HLA and Transplantation Flashcards
What is transplantation?
Transplantation is the act of transferring cells, tissues, or organs from one site to another.
Types of transplants
- Autograft
-> Tissue is derived from ‘self’, can be transplanted back to the same place or another site
-> Collection -> processing -> cryopreservation -> reinfusion - Isograft
-> Between genetically identical twins - Allograft
-> Tissue transferred from one individual to another (genetically non-identical, same species)
-> By far the most common type of transplant and includes: kidney, heart, pancreas, lung, liver, bowel, islet, bone, cornea, skin, tendon, cartilage, stem cell - Xenograft
-> Tissue transferred from one species to another (heart valves)
-> Whole organ xenografting limited by potential for hyperacute rejection
Immunological privileged sites
- Sites where grafts are not rejected (cornea, brain, eyes, testis and foetus)
- Successful transplant of corneal allograft from cadaveric donor - requires no assessment of HLA type and no administration of immunosuppressive drugs
- Lack of rejection is due to naturally immune-suppressive environment in anterior chamber of eye and lack of blood vessels in the cornea
- HLA is a major barrier to overcome in transplant
Human leukocyte antigens (HLA)
- 6 classical HLA loci: Class I (A, B, C) and class II (DR, DQ, DP) each encoded by separate genes
- These molecules allow tissues to be recognised as ‘self’ or ‘non-self’ by the host immune system and therefore, determines histocompatibility
HLA role and expression
- The primary function of these antigens is to serve as recognition molecules in the initiation of an immune response so they are very polymorphic
- HLA antigens present peptides from foreign substances to effector cells of the immune system (mainly T-cells)
- HLA Class I are expressed on nearly all cells and recognise pathogens that reside inside the cells (viruses)
- HLA Class II are only found in immune cells and recognise pathogens that reside outside the cell (bacteria)
- MHC Class I - All nucleated cells - A, B and C
- MHC Class II - Antigen-presenting cells - DR, DQ and DP
HLA nomenclature
- HLA-A defines the locus
- HLA-A24 shows the serologically defined antigen
- HLA-A*24 asterik denotes that the allele has been defined by molecular methods (low resolution)
- HLA-A*24:01 shows higher resolution, specific allele required for HSCT
HLA typing: serology
- All partial organ recipients and donors must have their major HLA loci determined to minimise the chance of rejection
- Also applies to bone marrow transplantation
- Terasaki trays are used which are plates with serum containing anti-HLA antibodies, patient cells and complement added, death occurs in wells where antibody reacts with patient sample
- Dyes show live cells (green) and dead cells (red)
HLA typing: molecular methods
- All molecular methods require the extraction of high quality genomic DNA
- In NHS laboratories this is achieved using a semi-automated system that extracts genomic DNA from whole blood
- Also possible to isolate DNA from buccal swabs, saliva samples and fingernails
- Sequence specific primer (SSP) PCR is often the first step in determining the HLA type
- SSP tests consist of multiple different PCR primers specific for known HLA polymorphisms
- Specific amplicons are produced if the primers are complementary to the DNA sample
HLA tissue typing: B27 and B57
- Individuals who are HLA-B27 have an increased risk for ankylosing spondylitis and other inflammatory disorders
- Suspected AS cases will be screened for HLA-B27 as 95% of AS sufferers are B27 positive
- HLA-B57 is associated with drug-induced inflammatory disorder
- All HIV positive patients in the UK are screened for HLA-B57 prior to beginning Abacavir (reverse transcriptase inhibitor) treatment
Anti-HLA antibody identification
- Recipients may have antibodies to antigens expressed on donor cells and this is a major risk factor for hyper-acute rejection
- They may arise from pregnancy, blood transfusion or previous transplantation
- CDC (Cytokine Dependent Cytotoxicity) cross match assays have been used successfully for several years, recipient sera is incubated with donor lymphocytes in presence of complement
Sources of donors
- Living
-> Family members/friends (subject to ABO/HLA compatibility)
-> Altruistic donation
-> Paired/pooled donations - Cadaveric
-> Those who are deemed to be ‘brain stem dead’ following testing
-> Donation after circulatory death (DCD) or non-heartbeating donors, usually occurs after admittance to A&E
Issues around consent
- 90% of the UK population support organ donation
- In practice, only 68% provide consent in the event of a family member being declared brain dead, possibly due to shock
- Opt-in and opt-out system
- Even with ‘deemed consent’, medics allow family input
Transplant allocation
- For kidney and pancreas, allocation is based on blood group match and HLA-A, B and DR, 000 mismatch are given priority
- Paediatric patients are always prioritised, then sensitisation, waiting time, age match and location are also considered
- These factors should prevent a patient waiting for many years, and older organs being given to younger patients and also reduce ischaemic time
- One HLA-A mismatch is considered, as is one HLA-B mismatch
- For heart and lung, the main factors are ABO match and HLA-DR
- There are far fewer of these transplants and short CIT is essential
- The size of the heart in relation to the donor is also important
Pre-transplant crossmatch
- For all transplants, a crossmatch is always performed immediately prior to surgery
- Recipients are screened prior to entry on the waiting list, but their antibody status may have changed (hyper-acute risk)
- For heart and lung, logistics may prevent the pre-transplant crossmatch, so a virtual crossmatch is performed instead
Graft rejection
- Can be hyper-acute (should never happen)
- Acute (sometimes happens despite best medical care)
- Chronic (always happens to some degree with a solid organ)
Hyper-acute rejection
- Mediated by the humoral response
- Usually seen within minutes of transplantation
- Results from pre-existing donor-specific antibodies in the recipient match or accidental ABO mismatch
- Antibodies activate the complement pathway, initiating the blood clotting cascade
Acute rejection
- Caused by activation of T-lymphocytes and occurs over several days
- Mismatched HLA causes cytotoxic T-cell attack of endothelial cells
- The recipient can also form de novo DSAs, causing antibody-dependent, cell-mediated cytotoxicity
- These processes can be avoided through HLA matching and immunosuppressants
Chronic rejection
- Multiple immune mechanisms, cell-mediated and humoral
- End result is vascular disease
- Alloantibodies bind to endothelial cells and recruit Fc-receptor bearing monocytes
- Inflammatory components in the vessel wall leads to damage, thickening of the vessel and narrowing of lumen, inadequate blood supply and damage
- Can involve lymphocytes, phagocytes, antibodies, complements
Immunosuppressive drugs
- Allogeneic transplantation requires some degree of immunosuppression, by drugs, if the transplant is to survive
- Medics aim to find a balance between reducing rejection whilst maintaining a reasonable immune system
Barriers to xenografting
Species - advantages - disadvantages
- Cow, horse, sheep - ? - Dis-concordant, large size
- Primate - Concordant - Endangered, ethical concerns, retrovirus
- Monkey - Concordant - Small size, retrovirus
- Dog - Size - Dis-concordant, ethical concerns
- Pig - Partly concordant - Require genetic engineering
- Genetic modification must be performed to avoid hyper-acute, acute and chronic rejection
- The key genetic modifications to date are:
-> Knock-out of porcine carbohydrate genes (GAL)
-> Induced expression of human complement and coagulation regulatory molecules
Donation after circulatory death (DCD)
- DCD (non-heartbeating donation) is the retrieval of organs from patients whose death is diagnosed and confirmed using cardio-respiratory criteria
- There are two types:
1. Controlled: takes place after death which follows the planned withdrawal of life
2. Uncontrolled: refers to organ retrieval after a cardiac arrest is unexpected and from which the patient cannot or should not be resuscitated - DCD has significantly increased in the UK and now represents about 40% of all deceased organs
Islet transplantation
Procedure
- Donor pancreas
- Ricordi Chamber: key islet isolation device
- Separated islets
- Islets are introduced into the liver
- Transplanted islets secreting insulin in the liver
Pros and cons
Advantages
- Can remove the need for insulin infection
- Prevents hypos
- Improves overnight controls
- Surgery is easy, quick and minimally invasive
Limitations
- Only 2% of the donor pancreas is islet material
- Repeated transplants are often necessary
- Shortage of donors
- Immunosuppression is required which leads to side effects
Advances in pancreatic islet transplantation. Sites for the treatment of diabetes.
- Anterior chamber of the eye
-> Vascularised
-> Innervated
-> Immune-privileged - Subcutaneous space
-> Ease of access and monitoring
-> Not immune privileged
-Intra-hepatic infusion
-> Clinically approved in humans
-> Hepatic microenvironment is not ideal
-Spleen
-> Highly vascularised
-> Drains into the hepatic portal vein - Epidydimal fat pad
-> Highly vascularised
-> Potentially immune privileged - Kidney capsule
-> Standard for mouse islet transplantation
-> Limited clinical translation - Bone marrow
-> Highly vascularised
-> Requires pre-conditioning for the site
Induced pluripotent stem cells (iPSC’s)… the future?
- Generation of autologous iPSCs
- Interspecies blastocyst complementation
- Removal of pancreas and isolation of islets
- Transplantation of islets into diabetic mice:
- Authors generated mouse-rat chimeric islets then transplanted them into a chemically-induced diabetic mouse model
- The mouse were normoglycaemic for over a year with no immunosuppression
Bone marrow transplantation
- Bone marrow transplant is the transfer of bone marrow cells from one human to another
- Bone marrow transplantation can be used to treat:
-> Leukaemia
-> Immunodeficiency/anaemias
Haematopoietic stem cells
- HSc can self-replicate and differentiate into any of the formed blood elements
- HSc can be harvested from cultures derived from bone marrow cells and are re-infused to reconstitute damaged bone marrow
- HSc is also found in small numbers in the peripheral blood
Sources of stem cells for transplant
- Patient - autologous
- Matched sibling - only 25% chance of a match, but outcomes are less favourable than a sibling donor
- Umbilical cord blood - restricted availability
Autologous HSCT
- Autologous: the transplanted stem cells have been previously taken from the patient
-> Bone marrow or peripheral blood stem cells
-> Less complicated - MHC compatible
-> Allows for high dose chemo and radiotherapy pre-transplantation which will irreversibly damage the bone marrow
Procedure
- Mobilise stem cells using growth factor and chemotherapy
- Stem cells collected from peripheral blood
- Stem cells frozen until required
- Conditioning chemotherapy to suppress the immune system
- Stem cells thawed and re-infused
- Support with blood products and antibiotics (approximately 2 weeks)
- Further follow-up as an outpatient for approximately 2-3 months with regular blood tests and medication
Allogeneic HSCT
- Recipient: Anti-cancer drug and radiation
- Donor: Healthy bone marrow cells removed
- Recipient: Healthy bone marrow cells infected
Cord blood vs. bone marrow
Cord blood
- Collection is non-invasive, painless and poses no risk to the donor
- Graft versus host disease (GVHD) is reduced to 10% due to the absence of antibodies in the stem cells
- Units are processed and ready for transplantation
- Significantly less expensive
Bone marrow
- Collection is invasive and painful. It must be performed in a hospital surgical setting
- Due to the maturity of the stem cells, it requires a greater HLA match to perform a transplant
- Serious GVHD occurs in 60% of all unrelated bone marrow transplants
- Bone marrow is dependent on donor participation
Graft versus host disease (GVHD)
Graft: section of transplanted or donated tissue
Host: tissues of person receiving the transplant
- GVHD occurs when immune cells from the donor (largely T-cells) attach to the recipients tissues
- GVHD is reduced by HLA matching as highly as possible
Graft vs leukaemia effect
- Occurs when immune cells from the donor (largely T-cells) eliminate residual leukaemia cells in the recipient
- GVHD and GVL often occur simultaneously
Post-transplant chimerism analysis
- The level of engraftment post-transplant must be measured - gives an indication of outcome
- For malignancies, 100% donor engraftment is required to prevent relapse
- For anaemias and enzyme deficiencies, mixed chimerism may be enough to correct the disorder
- Chimerism is often measured through single tandem repeat (STR) analysis
