Organ transplantation and the hla system lecture

Editor's Notes

• #3: Allograft transplantation is the removal of an organ from one body to another in circumstances where the recipient has an organ specific illnesses or damaged organ where there is no pharmacological therapy. Graft tissue can be sourced from cadavers or living donors depending on the organ required. The most frequently transplanted organ is the kidney for clinical indications such a diabetic nephritis. Graft tissue for a kidney transplant can be sourced from a cadaver or a living donor. Between April 2012 and march 20123, 4,212 transplants were carried out. Nearly a quarter of these were kidney transplants.Acceptance or rejection of a graft is dependent on the recipients immune response. In the same way that the body’s immune cells recognise pathogenic antigens displayed on MHC molecules, the MHC molecules on a foreign organ act as antigens and can provoke an immune reaction. Rejection may be mediated via HLA recognition and stimulation of B cells to produce antibodies targeted against graft tissue, or the interaction of recipient T cells with incompatible donor HLA molecules to produce a vigorous T cell response.
• #4: HLA antigens are glycoprotein complexes encoded for by approximately 200 genes that form The Major Histocompatibility complex on chromosome 6 in humans. The terms HLA and MHC can be used interchangeably.These genes are arranged into 3 clusters encoding 3 different polymorphic genes; the MCH class I alpha-chain genes MHC-A, MHC-B and MHC-C which are expressed on all nucleated cellsMHC class II alpha and beta chain genes which combine to form MHC-DR, MHC-DP and MHC-DQ expressed on Antigen Presenting Cells (APCs) and finally MHC class III genes encoding various soluble components of complement and other cytokines. We will focus on class I and II MHC molecules.
• #6: There is an enormous diversity exhibited by MHC molecules stemming from polymorphism, where multiple alleles exist at and both class I and class II loci. Several hundered allelic variations have been identified but we only express a small fraction of these; two alleles of each HLA antigen one inherited from each parent. The loci lie very close together and as such there is a very low combination frequency. Most individuals inherit one haplotype from each parent which are expressed in a co-dominant fashion.If a mother and father are homozygous at each allele, then their children will have one of two possible genotypes. In this situation the parents will always be able to act as a donor to their children, whereas the children will only be a 50% match. In most populations individuals are heterozygous and no two people have the same two haplotypes. Breeding between two individuals will result in children with four possible genotypes. This therefore means that there is a 25% chance that two siblings will inherit the same haplotype from each parent and will be histocompatible. Since each parent donates one haplotype, Parent to child grafts will always have one haplotype in common. UK statists show that the most common relationship between donor and recipient for kidney transplants is sibling to sibling, followed by child to parent. Only a small fraction of donors are unrelated to the recipient.
• #7: HLA nomenclature differs depending on the typing method used to identify it. Historically,HLA antigens were typed using serological methods and named in order of their discovery, for example A1, A2, etc. As more antigens were discovered, DNA sequencing methods were adopted instead however the HLA type of a donor is still often described in immunological terms, for example: A1, -, B8,39, DR1, 3, in which the individual is homozygous at locus A, heterozygous at locus B for alleles B8 and B39 and heterozygous at locus DR for alleles DR1 and DR3.In this example, the recipient has expresses the allele A24 which is not present on the donor tissue. In order for graft acceptance to occur, it is not necessary for the donor and recipient to have identical alleles but for the recipient to recognise the donor cells as self, despite there being additional antigens on the recipients cells.
• #9: Patients waitlisted for a transplant must undergo multiple tests; antibody screens to detect preformed antibodies towards HLA antigens, ABO blood typing, HLA tissue typing and once a donor has been selected, a final serological cross-match test to detect any donor specific antibodies. Donor and recipient tissues that are antigenically similar are said to be histocompatible. This is a preliminary cytotoxic assay called the Panel Reactive Antibody test that detects anti-HLA pre-sensitized antibodies in the recipient. The purpose of the test is to determine how easy or difficult it will be to find a compatible donor. It is expressed as a percentage that represents the percentage of the population that are unsuitable donors. A panel of 20-50 lymphocyte donors are selected from the donor population to represent the distribution of HLA alleles. The basic method requires the recipient serum to be mixed with the lymphocytes in individual wells along with complement and a vital dye. If the lymphocyte expresses a HLA antigen to which the recipient possesses an antibody, the complement membrane-attack complex forms on the lymphocyte cell membrane, causing the cells to take up the dye. If in a panel of 40 cells, 30 wells show cell death then the PRA is reported as 75%. This means that 75% of the population are not likely to be a suitable donor. PRA will constantly change due to the nature of an ever-changing heterogenous population, so it is impotant that this test is carried out regularly whilst the recipient is on the waiting list
• #10: PCR uses in vitro enzyme catalysed DNA synthesis to create millions of idential copies of DNA. If the base sequence of the DNA regions adjacent to the DNA to be amplified it enables the construction of synthetic oligonucleotide primers. PCR requires the target DNA to be denatured via heat and the primers to hybridize to the complementary single-stranded DNA. The primers are then extended by a heat stable DNA polymerase.A single PCR cycle consists of three steps:1- denaturation of double stranded DNA by heating to 98 degrees2 – the annealing of primers by reducing the temperature3 – extension of primers by DNA polymerase at 72 degrees. This step should last long enough to generate the PCR amplification product. It takes approximately one minute to generate a kilo base pair sequence.Each successive cycle generates an exponentially increasing number of DNA fragments as each newly synthesized strand can be used as a template for the next cycle.PCR products can be visualised by staining them with ethidium bromide and running them on an agarose gel using electrophoresis.Electrophoresis describes the movement of ions in n applied electrical field. Since DNA carries a negative charge, it moves across the gel from the cathode to the anode, with the smallest fragments moving the fastest through the agarose matrix. A water-soluble dye is usually added so the migration of the DNA sample through the gel can be followed visually.
• #12: Tissue typingHLA-proteins have had their sequences described and cloned - this enables clinicans to perform fast and accurate HLA typing using molecular methods such as SSP-PCR.In most PCR-based techniques, the PCR process is used only as an amplification step of DNA followed by a post-amplification step to discriminate between different alleles. SSP-PCR is slightly different. It stands for SEQUENCE SPECIFIC PRIMER POLYMERASE CHAIN REACTION. It allows for the discrimination of different alleles during the PCR process.SSP-PCR requires DNA to be isolated from both donor and recipient. Separately, it is amplified using PCR in multiple wells, each containing a specific primer for a particular HLA allele. When an individual has an allele that matches the primer in the reaction then amplification can occur. The contents of the wells are then run on an Agarose gel, with the amplification product appearing as a distinct band on the gel. Interpretation of the results is based on the presense or absense of an amplification product. Typically, atleast 96 reactions are required to define HLA-A, -B and –DR alleles. As a control against amplification failure, each PCR reaction must also contain a psotive control primer for a highly conserved gene such as Human Growth Hormone. When the products are run on agarose el electrophoresem a the positive control will show as a band at the same posotiion and of the same size in each lane.The fewer the mismatches, the better the chance of graft survival. The ideal scenario is to achieve a 12 antigen match between the donor and the recipient however when a fully HLA compatible donor is not available, transplantation may still be possible. In kidney transplants, the three most important antigens to match are HLA-A, HLA-B and HLA-DR, and the best chance for kidney transplant survival is a 6 antigen match for these loci.
• #14: Donor-recipient cross-matching checks for preformed donor specific antibodies. In general, anti-HLA antibodies belong to the IgG subclass.This assay is similar to that of the %PRA test. Donor lymphocytes are mixed with the recipients serum along with complement and a vital dye. In instances where the dye it taken up by the cell, the donor is declared non-suitable, even if they are HLA compatible. Donor specificpre-formed antibodies are the cause of hyper-acute transplant rejection, where the transplanted organ is rejected almost immediately. This can also be caused by ABO blood type mis-matching. The antibody-antigen complexes activate the recipients complement system, resulting in an influx of neutrophils. The neutrophils cause damage to the endothelial cells leading to inflammation, platelet activation and thrombosis, causing irreversible tissue damage and the graft will need to be removed.