during the first 2 months when acute rejection is most likely • A single dose of the antibody will saturate all the CD25 molecules in the body within 24 hours, the half-life of the antibody is 2 weeks, and its suppressive effect can last for more than a month. • Specific for activated T-cells, no wide-ranging effects and immunosuppression associated with many other drugs BLOCKING CYTOKINE SIGNALING CAN PREVENT ALLOREACTIVE T-CELL ACTIVATION Rapamycin (sirolimus) • Immunosuppressive macrolide (Streptomyces hygroscopicus) • Binds to FKBP12, but does not interfere with calcineurin • mTOR inhibitor, prevents signal transduction from the IL-2 receptor • More toxic than either cyclosporin A or tacrolimus but is a useful component of combination therapy • Does not impair the survival and functions of regulatory T-cells as much, which may promote immune suppression of allograft rejection • Dendritic cells, B-cells doi:10.1016/S0167-7799(98)01239-6 CYTOTOXIC DRUGS TARGET THE REPLICATION AND PROLIFERATION OF ALLOANTIGEN-ACTIVATED T-CELLS Cytotoxic drugs are administered only after transplantation Azathioprine: kidney transplantation • Prodrug, first converted in vivo to 6-mercaptopurine and then to 6thioinosinic acid • The latter inhibits the production of inosinic acid, an intermediate in the biosynthesis of adenine and guanine nucleotides • No selectivity, collateral damage to those tissues that are always engaged in cell division: bone marrow, intestinal epithelium, hair follicles: anemia, leukopenia, thrombocytopenia, intestinal damage, loss of hair Mycophenolate mofetil: • Penicillium stoloniferum • Similar effects to those of azathioprine • Metabolized in the liver to mycophenolic acid • Prevents cell division by inhibiting inosine monophosphate dehydrogenase, an enzyme necessary for guanine synthesis CYTOTOXIC DRUGS TARGET THE REPLICATION AND PROLIFERATION OF ALLOANTIGEN-ACTIVATED T-CELLS Cytotoxic drugs are administered only after transplantation Cyclophosphamide: • Originally developed as a chemical weapon (World War I.) • A pro-drug that is metabolized to phosphoramide mustard, a compound that alkylates and cross-links DNA molecules • Specifically damages the bladder, sometimes causing cancer or hemorrhagic cystitis. • Unlike azathioprine, cyclophosphamide is not particularly toxic to the liver, useful alternative for patients with liver damage • Cyclophosphamide is most effective when used in short courses of treatment Methotrexate: • Prevents DNA replication by inhibiting dihydrofolate reductase, an enzyme essential for the cellular synthesis of thymidine. SUMMARY OF IMMUNOSUPPRESSIVE DRUGS ACTING AT DIFFERENT STAGES IN THE ACTIVATION OF ALLOREACTIVE TCELLS METHODS TO INDUCE DONOR-SPECIFIC TOLERANCE Hematopoietic chimerism • Dizygotic twins who have had a common blood circulation during gestation are tolerant of each other’s tissues. • Combining solid organ transplantation with some mild form of hematopoietic cell transplant from the same donor could induce a more robust and stable tolerance and eliminate the necessity for long-term immunosuppression. • Promising results: renal/bone marrow allograft • Family members differing by one HLA haplotype • Non-myeloablative treatment: cyclophosphamide, cyclosporin, antiCD2 antibody, thymic irradiation • Immunosuppressive therapy: 9–14 months • Survival: up to 10 years Transfer of regulatory T-cells • Attempts to generate donor-specific regulatory T-cells in culture and to transfer these into graft recipients are ongoing. THE NEED FOR HLA MATCHING AND IMMUNOSUPPRESSIVE THERAPY VARIES WITH THE ORGAN TRANSPLANTED Anterior chamber-associated immune deviation (ACAID) • Immunological environment that suppresses inflammation while maintaining sufficient protection against pathogens. • Cornea lacks vasculature • Anterior chamber contains immunomodulatory factors (TGF-β) • Tolerogenic DCs, Tregs, IL-4, TGF-β. • Corneal transplants: 90% success in the absence of HLA matching or immunosuppressive therapy Liver • Specialized architecture and vasculature • Very low levels of HLA class I, no HLA class II • Daily exposure to the digestion products of a myriad of foreign proteins from the intestines with their characteristic anti-inflammatory environment • Liver is relatively refractory to rejection • HLA type or cross-match are not assessed before liver transplantation; ABO type assessed • Cyclosporin and tacrolimus has markedly improved the success of liver transplants At the other end of the spectrum from eye and liver is the bone marrow HEMATOPOIETIC STEM CELL TRANSPLANTATION HEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT) • Myeloablative therapy: combination of cytotoxic drugs and irradiation • Engraftment: pluripotent stem cells colonize the bones • When the immune system has been fully reconstituted (can take a year or more), the patient is a chimera (has a genetically different immune system) • • • • • HSCs are now obtainable from the blood G-CSF, GM-CSF: stem cells are mobilized from the bone marrow Leukapheresis Isolation: CD34 Between a quarter and half a billion CD34-positive cells are needed to ensure prompt engraftment MORE SHARED HLA ALLOTYPES, MORE ROBUST T-CELL RESPONSE THE SUCCESS OF HSCT CORRELATES WITH THE EXTENT OF THE HLA MATCH GRAFT-VERSUS-HOST DISEASE: ALLOREACTIVE DONOR TCELLS IN THE GRAFT ATTACK THE RECIPIENT’S TISSUES • The conditioning therapy which destroys bone marrow cells, also damages other tissues : skin, intestinal epithelium, hepatocytes • Cytokine storm, dendritic cell activation • Alloreactive T-cells from the transplant interact with the recipient’s dendritic cells • Methotrexate in combination with cyclosporin A • Markedly reduced GVHD: higher incidence of graft failure Minor histocompatibility antigens trigger alloreactive T-cells in recipients of HLAidentical transplants UMBILICAL CORD BLOOD AS THE SOURCE OF HEMATOPOIETIC STEM CELLS • Umbilical cord blood has the combined benefits of being rich in stem cells and having fewer alloreactive T-cells • There is less GVHD, and a greater HLA disparity can be tolerated. • Engraftment is slower • Limited volume: combined stem cells from two unrelated samples of cord blood • The patient’s reconstituting hematopoietic system becomes a chimera but in time the cells from one donor overwhelm the cells of the second • First use: 1988; child with Fanconi’s anemia • More than 25,000 cord blood transplants have been performed GENETIC DISEASES FOR WHICH HSCT IS A THERAPY HSCT is a therapy for many genetically determined immunodeficiencies, such as SCID HSCT IN TUMOR THERAPY Autologous HSCT • A sample of the patient’s bone marrow is taken before the remainder is ablated • The hematopoietic stem cells in are purified away from any tumor cells and are then given back to the patient • Perfectly histocompatibility: no GVHD, no immunosuppressive drugs • Limitation: the rate of relapse is significantly higher for autologous transplants than for allogeneic transplants GRAFT-VERSUS-LEUKEMIA (GVL) EFFECT Alloreactive T-cells in the graft help rid the patient of residual leukemia cells • Promotion of GVL reaction, less emphasis on tumor elimination by chemotherapy and irradiation • Less severe conditioning regimens: hematopoietic system is damaged but not destroyed, quicker recovery • One approach: transfusions of donor T- cells at a time after tranplantation when the inflammation caused by the conditioning regimen has subsided and the likelihood of GVHD is diminished NK CELLS ALSO MEDIATE GRAFT-VERSUS-LEUKEMIA EFFECTS Haploidentical transplant • GVHD prevention: T-cell depletion and infused antiT-cell antibodies • No further immunosuppressive transplantation treatment after • Reconstitution of NK cells occurs more rapidly than that of T-cells, alloreactive NK cells can emerge • The occurrence and specificity of the NK-cellmediated alloreactions are determined by the interactions of inhibitory KIR with HLA-B and HLA-C ligands and are predictable from the HLA types of the donor and recipient. • NK-cell alloreactions occur when the recipient’s HLA class I allotypes provide ligands for fewer types of inhibitory KIR than the donor’s HLA class I allotypes. • AML+, ALL• Alloreacive NK response wanes and is undetectable 4 months after transplantation. • With full reconstitution of the immune system, NKcells become tolerant of both recipient and donor cells. 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