1、HLA TYPING&ORGAN TRANSPLANTATION Scott Bainbridge Elena Crouson Israfiel MohammedSarah TuckerOrgan 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 AntigensStatistics on Organ Transplant
2、ation 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 waiting for a Kidney,17,000 waiting for a liver and 3,000 waiting for e
3、ither a heart or liver transplant.First Organ Transplantation In 1959,Joseph Murray and his colleagues in Boston successfully transplanted a Kidney that were donated by fraternal twins and it functioned for 20 years without immunosuppression drugs.First successful Liver transplant-In Denver on 7/23/
4、1967 First successful Heart transplant-In Cape Town,South Africa on 1/2/68 First successful Bone Marrow transplant-Minneapolis,MN on 8/25/68Why Is it Difficult?Organ transplantation is difficult because of the HLA antigens located on the cell surface.Human Leukocyte Antigen(HLA)also referred to as M
5、ajor Histocompatibility Complex(MHC)plays a role in intercellular recognition and discrimination between self and non-self.Location of HLA/MHC The 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 o
6、f MHC molecule.Types of MHC There are three classes of MHC molecules.Class I-encodes glycoproteins 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-cells Class II-encodes glycoproteins expressed primarily
7、on antigen-presenting cells,examples:macrophages,dendritic cells and B-cells,where they are present 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 inv
8、olved in inflammation.Different HLA Alleles Class I-HLA A 451 alleles HLA B 782 alleles HLA C 238 alleles Class II-HLA DR 525 alleles HLA DQ 105 alleles HLA DP 147 alleles HLA DM 11 alleles HLA DO 21 allelesRequirements for Transplant Each transplant center has different requirements for allele matc
9、hes.For the National Marrow 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 Therefor
10、e,it is required that 4 of the 6 antigens match for cord blood donation and 5 of the 6 antigens for adult donation.Requirements for TransplantNational Marrow Donor Program:Chance of a Match Mother/Father:25%chance of full match One Sibling:25%chance of full match Two Siblings:44%chance of full match
11、 Three siblings:58%chance of full match The chance to find donors may be better for more homogenous racial groups.HLA In Transplantation There are two characteristics of the HLA genes that make them special for organ transplantation:There high degree of polymorphism There strong immune reactions tha
12、t their products can produce in other individuals.HLA Pathways There are two pathways that can occur that cause problems in organ transplantation as a result of HLA antigens.Direct pathway-the alloreactive responses of recipient T-cells to donor APC expressing incompatible antigens.Indirect Pathway-
13、allogeneic HLA antigens are taken up and processed by recipient APC and presented in context with autologous HLA molecules to recipient T-cells.Problems from HLA antigens Engraftment-immunological rejection of donor hematopoietic cells by recipient T cells that recognize incompatible HLA determinant
14、s.Factors:pregnancy or transfusion HLA mismatching of the donor Using of less intense preparative regimen before transplant Suboptimal post-transplant immunosuppressive therapy Depletion of T lymphocytes from marrow grafts.Class I determinants govern graft acceptance Class II determinants play a rol
15、e in GVHD.Problems from HLA antigens Acute Graft versus Host Disease-immune reaction of mature donor T lymphocytes against HLA determinants of the recipient.The reaction is directed toward normal tissue including skin and gastrointestinal mucosa.HLA matched unrelated Bone Marrow transplant Acute GVH
16、D is 79%vs.35%HLA matched sibling BMT.Matching donor/recipient pairs for molecular typing has been showed to reduce the risk 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 antigensChronic GVHD-is the principle cause
17、 of morbidity and no relapse mortality for patients reaching day 100 after allogeneic transplant.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 f
18、actors.Overview Methods:Histocompatibility test,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 don
19、or/recipient transplant.Histocompatibility 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)HLA antigen typingTwo different methods
20、,serological and DNA sequencingSerological 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 and go to the bottommononuclear cells are less dense,and
21、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.Serological methodThis B cell enriched media is added to a microtiter p
22、late 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 complement is added to each well.If an antigen-antibody complex is present,complement will be activated,a
23、nd 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 method Cells are examined under a phase contrast microscope,and cells that are pink are positive.If 60%
24、or more of the cells are stained they are considered positive for the HLA antigen.DNA typing methodsGranulocytes and lymphocytes are separated 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 add
25、ed to the wells of a microtiter tray.Each well contains oligonucleotide primers complementary to a small segment of only one HLA allele.If the primer can attach,the HLA antigen is present on the cells.DNA typing methodsDNA polymerase and oligonucleotide triphosphates are added to each well and the p
26、late is incubated in a thermal cycler,which multiplies the sequence between the primers(same as PCR)The 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.Antibody screening for anti-HL
27、A antibodiesPurpose:to detect antibodies in the recipients serum 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-HLA antibodiesLeukocytes(neutrophils,monocytes,basophils,lymphocytes)are harveste
28、d from the blood of donors with 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-HLA ant
29、ibodiesIf 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.The higher
30、the number of different HLA antibodies the lower the probability of finding a match.Crossmatch testPurpose:to detect presence of preformed antibodies in recipient that are reactive against donor tissues.Crossmatch testPeripheral blood lymphocytes from the donor are separated into B and T lymphocyte
31、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 with monoclonal antibodies for T-cells.Crossmatch test B-cell crossmatch is per
32、formed using the same method as HLA typing T-cell crossmatch is performed using the same method as screening test Why do a crossmatch when screening seems sufficient?Antibodies against low-incidence antigens are likely to be missed.Acts as a mock transplantResultsPapers Results Focus on Four Aspects
33、 of Experiment HLA Typing serological methods for HLA Class I(A,B,C)alleles and using DNA methods for Class II alleles(DRB-1,DQB-1).Graft Failure Used statistical methods to determine percentage of graft failures based on types of mismatches present Acute Graft vs.Host Disease(GVHD)Also used statist
34、ical methods to study this.Survival Over the course of 8 years studied survival rates in patientsHLA Matching HLA Matching Tested 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 loci Of the transplant
35、s,83%were done using a Caucasian donor to Caucasian patient.85%of transplants had donor and recipient of same race.Graft Failure Graft 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 c
36、lass II mismatches present,graft failure is highSource:Blood,Vol.92,Issue 10,3515-3520,November 15,1998 Acute 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 GVHD What does the previou
37、s slide show?Risk of having grades III-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.Surv
38、ivalMultiple Class I Mismatches Lower 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 Mismatches Of seven patients with multiple mismatches,three died within 100 days-Other Four lived 3-8 years.Overall Messag
39、e Having 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.Survival(Cont.)What Do the Results Mean?Results ExplainedMismatches in certain classes of alleles resulted in either graft failure(Class I alleles)or acute GVHD(C
40、lass 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.To understand the differences between the loci in class I alleles will have to
41、 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 slightly Beneficial to certain ethnic groups which finding alleles that match completely might be difficult.Why the majority for this experiment
42、 were CaucasianThings to consider For this experiment,the researched focused on individuals with chronic myeloid leukemia.Ideal transplantation would be one where time between diagnosis and transplant is minimum.The results are not representative of what it could happen to patients with other types
43、of leukemia.Outcomes After Unrelated-Donor TransplantUnrelated marrow transplant Transplantation survival is increased by matching Class I and Class II alleles Multiple Class I disparities in the donor increase the risk of graft failure Multiple Class II disparities in the recipient increase the ris
44、k of GVHD Single disparities did not appear to compromise survivalBiological functions of Class I and Class II moleculesClass I Present peptides derived from endogenously synthesized proteins Responding T cells express CD8+Complex interactions with NK cellshttp:/ functions of Class I and Class II mo
45、leculesClass II Present peptides derived from exogenously synthesized proteins Responding T cells express CD4+http:/ DiseaseComplication of bone marrow transplants in which T cells from donor attack the hosts tissues.Acute GVHD and chronic GVHDGraft FailureAntigen-presenting cell trigger CD4 and CD8
46、 cellsLocal and systemic immune response developCytokine recruitment and activation of specific T cells,NK cells macrophage-mediated cytotoxicityAllograft destructionhttp:/cnserver0.nkf.med.ualberta.ca/cn/Schrier/Volume5/ch9/ADK5-09_1-3.pdfChimerism Implanted organ allografts become mixtures of dono
47、r and recipient cells.1968,karyotyping of livers transplanted to females from male cadavers Most livers remain male Kupffer cells were replaced with recipient female cellsLimitations on HLA-matching Under 40%of patients have matched siblings 25%chance of inheriting the same haplotypes Donor registri
48、es established for HLA-matched Finding a matched unrelated donor depends on HLA diversity which varies with race Chances decrease 60-70%in Caucasians,under 10%in ethnic minoritiesLimitations cont Time between registration and identification of donor Disease progression in patients Age Increase in mo
49、rbidity and mortality DNA-based matching Reduces the odds of finding a suitable matched donorRacial Distributions Data from the 8th International Histocompatibility Workshop Japanese registry would need 50,000 donors to provide the average Japanese patient an 80%chance to find at least one donor 1,0
50、00,000 for European patients 400,000 for North American CaucasiansMolecular Typing Methods Sequence Specific Primers(SSP)Sequence Specific Oligonucleotide Probe(SSOP)Analysis of allelic polymorphism at the DNA level Analyze Class II micropolymorphism down to a single a.a Sequence-Based Typing(SBT).p
