1、HLA TYPING & ORGAN TRANSPLANTATION Scott Bainbridge Elena Crouson Israfiel MohammedSarah TuckerHLA TYPING & ORGAN TRANSPLANTAOrgan Transplantation What is it?Organs or tissues from one human being (the donor) are put into another persons body (the recipient).Factors Effecting Transplantation?HLA Ant

2、igensOrgan Transplantation What is Statistics on Organ Transplantation There are more than 91,500 people on the organ transplantation waiting list.Each day 74 people receive an organ transplantation, but 18 people on the waiting list die because a donor is not available. There are 55,000 people wait

3、ing for a Kidney, 17,000 waiting for a liver and 3,000 waiting for either a heart or liver transplant.Statistics on Organ TransplantFirst Organ Transplantation In 1959, Joseph Murray and his colleagues in Boston successfully transplanted a Kidney that were donated by fraternal twins and it functione

4、d for 20 years without immunosuppression drugs. First successful Liver transplant- In Denver on 7/23/1967First successful Heart transplant- In Cape Town, South Africa on 1/2/68First successful Bone Marrow transplant- Minneapolis, MN on 8/25/68First Organ Transplantation InWhy Is it Difficult?Organ t

5、ransplantation is difficult because of the HLA antigens located on the cell surface. Human Leukocyte Antigen (HLA) also referred to as Major Histocompatibility Complex (MHC) plays a role in intercellular recognition and discrimination between self and non-self. Why Is it Difficult?Organ tranLocation

6、 of HLA/MHCThe MHC complex is a collection of genes arrayed within a long continuous stretch of DNA on chromosome 6. Each HLA type of associated with a different class of MHC molecule. Location of HLA/MHCThe MHC comTypes of MHCThere are three classes of MHC molecules. Class I- encodes glycoproteins

7、expressed on the surface of nearly all nucleated cell; the major function of the class I gene is presentation of peptide antigens to cytotoxic T-cellsClass II- encodes glycoproteins expressed primarily on antigen-presenting cells, examples: macrophages, dendritic cells and B-cells, where they are pr

8、esent processed antigenic peptides to T helper cells.Class III- encodes various secreted proteins that have immune function including components of the complement system; C2,C4, Factor B, &TNF, and molecules involved in inflammation. Types of MHCThere are three clDifferent HLA AllelesClass I- HLA A

9、451 alleles HLA B 782 alleles HLA C 238 allelesClass II- HLA DR 525 alleles HLA DQ 105 alleles HLA DP 147 alleles HLA DM 11 alleles HLA DO 21 allelesDifferent HLA AllelesClass I- Requirements for Transplant Each transplant center has different requirements for allele matches.For the National Marrow

10、Donor Program:In order to find a match doctors require that at least a minimum of 3 allele matches. HLA-A, HLA-B and HLA-DRB1. One set of the three antigens are inherited from your mother and the other set is inherited from your father. This makes 6 antigens to match Therefore, it is required that 4

11、 of the 6 antigens match for cord blood donation and 5 of the 6 antigens for adult donation. Requirements for Transplant EaRequirements for TransplantNational Marrow Donor Program:Requirements for TransplantNatChance of a Match Mother/Father: 25% chance of full match One Sibling: 25 % chance of full

12、 match Two Siblings: 44 % chance of full match Three siblings: 58% chance of full match The chance to find donors may be better for more homogenous racial groups. Chance of a Match Mother/FatheHLA In Transplantation There are two characteristics of the HLA genes that make them special for organ tran

13、splantation:There high degree of polymorphism There strong immune reactions that their products can produce in other individuals. HLA In Transplantation There aHLA PathwaysThere are two pathways that can occur that cause problems in organ transplantation as a result of HLA antigens. Direct pathway-

14、the alloreactive responses of recipient T-cells to donor APC expressing incompatible antigens.Indirect Pathway- allogeneic HLA antigens are taken up and processed by recipient APC and presented in context with autologous HLA molecules to recipient T-cells. HLA PathwaysThere are two pathProblems from

15、 HLA antigens Engraftment- immunological rejection of donor hematopoietic cells by recipient T cells that recognize incompatible HLA determinants. Factors: pregnancy or transfusionHLA mismatching of the donorUsing of less intense preparative regimen before transplantSuboptimal post-transplant immuno

16、suppressive therapyDepletion of T lymphocytes from marrow grafts.Class I determinants govern graft acceptanceClass II determinants play a role in GVHD. Problems from HLA antigens EngProblems from HLA antigensAcute Graft versus Host Disease- immune reaction of mature donor T lymphocytes against HLA d

17、eterminants of the recipient. The reaction is directed toward normal tissue including skin and gastrointestinal mucosa. HLA matched unrelated Bone Marrow transplant Acute GVHD is 79% vs. 35% HLA matched sibling BMT.Matching donor/recipient pairs for molecular typing has been showed to reduce the ris

18、k of acute GVHD, 48% vs. 70%If HLA-DRB1 and HLA-DQB1 are matched with recipient than it reduces the risk of acute GVHD. Problems from HLA antigensAcutProblems from HLA antigensChronic GVHD- is the principle cause of morbidity and no relapse mortality for patients reaching day 100 after allogeneic tr

19、ansplant. It may involve skin, oral mucosa, eyes, liver, gastrointestinal tract and lungs. It occurs in 35% to 70% of patients after unrelated donor BMT.Mortality rates range from 25% to 70% , depending on associated risk factors. Problems from HLA antigensChroOverviewMethods: Histocompatibility tes

20、t, consisting of three tests: HLA antigen typing, screening of the recipient for the anti-HLA antibodies and the lymphocyte crossmatch or compatability test. Results: More allele mismatch more complications Further Studies/Discussion: Outcomes of unrelated donor/recipient transplant.OverviewMethods:

21、 HistocompatibHistocompatibility testing consists of three testsHLA antigen typing (also called tissue typing)Screening of the recipient for anti-HLA antibodies (also called antibody screening)Lymphocyte cross matching (also called compatibility testing)Histocompatibility testing conHLA antigen typi

22、ngTwo different methods, serological and DNA sequencingHLA antigen typingTwo differenSerological methodLymphocytes are harvested from the blood by density gradient centrifugationA solution of Ficoll-Hypaque is layer underneath the whole blood, and the tube is centrifugedRed blood cells are denser an

23、d go to the bottommononuclear cells are less dense, and are found in the middle, just underneath the plateletsThe mononuclear layer is removed, and washed. T-cells are removed usually by binding to magnetic beads coated with T-cell antibodies, and are washed away, leaving only the B-cells. Serologic

24、al methodLymphocytes Serological methodThis B cell enriched media is added to a microtiter plate with each well containing a different antibody to a certain HLA antigen. If a certain MHC cell is present, the antibodies will bind, forming an antigen-antibody complex.After incubation, rabbit complemen

25、t is added to each well. If an antigen-antibody complex is present, complement will be activated, and will destroy the cells with an antigen-antibody complex.After incubation formalin is added to fix the cells and stop the complement reaction. Eosin Y is added to stain any dead cells.Serological met

26、hodThis B cell Serological methodCells are examined under a phase contrast microscope, and cells that are pink are positive. If 60% or more of the cells are stained they are considered positive for the HLA antigen.Serological methodCells are exDNA typing methodsGranulocytes and lymphocytes are separ

27、ated from blood by lysis of the red blood cells using ammonium chloride and centrifugation. DNA is extracted from the white cells by chloroform and ethanol and added to the wells of a microtiter tray. Each well contains oligonucleotide primers complementary to a small segment of only one HLA allele.

28、 If the primer can attach, the HLA antigen is present on the cells.DNA typing methodsGranulocytesDNA typing methodsDNA polymerase and oligonucleotide triphosphates are added to each well and the plate is incubated in a thermal cycler, which multiplies the sequence between the primers (same as PCR)Th

29、e DNA is removed and run on agarose gel by electrophoresis. Since the DNA was amplified, if there is any DNA detected, HLA is present. If no DNA is seen, HLA is not present. DNA typing methodsDNA polymeraAntibody screening for anti-HLA antibodiesPurpose: to detect antibodies in the recipients serum

30、that react with HLA antigens. We know what HLA type the person is, but we dont know what antibodies they have to other HLA typesAntibody screening for anti-HLAntibody screening for anti-HLA antibodiesLeukocytes (neutrophils, monocytes, basophils, lymphocytes) are harvested from the blood of donors w

31、ith a known HLA type and are added to a microtiter plate.Serum from the recipient is added to each well.After incubation, cells are washed to remove any unbound proteinsAnti-human Ab is added, incubated, and then rabbit complement is added. Antibody screening for anti-HLAntibody screening for anti-H

32、LA antibodiesIf an antibody against HLA is present, it will bind to the cells, and antigen-antibody complexes will bind to the anti-human Ab, which will then activate complement. Eosin Y is added, cells are examined under a microscope. Pink stained cells indicates the presence of anti-HLA antibodies

33、. The higher the number of different HLA antibodies the lower the probability of finding a match.Antibody screening for anti-HLCrossmatch testPurpose: to detect presence of preformed antibodies in recipient that are reactive against donor tissues. Crossmatch testPurpose: to detCrossmatch testPeriphe

34、ral blood lymphocytes from the donor are separated into B and T lymphocyte populationsT-cells are purified by magnetic beads coated with monoclonal antibodies for B-cells. The B-cells bind and are removed by magnetic force. B-cells are purified in the same manner, but the magnetic beads are coated w

35、ith monoclonal antibodies for T-cells. Crossmatch testPeripheral blooCrossmatch testB-cell crossmatch is performed using the same method as HLA typingT-cell crossmatch is performed using the same method as screening testWhy do a crossmatch when screening seems sufficient? Antibodies against low-inci

36、dence antigens are likely to be missed. Acts as a mock transplantCrossmatch testB-cell crossmatResultsPapers Results Focus on Four Aspects of ExperimentHLA Typing serological methods for HLA Class I (A, B, C) alleles and using DNA methods for Class II alleles (DRB-1, DQB-1). Graft FailureUsed statis

37、tical methods to determine percentage of graft failures based on types of mismatches presentAcute Graft vs. Host Disease (GVHD)Also used statistical methods to study this.SurvivalOver the course of 8 years studied survival rates in patientsResultsPapers Results Focus oHLA MatchingHLA MatchingTested

38、300 pairs in study142 matched for the HLA Alleles (A, B, C, DRB1, DBQ1)158 mismatched pairs- 83 mismatched at a single locus- 75 mismatched at two or more lociOf the transplants, 83% were done using a Caucasian donor to Caucasian patient. 85% of transplants had donor and recipient of same race. HLA

39、MatchingHLA MatchingGraft FailureGraft failure was high when one or more HLA class I allele mismatches were present. Class II allele mismatches did not lead to graft failure. However when both class I and class II mismatches present, graft failure is highSource: Blood, Vol. 92, Issue 10, 3515-3520,

40、November 15, 1998 Graft FailureSource: Blood, VoAcute GVHDFig 1. Cumulative incidence estimates of grades III-IV acute GVHD according to recipient disparity. Source: Blood, Vol. 92, Issue 10, 3515-3520, November 15, 1998 Acute GVHDAcute GVHDWhat does the previous slide show? Risk of having grades II

41、I-IV GVHD depend mainly on the class of mismatched allele and the number of mismatches.Class II allele mismatches and Class I/II mismatches combined have the highest probability for GVHD. Also more than one class I mismatch leads to an increased risk for GVHD. Acute GVHDWhat does the previoSurvivalM

42、ultiple Class I MismatchesLower chance of survival amongst patients that have more than one class I mismatch or have both class I and class II mismatches. Multiple Class II MismatchesOf seven patients with multiple mismatches, three died within 100 days- Other Four lived 3-8 years.Overall MessageHav

43、ing a single mismatch in either Class I or Class II can survive, but when there is more than one mismatch it could lead to fatalities.SurvivalMultiple Class I MismaSurvival (Cont.)Survival (Cont.)What Do the Results Mean?What Do the Results Mean?Results ExplainedMismatches in certain classes of alle

44、les resulted in either graft failure (Class I alleles) or acute GVHD (Class II alleles)The two classes of alleles are distinguished on a molecular level. In addition, HLA class I molecules will interact with natural killer (NK) cells. This contributed to death in individuals within an 8 year span. T

45、o understand the differences between the loci in class I alleles will have to be done in future results.This is because donors with a single HLA-A, B, or C mismatch was too small to do comparisons.Having one mismatch affected survival slightlyBeneficial to certain ethnic groups which finding alleles

46、 that match completely might be difficult.Why the majority for this experiment were CaucasianResults ExplainedMismatches inThings to considerFor this experiment, the researched focused on individuals with chronic myeloid leukemia.Ideal transplantation would be one where time between diagnosis and tr

47、ansplant is minimum. The results are not representative of what it could happen to patients with other types of leukemia.Things to considerFor this expOutcomes After Unrelated-Donor TransplantOutcomes After Unrelated-DonorUnrelated marrow transplantTransplantation survival is increased by matching C

48、lass I and Class II allelesMultiple Class I disparities in the donor increase the risk of graft failureMultiple Class II disparities in the recipient increase the risk of GVHDSingle disparities did not appear to compromise survivalUnrelated marrow transplantTraBiological functions of Class I and Cla

49、ss II moleculesClass I Present peptides derived from endogenously synthesized proteinsResponding T cells express CD8+Complex interactions with NK cells/jkimball.ma.ultranet/BiologyPages/H/HLA.html#cd8Biological functions of Class Biological functions of Class I and Class II moleculesClass IIPresent

50、peptides derived from exogenously synthesized proteinsResponding T cells express CD4+/jkimball.ma.ultranet/BiologyPages/H/HLA.html#class_IIBiological functions of Class Graft-versus-Host DiseaseComplication of bone marrow transplants in which T cells from donor attack the hosts tissues.Acute GVHD an

51、d chronic GVHDGraft-versus-Host DiseaseComplGraft FailureAntigen-presenting cell trigger CD4 and CD8 cellsLocal and systemic immune response developCytokine recruitment and activation of specific T cells, NK cells macrophage-mediated cytotoxicityAllograft destructionhttp:/cnserver0.nkf.med.ualberta.

52、ca/cn/Schrier/Volume5/ch9/ADK5-09_1-3.pdfGraft FailureAntigen-presentinChimerismImplanted organ allografts become mixtures of donor and recipient cells. 1968, karyotyping of livers transplanted to females from male cadaversMost livers remain maleKupffer cells were replaced with recipient female cellsChimerismImplanted organ allogLimitations on HLA-matchingUnder 40% of patients have matched siblings25% chance of inheriting the same haplotypesDonor registries established for HLA-matchedFindin