transplantation immunology

week 9 immunology

cornerstones of organ transplantation

• vascular anastomosis

• short-term hypothermic organ preservation

• inhibition of immune rejection

  • concept of acqquired immunologic tolerance

  • hyperacute, accelerated, acute and chronic rejection

medical and immunological considerations in transplantation immunology

• acute and chronic graft rejection

• immune signals

• sensing of danger and stranger molecule

• ischaemia and reperfusion

• recognition of non self antigens

• polymorphism of MHC genes

• adaptive memory immunity and innate trained immunity

graft classification

• autograft

• allograft

• isograft

• xenograft

autograft

• self-tissue transferred from one site to another in the same individual from one site to another in the same individual (usually accepted)

• tissue transferred between different individuals in the same species: usually rejected unless given immunosuppressive therapy

allograft

• tissue transferre between genetically different individuals in same species: usually rejected unless given immunosuppressive therapy

isograft

• tissue transferred between genetically identical individuals: usually successful

xenograft

• tissue transferred between different species

  • hyperacute rejection

• better availability, easier to breed if animal organs are used

cardiac xenotransplantation

• CRISPR

• 2022

• survived for 2 months after transplant

• possible problem:

  • organ rejection

  • anti-pig ABs may have attacked heart

  • pig virus

  • pt may have been too sick

graft acceptance and rejection depends on 2 factors

1. genetic relationship determines if graft is accepted

2. whether graft consists of isolated cells or tissue or an intact organ

   • autografts have low failure rate

   • cornea transplants common as anyone can donate them and they are privileged sites (not exposed to immune system)

autograft acceptance (skin graft)

• day 1: grafted epidermis, blood vessels

• days 3-7: revascularisation

• days 7-10: healing

• days 12-14: resolution

allograft rejection

• rate of allograft rejection varies according to the tissue involved (skin grafts are rejected faster than other tissues)

• first set rejection:

  • days 3-7: revascularisation

  • days 7-10: cellular infiltration

  • days 10-14: thrombosis and necrosis

• second set rejection:

  • days 3-4: cellular infiltration

  • days 5-6: thrombosis and necrosis: immunologic memory

first set rejection: skin graft

day 1: grafted epidermis

days 3-7: revascularisation

days 7-10: cellular infiltration

days 10-14: thrombosis and necrosis

second set rejection: skin graft

• day 1: grafted epidermis

• days 3-4: cellualr infiltration

• days 5-6: thrombosis and necrosis

graft rejection displays…

immunological memory

corneal allograft rejection

• 3 years status post penetrating keratoplasty

• graft rejection mat transpire despite prophylactic efforts

skin graft rejection is the result of T cell mediated anti-graft response

• grafts differing at the MHC are rejected at 10-13 days after grafting

• naive mice that are given T cells from a sensitised donor behave as if they had already been grafted

immunological mechanism of a graft rejection

• adaptive immune responses to a graft’s foreign proteins are the major barrier to effective tissue transplantation

• mediated by either CD8+ or CD4+

• antigens that differ between members of the same species are knowna s alloantigens → alloreactive response

• different MHC alleles

major histocompatibility complex (MHC)

• human leukocyte antigen (HLA) system in humans

• histocompatibility antigens are encoded on more than 40 loci

• most vigorous allograft rejection reactions are on MHC

• 3 major class I alleles:

  • HLA-A

  • HLA-B

  • HLA-C

• 3 major class II alleles:

  • HLA-DR

  • HLA-DQ

  • HLA-DP

• polymorphisms in HLA, esp HLA-A, HLA-B and DR loci are most important biological barriers to a successful transplantation

• as a closely HLA-matched graft is less likely to be recognised and rejected, HLA mismatching has a substantial impact on graft survival

major and minor histocompatibility molecules serve as alloantigens in graft rejection

• even complete matching at the MHC doesn’t ensure graft survival

• although synergic grafts are not rejected, MHC identical grafts from donors (that differ at other loci- minor H antigen loci) are rejected

• rejected more slowly than MHC disparate grafts

minor histocompatibility antigens

• if a polymorphic protein differs between the graft donor and recipient, can give rtise to an antigenic peptide

• can be recognised by recipient’s T cell as non self and elicit an immune response

• such antigens are minor H antigens

mechanisms of rejection

• immune response to a transplanted organ consists of both cellular (lymphocyte mediated) and humoral (AB mediated) mechanisms

• T cells are central in graft rejection

• rejection reaction consists of sensitisation stage and effector stage

  • direct mode of recognition

  • indirect mode of recognition

  • recognition by AB

direct and indirect pathways of allorecognition contribute to graft rejection

• direct allorecognition

  • organ grafts carry with them APCs of donor origin/passenger leukocytes

  • APCs leave graft and migrate to secondary lymphoid tissue of recipient where they activate host T cells that bear corresponding TCRs

  • associated with acute rejection

• indirect allorecognition

  • uptake of allogeneic proteins by recipient’s own APCs and their presentation to T cells by self MHC molecules

  • peptides derived from both foreign MHC molecules themselves and minor histocompatibility antigens can be presented by indirect allorecognition

  • ABs are produced against non self antigens from same species and are known as alloABs

ABs in graft rejection

• pre-existing alloABs against blood group antigens and polymorphic MHC antigens can cause rejection of within minutes of transplantation

• ABs react with antigens on vascular endothelial cells of the graft and initiate complement and blood clotting cascades

• vessels fo the graft become blocked, causing its rapid destruction

to reduce graft rejection

• ABO matching

• HLA tissue typing

• cross matching

types of graft rejection

sensitisation stage

• CD4 and CD8 T cells recognise alloantigen expressed on cells of foreign graft (including major and minor histocompatibility alloantigens)

• → Th cell activation by APCs (dendritic cells)

• ‘passenger leukocytes’: population of donor APCs that migrate from graft to lymph node, express allogeneic MHC antigens of the donor graft

• direct vs indirect pathway of allorecognition, each leading to generation of different sets of allospecific T cell clones

effector stage

• hallmark of graft rejection involving cell-mediated reactions is an influx of T cells and macrophages into graft

• recognition of foreign class I alloantigens of the graft by host CD8+ cells can lead to CTL-mediated killing

• in some cases, CD4+ T cells that function as class II MHC-restricted cytotoxic cells mediate graft rejection

• cytokines secreted by Th cells play a central role

effector mechanisms involved in graft rejection

• IL-2, IFN-gamma, TNF-beta are important mediators in graft rejection

• IFN-gamma promotes the influx of macrophages into graft and their subsequent activation into more destructive cells

• TNF-beta has a direct cytotoxic effect on cells of a graft

• during rejection episode, cytokine levels increase

mechanisms of target cell destruction

• direct killing by Tc cells and indirect tissue damage through release of cytokines such as IFNγ and TNF from Th-1

• direct killing by NK cells enhanced by interferon

• attack by AB dependent cellular cytotoxicity (ADCC)

• phagocytosis of target coated with AB (heightened by bound C3b)

• sticking of plts to AB bound to the surface of graft vascular endothelium leading to microthrombi formation

• complement mediated cytotoxicity

• macrophages activated non-specifically by agents such as IFNγ and possibly C3b can be cytotoxic for graft cells, perhaps through extracellular action of TNF and 02 radicals generated at cell surface

graft versus host disease (GvHD)

• T cells recognise alloantigens in recipient tissues

• mature T cells of graft are immunocompetent, reactive cells

• frequently occurs following HSC transplantation (bone marrow, cord blood)

• can often be ameliorated by removing T cells from donor bone marrow before transplantation

GVH pathogenesis

• involves secretion of IL-1β, TNF and IFN-γ from damaged host tissue

• both donor and recipient dendritic cells activate donor Th1 cells to secrete IL-2 and more IFN-γ

• host is attacked by donor CTls and NKs (Fas-FaL perforin/granzyme B pathways) inducing apoptosis

• Treg cells may be harnessed to limit GVH

GvH disease in humans: donor cells react against recipient

• GvHD can affect many different parts of the body

• skin, eyes, stomach and intestines are affected most often

• can range from mild to life threatening

• chronic GVH: has a somewhat good prognosis if ilimited to skina and liver, not if multiple organs are involved

graft-versus leukaemic effect (GVL)

• mild to moderate GVHD can also be beneficial when donor immune cells attack recipient tumour cells that have surivived the aggressive chemo and radiation

• in case of leukaemia: graft versus leukaemia

• though removing donor T cells from graft reduces risk of GVHD, this may not be best approach for marrow transplants used in antineoplastic therapy

foetus is an allograft that is tolerated

• high likelihood that mother and father have different HLA types

• foetus expresses on HLA haplotype of maternal and one of paternal origin

• paternal HLA class I and class II molecules expressed by foetus are alloantigens

• foetus protected from pre-existing alloreactive ABs or T cells

• no sign of immunological rejection if mother has more than one child with same father

foetus as an allograft

• maternal blood with immunocompetent lymphocytes circulate in contact with foetal trophoblast

• foetomaternal tolerance

• trophoblast doesn’t express MHC class I and II

• nutrient depletion reduces T cell responsiveness

• cytokine milieu (TGF-β, IL-10) suppresses development fo effector T cells in favour of iTreg cells

• stromal cells of maternal uterine tissue that directly interacts with placenta represses local expression of key T cell attracting chemokines

blood typing

• blood type O is considered universal donor

• blood type AB is called universal recipient because they can receive an organ or blood from people with any blood type

HLA tissue typing

• phenotypic methods

  • serology (microcytoxicity)

  • tissue typing: mixed lymphocyte reaction

    • phenotypic methods have been phased out and replaced with molecular methods based on DNA analysis

• genotypic methods

  • PCR based techniques for detecting HLA genes

  • PCR-RFLP (restriction fragment length polymorphism)

  • variable number tandem repeat (VNTR) typing

  • short tandem repeat typing

HLA typing by microcytotoxicity assay

• peripheral WBC of donor and recipient are incubated with allele-specific ABs and with complement

• lysis of cells bearing antigen is assessed by addition of specific dyes

HLA typing by mixed lymphocyte reaction

• irradiated stimualtor lymphocytes from donor are incubated with responding T cells from recipient

• proliferation of recipient T cells is measured by uptake into cell DNA and indicates degree of compatibility

crossmatching- complement dependent cytotoxicity

• cell based assay

• can determine presence of donor specific anti-HLA ABs in serum of recipient

• B and T cells are separately tested against serum from recipient

  1. serum from recipient is added to donor lymphocytes (T or B) in presence of complement

  2. negative test: donor specific anti-HLA ABs are absent, no complement activation

  3. positive test: donor specific ABs bind to lymphocytes, complement activationa and cell lysis

crossmatching: flow cytometry

• cell based

• donor lymphocytes are mixed with recipient’s serum

• lymphocytes bind to donor specific ABs and are quantified by flow cytometer

• quantification:

  • measurement of the fluorescence intensity as a ratio of control

  • serial dilutoions of recipient’s serum are made to react with donor lymphocytes and the minimum dilution which yield’s a negative result gives a measurable estimate

virtual crossmatching

• bead technology beads are impregnated with different ratio of 2 fluorochromes resulting in a signal that is unique to specific bead

• serum HLA ABs will react with HLA antigens on bead

• beads are washed and incubated with a second AB

immunosuppression therapy

• induction therapy: immunosuppression is started at the time of transplantation to prevent an immune response against the graft

  • anti-t CELL ABs and/or IL-2 receptor antagonists

• maintenance therapy: transplant recipients usually need to be maintained on immunosuppressive drugs for the rest of their lives

  • calc inhibitors, purine metabolism inhibitors and mTOR inhibitors are used, often together with steroids

• treatment of rejection episodes:

  • humoral rejection can be treated with IV Ig, plasmapheresis and anti CD20 AB

  • variety of immunosuppressive anti-T cell agents are also commonly employed

mode of action of immunosuppressants

• rabbit anti-thymoglobulin and anti-CD52 monoclonal ABs are used to deplete T cells and other leukocytes before transplantation

• anti-CD3 monoclonal AB prevents generation of signalling by T cell receptor complex

• cyclosporin and tacromilus interfere with translocation of nuclear factor of activated T cells to nucleus by inhibiting calcineurin

• CTLA-4-Fc fusion protein belatacept binds B7 and prevents generation of co-stimualtion via CD28

• anti-CD25 AB binds to high affinity IL-2 receptor on partially activated T cells and prevents IL-2 signalling

• sirolumus interferes with activation of the mTOR cascade which is required for differentiation of effector T cells

cyclosporin and rapamycin act at different stages of T cell activation

costimulatory blockage: inducing tolerance

1. T cell activation requires co-stimulatory signals (engagement of CD28 on the T cell surface by the B7 molecules on surface of APC)

2. CTLA-4 binds to CD80-86 with higher affinity than CD28 and therefore soluble CTLA-4-Ig fusion protein blocks these co-stimulatory signals, resulting in T cell anergy

   • monoclonal AB to CD40L on T cell would block co stimulatory signals normally provided by CD40 on the APC

stem cell therapy

• creating an ideal transplant entirely from cells of the recipient would eliminate the need for immumosuppression

• possible to isolate stem cells from various adult organs (incl bone marrow)

application in medicine

• privileged sites

  • transplants at certain anatomical sites are generally accepted without any immune rejection

    • absence of lymphatic drainage is probably critical common factor

  • haemapoietic stem cell transplants (bone marrow transplant)

    • 3 sources of stem cells are used, listed in order of decreasing mature T cell contamination:

      • peripheral blood (enriched by cytokine administration)

      • bone marrow

      • cord blood