Describe and discuss the categories ofsolid organ allograft rejection, and the means by which they may be limitedFor patientswith end stage organ failure the gold standard of treatment is anallotransplant of the failing organ. Kidneys, pancreas, heart, lung, liver andintestines are all routinely transplanted. Restrictions to solid-organtransplant include a lack of suitable organs available, and the risk ofrejection of the transplanted organ. An immunological basis for allograftrejection was first proposed in the 1930s by Gorer gorer. Medewar furthercemented the idea by showing that rejection of skin grafts displayedspecificity and memory for the donor tissue with second allografts from thesame donor being rejected faster than the previous allograft.
Further work in the 1960s There are threemain classes of antigens that are involved in initiating the immune responsethat leads to rejection. Those are the major histocompatibility complex (MHC)antigens, the minor histocompatibility antigens and blood group antigens. While MHC antigens (human leukocyte antigens,HLA) are considered the primary transplantation antigens, blood group antigensare the first consideration for any transplant. This essay will focus on theimpact MHC antigens have on solid organ allograft rejection. Rejection ClassificationRejection occursdue to an immune response being mounted against the transplanted organ. Thereare three types of transplant; autografts, allografts and xenografts (1,2). Autografts describe thetransplant of tissue or organs from one part of the body to another (1). As autografts are recognisedby the immune system as ‘self’, i.
e. not foreign, there is no risk ofrejection. Allografts are defined as transplants of tissues or organs betweentwo individuals of the same species. Allograft rejection is defined as animmune response mounted by a recipient against donor antigens that result in allograftloss. Rejection of allografts can be classified as hyperacute, accelerated, acuteand chronic (1,2).
Classification criteria arebased upon histopathology and time of rejection post-transplant rather than byrejection mechanism. Hyperacuterejection can occur within minutes or up to 24 hours post-transplant, with thecausative agent being the presence of pre-existing antibodies against HLA. Clinically,hyperacute rejection is characterised by endothelial cell injury, plateletmargination, complement activation and thrombosis within allograft vasculaturefollowing anastomosis Gautreaux.
The transplanted organ will sufferirreparable ischemic damage following hyperacute rejection. While there is noeffective treatment for hyperacute rejection, with appropriatepre-transplantation testing hyperacute rejection is now extremely rare,particularly in kidney transplants. Accelerated vascular rejection is similarto hyperacute rejection in that it is aggressive, mediated by pre-existing antibodies(albeit at a lower circulating titre than those found in hyperacute), and hasno standard therapy. The timeline for accelerated rejection is within two weeksof transplant. Acute rejectionis defined as allograft failure within 6 months of transplantation and can beeither T-cell (cellular rejection) or antibody mediated (AMR). The clinicalcharacteristic of acute rejection is necrosis of allograft endothelial cellsGautreaux.
While hyperacute rejection is shown histologically via vasculaturethrombosis, acute rejection more typically presents with vasculitis. Acute cellularrejection (ACR) is the most common form of early onset acute rejection and can appearfrom 5-7 days post-transplant. ACR is mediated by lymphocytes, specifically T-and NK-cells, that infiltrate the allograft parenchyma. Untreated ACR can leadto irreversible histological damage to the transplanted organ through theactivity of complement.
Long-term, continuous ACR may lead to chronicdeterioration that eventually results in chronic rejection. Chronicrejection is the greatest barrier to long-term allograft and patient survivaland is defined as delayed loss of allograft function months or even yearsfollowing transplant. Chronic rejection describes the long-term deteriorationof tissue due to continual immune activation against the allograft. In cardiac and kidney transplants chronic rejectionhistologically manifests as a narrowing of the allograft arteries. Thisarterial narrowing is termed obliterative arteriopathy and results from abuild-up of smooth muscle cells and connective tissue within the vascular lumenGautreaux.
Obliterative arteriopathy isnot such a risk in lung transplants as the lung is not particularlyvascularised. Graft versushost disease (GvHD) is unlike the other forms of rejection in that it is donorimmune cells that recognise the recipient as foreign and mount a response. ForGvHD to occur viable donor lymphocytes must be transplanted alongsidenon-immune cells.
As such GvHD is commonly associated with haematopoietic stemcell transplantation however can also occur in patients who have intestinal orliver transplants. This is because both bowel and liver possess an abundance ofimmune cells that are transplanted into the recipient alongside the requiredorgan. Clinically GvHD presents with a wide range of symptoms affecting skin,liver and intestine Mazariegos. Symptomsmay include skin rash or blistering, GI tract ulceration, liver dysfunction ormouth and tongue lesions Mazariegos.
GvHD following solid organ transplant isassociated with a significant mortality. Studies by Hawksworth et.al. andClouse et.al. showed that intestinal transplant patients had a survival rate of<30% following onset of GvHD Hawksworth/clouse. The study by Clouse et.al.
showed that adult intestinal patients had a higher mortality rate compared withpaediatric patients (83% vs 57%). Mechanisms of rejectionRejectionwas first thought to be solely humoral or antibody mediated however furtherwork proved that cellular immunity also had a role to play. This next sectionwill discuss how the immune system is involved in allograft rejection andoutline the expected stages. Thefirst step in activation of the immune system is trauma to the donor organ. Braindeath in a donor initiates neuroendocrine signalling and hemodynamic responseswhich may activate the innate immune response.
Harvesting and storage of theorgan requires cooling and perfusion with a preservation solution which mayresult in ischaemic injury upon refusion following transplant. Donor organtrauma activates inflammation causing generation of pro-inflammatory cytokinessuch as IL-1, IL-6 and TNF-? Ball. Cellular damage can alsoresult in complement activation and initiate the coagulation cascade.
Complementactivation has been shown to be detrimental to graft outcome, particularly inkidney transplants. Animal studies have suggested that treating donors withcomplement inhibitors can improve kidney and cardiac graft function followingtransplantation Damman, Atkinson. Theinnate response is activated rapidly and non-specifically in response topattern recognition Janeway, Land.
There are two classes of patternrecognition – pathogen-associated molecular patterns (PAMPS) anddamage-associated molecular patterns (DAMPS) Cozzi. PAMPS initiate an innate response when thebody is exposed to pathogens while DAMPS are released following tissue damageand thus are of interest when considering mechanisms of allograft rejection.DAMPS are molecules that are sequestered within cells under normal conditionsbut are released from cells following damage. Examples of DAMPS includeheat-shock proteins, ATP or nuclear proteins Cozzi, Ball. Cells of the innatesystem recognise DAMPS through the presence of pattern recognition receptors(PRRs). PRRs are found both at the cell surface of innate immune cells andwithin their cytoplasm. PRRs include molecules such as C-reactive protein,toll-like receptors (TLRs) and Nod-like receptors (NLRs). Activation of innateimmune cells such as macrophages and dendritic cells (DCs) occurs throughbinding of DAMPS and PRRs.
Macrophages and DCs are antigen presenting cells(APCs) and as such are essential mediators for activating the adaptive immuneresponse. T-cells are thefirst aspect of the adaptive immune system to be activated. The first stage ofT-cell activation following transplantation is antigen presentation. Followingan allograft transplant antigen presentation to T-cells can occur via twodistinct pathways – direct and indirect. In the direct pathway recipientT-cells recognise and bind directly to donor antigen presented by donor APCs. Theindirect pathway involves recipient T-cells recognising donor peptides that arepresented by the recipients’ APCs. For the indirect pathway recipient APCs mustprocess and present peptides from the donor antigen.
Recent work has identified a theoretical thirdpathway that has been termed semi-direct Carty, Menon. In the semi-directpathway donor antigens are physically relocated to the recipients’ APCscellular membrane where they are recognised by recipient T-cells. Thisrelocation is theorised to occur via either cell-cell contact or the releaseand uptake of MHC through exosomes Carty. Figure X shows a schematic representation of these threepathways. Antigenpresentation occurs via MHC. There are two classes of MHC, class I (HLA-A, B,C) and class II (HLA-DP, DQ, DR).
Class I and class II MHC molecules arerecognised by specific T-cell receptors (TCR). MHC class I molecules interactwith CD4 while MHC class II molecules bind to CD8. Antigen presentation via MHCand TCR alone is not enough for T-cell activation and subsequent allograftrejection, an additional two signals are required. Signal two is known ascostimulation and occurs between paired ligands and receptors present on thecell surface of T-cells and APCs. Thereare two sets of costimulatory pairs; the B7 family and the TNF/TNF receptorfamily.
In the B7 family CD28 is expressed by T-cells while APCs expressCD80/86 also known as B7.1/B7.2.
The TNF/TNF receptor family is characterisedby CD40, expressed by APCs, and CD40L (also known as CD154) present on T-cells.The third and final signal is production of IL-2 and IL-2 receptor Janeway.IL-2 is required for clonal proliferation of the activated T-cell.
The three signals required forT-cell activation are summarised in Figure X. T-cell mediatedrejection involves both CD4+ and CD8+ T-cells, which act via distinct pathways.CD8+ T-cells develop into cytotoxic T-cells (CTL). CTL cause graft damage bycausing cell lysis through production of cytotoxic granules containing perforinand granzyme B. These granules lyse the cell membrane and trigger apoptosis.CTL may also initiate apoptosis through the binding of Fas ligand expressed bythe CTL to Fas present on the surface of allograft cells Cozzi. CD4+ T-cellsare often termed helper T-cells and have several categories represented by Th1,Th2, Th17 and Tregs (regulatory T–cells)Cozzi. Th1 and Th17 helper T-cellsproduce pro-inflammatory cytokines while Th2 produce anti-inflammatorycytokines.
Tregs act to try and mitigate allograft rejection by regulatingproduction of pro-inflammatory cytokines by Th1. Cytokines produces by Th1T-cells recruit and activate monocytes and macrophages which prolong theinflammatory response within the allograft. CD4+ Th2 T-cells are also involvedin the activation of the humoral or antibody response. Unlike T-cells,B-cells do not require foreign antigens to be processed prior to recognition.B-cell receptors (BCR) present on the cell surface can identify antigens intheir native configurationIn hyperacuterejection circulating antibodies recognises donor HLA or ABO blood groupantigens expressed on the surface of endothelial cells.
The binding of antibodyto antigen activates the complement system leading to endothelial cell damageand cell lysis. In addition to antibody mediated damage there is anaccumulation of granulocytes and platelets which leads to the formation ofthrombi within the allograft vasculature. The presence of thrombi leads toischaemia and infarction of the allograft.
Acceleratedrejection, like hyperacute, is antibody mediated. Unlike hyperacute rejectionthere is a slight delay in the onset of accelerated rejection. In acceleratedrejection the allograft may function normally for the first few days but thenthere is rapid deterioration in allograft function. There may be two reasonsfor this delay, first any circulating antibodies may be present at a lowertitre than those involved with hyperacute rejection. Secondly, while thetransplanted patient may be sensitised against a specific HLA there may be nocirculating antibodies. Thus, the delay may be due to the immune systemrequiring time to induce memory B-cells to produce the appropriate antibody.
Preventing rejection Since the firstsuccessful solid-organ transplant in 1954, advances in laboratory testing,surgical techniques and immunosuppressive therapies have increased theavailability of solid organ transplantation while decreasing allograftrejection. However, these advances have not completely eradicated allograftrejection, and in the case of immunosuppression introduce new risks. Thefollowing topics discuss methods currently in use to reduce the risk of allograftrejection. HLA typing: As describedabove HLA are considered the primary transplant antigens.
Typing of HLA forboth recipient and donor is therefore an essential step to reduce transplantinga poorly matched organ and thus reducing the potential for rejection. For eachclass I locus (A, B and C) an individual inherits two alleles, one from eachparent. This means that each individual can have a maximum of six class Iantigens present on their cell surfaces. Each class II MHC (DP, DQ, DR)molecule are composed of two antigens, an alpha and a beta chain Janeway. Aswith class I MHC an individual inherits two alleles meaning a maximum 12 classII molecules may be expressed Janeway.
The genes that encode for HLA arehighly polymorphic allowing for an incredible number of alleliccombinations, meaning that the chances of two unrelated individuals having anidentical HLA complement are remote. The greater the number of mismatchesbetween donor and recipient the more likely it is that the allograft will berejected. Historically HLAtyping was performed using serological methods, most commonly themicrolymphocytotoxicity assay. Serology typing has several limitationsincluding the requirement for a large number of pure lymphocytes. This isparticularly true for class II typing as these proteins are only expressed onB-cells, which comprise less than 20% of the total lymphocyte population. Thiscauses difficulties for effective class II typing. To overcome this limitationa large volume of blood is required which in turn has its own limitations. Otherlimitations include the requirement for 100% cell viability, which can beaffected by isolation methods, patient health status, or other medications, andvariability in complement.
Today, the majority of H&I laboratories use molecularbased methods for typing. There are three molecular methods in use; sequencespecific primer (SSP), sequence specific oligonucleotide (SSO) and sequencingbased typing (SBT). Familialallografting: The possibilityof allograft mismatching is reduced if donors and recipients are geneticallyrelated. As described above HLA alleles are inherited in a block known as ahaplotype. Haplotype inheritance gives a50% chance of a complete match between a parent and child while there is a 25%chance of a complete haplotype match occurring between siblings. Even with atransplant between complete HLA matched siblings rejection may still occur dueto differences in minor histocompatibility antigens.
Minor histocompatibility antigens include Antibodyscreening: Individuals candevelop HLA antibodies in response to three events; previous transplant(s) withHLA mismatches, pregnancy and blood transfusion. Antibody development throughthese events is known as sensitisation. Individuals awaiting a transplant arescreened regularly for the presence of HLA antibodies. Antibody screening isspecific for HLA antibodies and is performed for two purposes. The first is to determine if HLA antibodiesare present, and if so what are their specificities. This is an essential stepto determine which donor HLA are unacceptable for potential transplant.
Second,antibody screening provides the information required to determine a patientscalculated reaction frequency (cRF) Gautereaux. Each individual’s cRF is calculated from theunacceptable HLA identified by screening. Unacceptable HLA specificities arecompared with a pool of 10,000 donors with a matching ABO blood group, and thecRF is expressed as a percentage of HLA incompatible donors as calculated fromthe comparison. With accurate antibody screening the cRF reflects the chancesof any sensitised patient receiving a HLA compatible transplant.
It must be kept in mind though that a negativeantibody screening is indicative of a lack of circulating HLA antibodies therefore regular screening is essentialto correctly identify all potential HLA antibodies present. Antibody screeningmay also be performed post-transplant for patients who underwentdesensitisation treatment prior to transplant. As these patients are at a highrisk for AMR, regular monitoring of antibody levels is essential. Crossmatching:Crossmatching isused to determine whether there are circulating HLA antibodies present in therecipients serum that would react with HLA present cells from a potential donorand is performed prior to a transplant to ensure that the donor organ isappropriate for the recipient. There are two common methods used forcrossmatching, complement dependent cytotoxicity (CDC) and flow cytometry. Flowcytometry is considered to be a more sensitive method compared with CDCGautreaux. A negative crossmatch is a positive indication for a transplant togo ahead. Positive crossmatches are generally contraindicative for transplanthowever the type of positive crossmatch can be indicative of clinical outcome.
Poorer clinical outcomes are associated with a positive T-cell crossmatchcompared with a B-cell positive. Smith et.al.report that 1-year allograft survival for cardiac and cardiac-lung transplants isless than 30% in cases where there was a positive T-cell crossmatch Smith. While it may beconsidered best practice to perform crossmatching prior to transplant, cardiacand liver transplants are often performed before crossmatch results areavailable due to cold ischaemia time (CIT) limitations.
A prolonged CIT canincrease the risk of delayed allograft function as well as reducing thelong-term survival of the organ. A study performed by Taylor et.al. in 2000, with a follow up studyperformed 10-years later, showed that use of a virtual crossmatch – where exactknowledge of the HLA antibody status of the recipient and the HLA typing of thedonor are both available – could accurately predict whether a crossmatch wouldbe negative Taylor.
This predictive or virtual crossmatch was used inpatients who had a minimum of 6 months negative antibody screening and who hadhad no recent sensitisation events. Theauthors concluded that a virtual crossmatch could be safely performed inpatients who meet these criteria, reduced CIT and may reduce delayed graftfunction Taylor. Regardless of whether the performed crossmatch is laboratorybased or virtual it is still a crucial element in preventing allograftrejection. Plasmapheresisand Immunoadsorption:An estimated 25%of patients awaiting a solid organ transplant are highly sensitised againstHLA. The presence of HLA antibodies may be contra-indicative for a transplantto go ahead. A highly sensitised individual may have an extended wait of thetransplant list awaiting a suitable organ compared with a non-sensitisedindividual. One potential technique that may reduce their waiting time for amatching organ is antibody depletion via plasmapheresis or immunoadsorption.
Plasmapheresisis a process that removes all blood proteins and is therefore non-specific forremoval of HLA antibodies cozzi. Immunoadsorption is a more tailored methodthat gives broad HLA antibody removal using specific adsorbers Cozzi. Bothplasmapheresis and immunadsorption are used to directly preventantibody-mediated rejection.
Bestresults for plasmapheresis and immunoadsorption are noted in cases with livingdonors as recipients can be treated prior to transplantation, reducing thechances of AMR occurring. Immunosuppression:Immunosuppressivetherapies utilise one of three mechanisms; 1) lymphocyte depletion, 2) blockinglymphocyte response and 3) diversion of lymphocytes. The majority of currentlyavailable immunosuppressive agents are designed to either block lymphocyteresponse or disrupt the cell cycle resulting in lymphocyte depletion. There area wide variety of immunosuppressive drugs including small-molecule drugs,monoclonal and polyclonal antibodies, intravenous immunoglobulin (IVIG) andcorticosteroids. Use of immunosuppressionthat causes lymphocyte depletion reduces the risk of acute rejection, but canincrease the risk of infection and development of post-transplantlymphoproliferative disorder.Immune tolerance:Immunosuppressionis a significant burden both for recipients of a transplant and for thehealthcare provider.
For the transplant patient they face a lifetime drugregime that increases their risk of infection and developing cancer. Forhealthcare providers the burden is financial. Immune tolerance, defined as alack of immune response to specific donor tissues is therefore a future aim forsolid organ transplants (6). Currently the aim is toachieve operational tolerance, where recipients have long-term (more than 1year) allograft survival following transplant with no immunosuppression (6). The immune tolerance network (ITN) have been involvedin several clinical trials Chimerism as amethod for preventing allograft rejection was first demonstrated in 1953 inmice Billingham. Despite use ofthese methods rejection is still a significant risk for any solid organtransplant.
Therefore, there must be effective treatment options available. Treatment options for allograft rejectionWith anincreased understanding of how cellular and antibody rejection occurs there hasbeen development of several therapeutic agents that can target specific facetsof the immune system. As stated previously, due to the rapid onset, there is notreatment available for hyperacute rejection. While there is no standardanti-rejection therapy for accelerated rejection, some successes in preventingallograft rejection have been achieved with plasmapheresis, IVIG,anti-thymoglobulin (ATG) and eculizumab therapies. Immunosuppressive therapies currentlyfocus on disrupting the adaptive immune response involved in acute and chronicrejection. The following section discusses available treatment options and howthey exert their immunosuppressive effect. Targetingcellular rejection:T-cell mediated,or cellular rejection is heavily involved in early onset acute rejection. Themajority of immunosuppressants currently used target one of the three signalsrequired for T-cell activation.
ACR (acutecellular rejection) has been shown to respond to treatment with corticosteroidsKasiske. In cases where corticosteroid treatment is ineffective, or ACR isrecurrent, then treatment with an anti-T-cell antibody such as OKT3(muromonab), ATG or ALG may prevent allograft loss Kasiske. Targetingantibody-mediated rejection:There areseveral treatment options available to treat acute AMR. However, there is, asyet, no successful treatment available for chronic AMR.
Treatment options caninclude suppression of the T-cell response, elimination of circulatingantibodies, suppression or deletion of B cells or blockage of the complementcascade. Table Xsummarises the potential therapies that can mediate these effects. Strategiesfor treating AMR may include using a combination of therapies. AMR Treatment Immunotherapy Suppression of T cell response ATG CNI, MMF Elimination of circulating antibodies Plasmapheresis/Immunadsorption IVIG Splenectomy Suppression/Deletion of B cells Rituximab Bortezimib Belatacept Thymoglobin Complement cascade blockage Ecluzimab