Transplants

Transplants

Why transplant?
1. Cure for organ specific disease
2. Skin
  1. Burns victims
  2. Temporary grafts of non-viable tissue
3. Blood
  1. Transfused from living donor
  2. ABO and Rh matching required
  3. Complications extremely rare
4. Pancreas
  1. From cadaver
  2. Islet cells from organ sufficient
5. Kidney
  1. From live donor or cadaver
  2. ABO and MHC matching useful
  3. Immunosuppression usually required
6. Bone marrow
  1. Needle aspiration from living donor
  2. Implanted by IV injection
  3. ABO and MHC matching required
7. Liver
  1. From cadaver
  2. Surgical implantation complex
  3. Resistant to hyperacute rejection
8. Heart
  1. From brain dead donor
  2. MHC matching useful but often impossible
  3. Risk of coronary artery damage
9. Lung
  1. From brain dead donor
  2. Procedure recently developed
History: skin transplantation
1. Early experimentation had little success
2. Skin grafting was attempted on burnt patients in WW2
3. Skin could be grafted from one part of the body to another
4. Skin could not be grafted from one individual to another unless they were identical twins
5. Athymic children have no lymphocytes so could not make immune responses and could accept any skin graft
Genetic basis of transplant rejection
1. Inbred mouse strains means all genes are identical
2. Transplantation of skin between strains showed that rejection or acceptance was dependent on the genetics of each strain
3. Skin from inbred mouse grafted onto the same strain of mouse is accepted
4. Skin from inbred mouse grafted onto a different strain of mouse is rejected
5. Mouse grafted with different skin, rejected and lymphocytes taken and injected into a new mouse
  1. Same graft transplanted to injected moues is again rejected but much faster: secondary rejection
    1. If injected mouse is grafted with a different skin, primary rejection occurs
6. Rejection response shows all hallmarks of an adaptive immune response
  1. Property of lymphocytes
  2. Specific
  3. Escalating response
  4. Memory
  5. Primary rejection: slow (naive)
  6. Secondary rejection: fast (memory)
7. Isograft: twin to twin
8. Autograft: me to me
9. Allograft: person to person
10. Xenograft: species to species:
Role of T cells in graft rejection
1. CD4 T cells are important for rejecion
2. Anti CD4/CD8 is used to determine levels after grafting
3. A mouse with no CD4 cells (nude mouse) will tolerate a skin graft
  1. A mouse with CD4 T cells will undergo actue skin rejection after a skin graft
4. CD8 T cells will tolerate the graft only if they are sensitised by CD4 T cells
Transfusion vs. Transplantation
1. Transfusion
  1. Transfer of blood
  2. Ab mediated reactions
2. Transplantation
  1. Transfer of tissue or organ
  2. T cell mediated reactions
  3. Transplantation antigens
    1. ABO: limited polymorphism
    2. MHC: high polymorphism
      1. Recipient immune system destroys MHC in graft
    3. non-MHC antigens: limited polymorphism
    4. Xenoantigens: high polymorphism
    5. Alloantigens: molecules that are recognised as foreign on allografts
    6. Alloreactive: lymphocytes that interact with alloantigens
MHC
1. In mice MHC is called H2
2. Rapid graft rejection between strains segregated with antigen-2 encoded as part of MHC halotype
  1. A set of genes inherited as a unit
3. Inbred mice identical at H2 did not reject skin grafts from each other
  1. MHC genetics in mice are simplified by inbred strains
4. In humans, only monozygous twins have identical MHC
  1. The human population is extensively outbred
  2. MHC genetics in humans is extremely complex
    1. MHC is polygenetic: many genes encode different MHC
    2. MHC is polyalleic: many gene alleles at each locus
    3. MHC is polymorphic: many variations in amino acid sequence
Transplant rejection
1. The failure of a recipient's body to accept transplanted tissue of organ as the result of immunological incompatibiilty
2. Association with inflammation and lymphocyte infiltration
3. Autograft acceptance
  1. Grafted epidermis
    1. Day 3-7: revascularisation
      1. Days 7-10: healing
        Days 11-14: resolution
4. First set rejection
  1. Grafted epidermis
    1. Days 3-7: revascularisation
      1. Days 7-10: cellular infiltration
        Days 11-14: thrombosis and necrosis
5. Second set rejection
  1. Grafted epidermis
    1. Days 3-4: cellular infiltration
      1. Days 5-6: thrombosis and necrosis
6. Hyperacute rejection
  1. Occurs within hours after transplantation
  2. Immediate graft rejection
  3. Primary mechanism: humoral mediated rejection
    1. Preformed antibodies from previous transplants or multiple pregnancies
    2. Caused by transplanting organ with incompatible blood type
  4. Prevented by selecting donors with compatible blood types
  5. Outcome is irreversible and untreatable
    1. Transplanted organ must be removed
  6. Example: kidney graft
    1. Pre-existing antibodies are carried to graft
    2. Antibodies bind to antigens of renal capillaries and activate complement
    3. Complement split products attract neutrophils which release lytic enzymes
    4. Neutrophil lytic enzymes destroy endothelial cells; platelets adhere to injured tissue, causing vascular blockage
7. Acute rejection
  1. Occurs within weeks/months after transplant
  2. Class I and II antigens on the cells of the transplanted graft activate cellular mediated rejectionn
  3. Treatable and reversible
  4. Example: skin graft
    1. Skin graft with Langerhans cells
    2. Langerhans cells migrate to local lymph node where they activate effector cells
    3. Effector cells migrate to graft via blood
    4. Graft destroyed by effector cells
    5. Initiation of graft rejection involves migration of donor APC from the graft to the local lymph node
8. Chronic rejection
  1. Develops over months/years
  2. Combination of cellular and humoral
  3. Results in diffuse scarring tissue and stenosis of vasculature of organ
  4. Untreatable and eventually leads to graft loss
9. Problems with pre-existing antibodies
  1. Allotransplants
    1. Natural antibodies to ABO blood group antigens
    2. Anti-MHC antibodies raised during previous transfusion, transplant or pregnancy
    3. Solution: test recipient serum for ABO compatibiilty and negative crossmatch
  2. Xenotransplants
    1. Natural antibodies to Gala1-3Gal epitope present in non-primate mammals
    2. Solution: agalactosyl transferase knockout pig
  3. Sensitisation
    1. 'Passenger' leukocytes drain out of the graft and into the recipient lymph nodes
      1. Recipient CD4 lymphocytes recognise MHC II
  4. Effector
    1. Allospecific T cells differentiate into mature helper and cytotoxic T lymphocytes
      1. Alloreactive effector cells migrate back to the graft: MHC disparate graft is destroyed
  5. CD8 T cells lyse endothelial cells
    1. CD4 T cells can recruit and activate macrophages-graft injury by a delayed type hypersensitivity reponse
      1. Antibodies activate complement and injure graft vasculature
10. Recognition of alloanigens in grafted organs
  1. Direct recognition: donor APCs migrate to local lymph node and stimulate alloreactive recipient T cells
    1. Donor MHC/peptide complexes are directly recognised by recipient TCR
  2. Indirect recognition: recipient APCs process and present peptides derived from graft
    1. Fragments of donor cells can be processed and presented by recipient APC and presented to T cells
11. Allorecognition
  1. Sequences of donor MHC II molecules are frequently found in self MHC peptide grooves
  2. This is thought to play a role in the later stages of the rejection process (chronic rejection)
Minor antigen incompatibility
1. Complete MHC matching does not ensure graft survival
2. Responses to minor antigens are much less potent than responses to MHC because the frequency of the responding T cells is much lower
Strength of the response
1. MHC II differences
  1. High strength
  2. Present on APCs and present peptides to CD4 T cells
2. MHC I differences
  1. Mid strength
  2. Present on all nucleated cells and highly polymorphic
3. Minor Histocompatibility complex antigens
  1. Low strength
  2. Minor polymorphisms
Alloreactive T cells
1. High precursor frequency
2. High determinant density
  1. To activate antigen specific T cells- 10-100 MHC molecules are needed to present antigenic peptide
  2. All foreign MHCs can act as ligands for the alloreactive TCR meaning there are more ligands for TCR
  3. High concentration of ligand could stimulate a broader range of T cells with lower affinity
3. Multiple binary complexes
  1. Donor allogenic MHC bind different spectrum of cellular peptides
  2. Foreign MHC + self peptide could resemble self MHC and foreign peptide
  3. Numerous different clones are activated by the allogenic MHC/peptide complexes
4. Foetus is a natural allograft tolerated by the mother
  1. Trophoblast cells of the placenta lack expression of MHC molecules
  2. Secretion of TH2 inducing cytokines
Tissue typing
1. Differences in MHC antigens are responsible for most intense acute graft rejection
2. Screens recipients and donors for their MHC type
3. Aim: to match donor to recipient
4. Serological techniques
  1. Microtoxicity test
    1. Cells from recipients and potential donors are tested against a series of different antibodies anti MHC I and II in the presence of complement
    2. Cytotoxicity is assessed as uptake of dye by the lysed cells
  2. MHC typing
    1. Anti-MHC antibodies attach to MHCs on lymphocyte
      1. Complement and trypan blue dye added
        Cell damaged by complement takes up dye
  3. Cytotoxic cross match
    1. Presence of anti-donor antibodies is detected by the ability of the recipient serum to lyse donor cells
    2. Cannot distinguish between MHC I and II antibodies
    3. Cannot distinguish between IgM and IgG
  4. Flow cytometric cross match
    1. Presence of anti-donor MHC antibodies is detected by the ability of recipient serum to bind to donor cells
    2. Very sensitive, rapid, specific technique
  5. Mixed lymphocyte reaction (MLR)
    1. In vitro model of direct T cell recognition of allogenic MHC
    2. Predictive test of cell mediated graft rejection
    3. Donor cells irradiated
      1. If recipient cells lack MHC II sharing with donor then recipient cells will be activated and proliferated
        Radioactivity of donors will be incorporated into cell nuclear DNA
        Graft will be rejected
    4. Time consuming
5. Molecular technqiues
  1. Restriction fragment length polymorphism
    1. Cleave DNA with restriction enzymes
      1. Separate fragments on agarose gel
        Probe with labelled cDNA
        PCR
  2. Sequence specific oligonucleotide typing (SSO)
    1. Amplify group of alleles
    2. Sequence specific oligonucleotide probes used to detect polymorphic sequences in the amplified DNA
  3. Advantages
    1. Accuracy
    2. Cell type, viability, surface expression are unimportant
    3. DNA probes are easier to make than continuous screening for allo-antisera
    4. Easy to assay large batches
    5. Reproducible
6. Test for MHC antigens
  1. Serological detection
    1. Measures difference between donor and recipient antigens
    2. Monoclonal antibodies used for defining MHC antigens
  2. Dectection of transplantation antigens by mixed leukocyte reaction
    1. Leukocytes from donor and recipient are cultured together for several days
    2. See if recipient lymphocytes will react against donor MHC antigens
    3. Reaction intensity depends on degree of MHC differences
    4. Lengthy procedure
  3. Genotyping of transplantation epitopes
    1. Type epitopes on MHC molecules rather than entire molecule
    2. Typing on genomic level
      1. Detects differences between amino acids
    3. More accurate than serological
Transplant promises
1. Improvement in quality of life
2. Highly successful surgical treatment
Bone marrow transplant
1. Provides a functional immune system
  1. Individuals with SCID
2. Replaces a defective haemopoeitic system
  1. Cure patients with life threatening disorders such as thalaseemia
3. Restoring haemopoeitic system of cancer patients
  1. Chemotherapy can destroy system
4. 10% donor bone marrow is enough to restore system
5. Haemopoietic stem cells find their own way to bone marrow after IV injection
Immunocompromised host
1. Immunocompetent lymphoid cells are transplanted in an immunological incompetent host
2. Host appears foreign to the graft
3. Pre treatment with chemotherapy
  1. Eliminates malignancy
  2. Provides immune supression to prevent rejection of new stem cells
  3. Creates space for new stem cells
4. Conditioning
  1. Total body irradiation or chemotherapy can cause extensive damage to host tissue
    1. Allows translocation of microbial products
      1. Stimulates secretion of pro-inflammatory cytokines
        Activated macrophages produce chemokines that activate neutrophils which increase inflammation
        Increases expression of MHC and adhesion molecule on host, enhancing their antigen presenting capacity
5. Induction
  1. Activation of donor T cells
    1. Drain GVHD target organs
      1. IFN production
        MHC on APC and antigen presentation
        CD8/CD4 expression and NK cells
      2. Symptoms of GVHD
        Pruritic rash often on palms, soles and ears progressing to total body erythroderma
        Gastrointestinal symptoms: anorexia, nausea, diarrhoea and abdominal pains, liver dysfunction and selective epithial damage or target organs
        Clinical result: severe immunodeficiency and immunocompetence

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