Describe As autografts are recognised by the immune

Describe and discuss the categories of
solid organ allograft rejection, and the means by which they may be limited

For patients
with end stage organ failure the gold standard of treatment is an
allotransplant of the failing organ. Kidneys, pancreas, heart, lung, liver and
intestines are all routinely transplanted. Restrictions to solid-organ
transplant include a lack of suitable organs available, and the risk of
rejection of the transplanted organ. An immunological basis for allograft
rejection was first proposed in the 1930s by Gorer gorer. Medewar further
cemented the idea by showing that rejection of skin grafts displayed
specificity and memory for the donor tissue with second allografts from the
same donor being rejected faster than the previous allograft. Further work in the 1960s

There are three
main classes of antigens that are involved in initiating the immune response
that 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 antigens
are the first consideration for any transplant. This essay will focus on the
impact MHC antigens have on solid organ allograft rejection.

Rejection Classification

Rejection occurs
due to an immune response being mounted against the transplanted organ. There
are three types of transplant; autografts, allografts and xenografts (1,2). Autografts describe the
transplant of tissue or organs from one part of the body to another (1). As autografts are recognised
by the immune system as ‘self’, i.e. not foreign, there is no risk of
rejection. Allografts are defined as transplants of tissues or organs between
two individuals of the same species. Allograft rejection is defined as an
immune response mounted by a recipient against donor antigens that result in allograft
loss. Rejection of allografts can be classified as hyperacute, accelerated, acute
and chronic (1,2). Classification criteria are
based upon histopathology and time of rejection post-transplant rather than by
rejection mechanism.

Hyperacute
rejection can occur within minutes or up to 24 hours post-transplant, with the
causative agent being the presence of pre-existing antibodies against HLA. Clinically,
hyperacute rejection is characterised by endothelial cell injury, platelet
margination, complement activation and thrombosis within allograft vasculature
following anastomosis Gautreaux. The transplanted organ will suffer
irreparable ischemic damage following hyperacute rejection. While there is no
effective treatment for hyperacute rejection, with appropriate
pre-transplantation testing hyperacute rejection is now extremely rare,
particularly in kidney transplants. Accelerated vascular rejection is similar
to hyperacute rejection in that it is aggressive, mediated by pre-existing antibodies
(albeit at a lower circulating titre than those found in hyperacute), and has
no standard therapy. The timeline for accelerated rejection is within two weeks
of transplant.

Acute rejection
is defined as allograft failure within 6 months of transplantation and can be
either T-cell (cellular rejection) or antibody mediated (AMR). The clinical
characteristic of acute rejection is necrosis of allograft endothelial cells
Gautreaux. While hyperacute rejection is shown histologically via vasculature
thrombosis, acute rejection more typically presents with vasculitis.

Acute cellular
rejection (ACR) is the most common form of early onset acute rejection and can appear
from 5-7 days post-transplant. ACR is mediated by lymphocytes, specifically T-
and NK-cells, that infiltrate the allograft parenchyma. Untreated ACR can lead
to irreversible histological damage to the transplanted organ through the
activity of complement. Long-term, continuous ACR may lead to chronic
deterioration that eventually results in chronic rejection.

Chronic
rejection is the greatest barrier to long-term allograft and patient survival
and is defined as delayed loss of allograft function months or even years
following transplant. Chronic rejection describes the long-term deterioration
of tissue due to continual immune activation against the allograft.  In cardiac and kidney transplants chronic rejection
histologically manifests as a narrowing of the allograft arteries. This
arterial narrowing is termed obliterative arteriopathy and results from a
build-up of smooth muscle cells and connective tissue within the vascular lumen
Gautreaux. Obliterative arteriopathy is
not such a risk in lung transplants as the lung is not particularly
vascularised. 

Graft versus
host disease (GvHD) is unlike the other forms of rejection in that it is donor
immune cells that recognise the recipient as foreign and mount a response. For
GvHD to occur viable donor lymphocytes must be transplanted alongside
non-immune cells. As such GvHD is commonly associated with haematopoietic stem
cell transplantation however can also occur in patients who have intestinal or
liver transplants. This is because both bowel and liver possess an abundance of
immune cells that are transplanted into the recipient alongside the required
organ. Clinically GvHD presents with a wide range of symptoms affecting skin,
liver and intestine Mazariegos.  Symptoms
may include skin rash or blistering, GI tract ulceration, liver dysfunction or
mouth and tongue lesions Mazariegos. GvHD following solid organ transplant is
associated with a significant mortality. Studies by Hawksworth et.al. and
Clouse 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 with paediatric patients (83% vs 57%). Mechanisms of rejection Rejection was first thought to be solely humoral or antibody mediated however further work proved that cellular immunity also had a role to play. This next section will discuss how the immune system is involved in allograft rejection and outline the expected stages. The first step in activation of the immune system is trauma to the donor organ. Brain death in a donor initiates neuroendocrine signalling and hemodynamic responses which may activate the innate immune response. Harvesting and storage of the organ requires cooling and perfusion with a preservation solution which may result in ischaemic injury upon refusion following transplant. Donor organ trauma activates inflammation causing generation of pro-inflammatory cytokines such as IL-1, IL-6 and TNF-? Ball. Cellular damage can also result in complement activation and initiate the coagulation cascade. Complement activation has been shown to be detrimental to graft outcome, particularly in kidney transplants. Animal studies have suggested that treating donors with complement inhibitors can improve kidney and cardiac graft function following transplantation Damman, Atkinson.    The innate response is activated rapidly and non-specifically in response to pattern recognition Janeway, Land. There are two classes of pattern recognition – pathogen-associated molecular patterns (PAMPS) and damage-associated molecular patterns (DAMPS) Cozzi.  PAMPS initiate an innate response when the body is exposed to pathogens while DAMPS are released following tissue damage and thus are of interest when considering mechanisms of allograft rejection. DAMPS are molecules that are sequestered within cells under normal conditions but are released from cells following damage. Examples of DAMPS include heat-shock proteins, ATP or nuclear proteins Cozzi, Ball. Cells of the innate system recognise DAMPS through the presence of pattern recognition receptors (PRRs). PRRs are found both at the cell surface of innate immune cells and within their cytoplasm. PRRs include molecules such as C-reactive protein, toll-like receptors (TLRs) and Nod-like receptors (NLRs). Activation of innate immune cells such as macrophages and dendritic cells (DCs) occurs through binding of DAMPS and PRRs. Macrophages and DCs are antigen presenting cells (APCs) and as such are essential mediators for activating the adaptive immune response. T-cells are the first aspect of the adaptive immune system to be activated. The first stage of T-cell activation following transplantation is antigen presentation. Following an allograft transplant antigen presentation to T-cells can occur via two distinct pathways – direct and indirect. In the direct pathway recipient T-cells recognise and bind directly to donor antigen presented by donor APCs. The indirect pathway involves recipient T-cells recognising donor peptides that are presented by the recipients' APCs. For the indirect pathway recipient APCs must process and present peptides from the donor antigen.  Recent work has identified a theoretical third pathway that has been termed semi-direct Carty, Menon. In the semi-direct pathway donor antigens are physically relocated to the recipients' APCs cellular membrane where they are recognised by recipient T-cells. This relocation is theorised to occur via either cell-cell contact or the release and uptake of MHC through exosomes Carty. Figure X shows a schematic representation of these three pathways. Antigen presentation 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 are recognised by specific T-cell receptors (TCR). MHC class I molecules interact with CD4 while MHC class II molecules bind to CD8. Antigen presentation via MHC and TCR alone is not enough for T-cell activation and subsequent allograft rejection, an additional two signals are required. Signal two is known as costimulation and occurs between paired ligands and receptors present on the cell surface of T-cells and APCs.  There are two sets of costimulatory pairs; the B7 family and the TNF/TNF receptor family. In the B7 family CD28 is expressed by T-cells while APCs express CD80/86 also known as B7.1/B7.2. The TNF/TNF receptor family is characterised by 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 for T-cell activation are summarised in Figure X. T-cell mediated rejection 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 by causing cell lysis through production of cytotoxic granules containing perforin and granzyme B. These granules lyse the cell membrane and trigger apoptosis. CTL may also initiate apoptosis through the binding of Fas ligand expressed by the CTL to Fas present on the surface of allograft cells Cozzi. CD4+ T-cells are 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-cells produce pro-inflammatory cytokines while Th2 produce anti-inflammatory cytokines. Tregs act to try and mitigate allograft rejection by regulating production of pro-inflammatory cytokines by Th1. Cytokines produces by Th1 T-cells recruit and activate monocytes and macrophages which prolong the inflammatory response within the allograft. CD4+ Th2 T-cells are also involved in 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 in their native configuration In hyperacute rejection circulating antibodies recognises donor HLA or ABO blood group antigens expressed on the surface of endothelial cells. The binding of antibody to antigen activates the complement system leading to endothelial cell damage and cell lysis. In addition to antibody mediated damage there is an accumulation of granulocytes and platelets which leads to the formation of thrombi within the allograft vasculature. The presence of thrombi leads to ischaemia and infarction of the allograft. Accelerated rejection, like hyperacute, is antibody mediated. Unlike hyperacute rejection there is a slight delay in the onset of accelerated rejection. In accelerated rejection the allograft may function normally for the first few days but then there is rapid deterioration in allograft function. There may be two reasons for this delay, first any circulating antibodies may be present at a lower titre than those involved with hyperacute rejection. Secondly, while the transplanted patient may be sensitised against a specific HLA there may be no circulating antibodies. Thus, the delay may be due to the immune system requiring time to induce memory B-cells to produce the appropriate antibody. Preventing rejection Since the first successful solid-organ transplant in 1954, advances in laboratory testing, surgical techniques and immunosuppressive therapies have increased the availability of solid organ transplantation while decreasing allograft rejection. However, these advances have not completely eradicated allograft rejection, and in the case of immunosuppression introduce new risks. The following topics discuss methods currently in use to reduce the risk of allograft rejection. HLA typing: As described above HLA are considered the primary transplant antigens. Typing of HLA for both recipient and donor is therefore an essential step to reduce transplanting a poorly matched organ and thus reducing the potential for rejection. For each class I locus (A, B and C) an individual inherits two alleles, one from each parent. This means that each individual can have a maximum of six class I antigens 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. As with class I MHC an individual inherits two alleles meaning a maximum 12 class II molecules may be expressed Janeway. The genes that encode for HLA are highly polymorphic allowing for an incredible number of allelic combinations, meaning that the chances of two unrelated individuals having an identical HLA complement are remote. The greater the number of mismatches between donor and recipient the more likely it is that the allograft will be rejected. Historically HLA typing was performed using serological methods, most commonly the microlymphocytotoxicity assay. Serology typing has several limitations including the requirement for a large number of pure lymphocytes. This is particularly true for class II typing as these proteins are only expressed on B-cells, which comprise less than 20% of the total lymphocyte population. This causes difficulties for effective class II typing. To overcome this limitation a large volume of blood is required which in turn has its own limitations. Other limitations include the requirement for 100% cell viability, which can be affected by isolation methods, patient health status, or other medications, and variability in complement. Today, the majority of H&I laboratories use molecular based methods for typing. There are three molecular methods in use; sequence specific primer (SSP), sequence specific oligonucleotide (SSO) and sequencing based typing (SBT). Familial allografting: The possibility of allograft mismatching is reduced if donors and recipients are genetically related. As described above HLA alleles are inherited in a block known as a haplotype.  Haplotype inheritance gives a 50% 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 a transplant between complete HLA matched siblings rejection may still occur due to differences in minor histocompatibility antigens.  Minor histocompatibility antigens include   Antibody screening: Individuals can develop HLA antibodies in response to three events; previous transplant(s) with HLA mismatches, pregnancy and blood transfusion. Antibody development through these events is known as sensitisation. Individuals awaiting a transplant are screened regularly for the presence of HLA antibodies. Antibody screening is specific for HLA antibodies and is performed for two purposes.  The first is to determine if HLA antibodies are present, and if so what are their specificities. This is an essential step to determine which donor HLA are unacceptable for potential transplant. Second, antibody screening provides the information required to determine a patients calculated reaction frequency (cRF) Gautereaux.  Each individual's cRF is calculated from the unacceptable HLA identified by screening. Unacceptable HLA specificities are compared with a pool of 10,000 donors with a matching ABO blood group, and the cRF is expressed as a percentage of HLA incompatible donors as calculated from the comparison. With accurate antibody screening the cRF reflects the chances of any sensitised patient receiving a HLA compatible transplant.  It must be kept in mind though that a negative antibody screening is indicative of a lack of circulating HLA antibodies therefore regular screening is essential to correctly identify all potential HLA antibodies present. Antibody screening may also be performed post-transplant for patients who underwent desensitisation treatment prior to transplant. As these patients are at a high risk for AMR, regular monitoring of antibody levels is essential. Crossmatching: Crossmatching is used to determine whether there are circulating HLA antibodies present in the recipients serum that would react with HLA present cells from a potential donor and is performed prior to a transplant to ensure that the donor organ is appropriate for the recipient. There are two common methods used for crossmatching, complement dependent cytotoxicity (CDC) and flow cytometry. Flow cytometry is considered to be a more sensitive method compared with CDC Gautreaux. A negative crossmatch is a positive indication for a transplant to go ahead. Positive crossmatches are generally contraindicative for transplant however the type of positive crossmatch can be indicative of clinical outcome. Poorer clinical outcomes are associated with a positive T-cell crossmatch compared with a B-cell positive. Smith et.al. report that 1-year allograft survival for cardiac and cardiac-lung transplants is less than 30% in cases where there was a positive T-cell crossmatch Smith. While it may be considered best practice to perform crossmatching prior to transplant, cardiac and liver transplants are often performed before crossmatch results are available due to cold ischaemia time (CIT) limitations. A prolonged CIT can increase the risk of delayed allograft function as well as reducing the long-term survival of the organ. A study performed by Taylor et.al. in 2000, with a follow up study performed 10-years later, showed that use of a virtual crossmatch – where exact knowledge of the HLA antibody status of the recipient and the HLA typing of the donor are both available – could accurately predict whether a crossmatch would be negative Taylor. This predictive or virtual crossmatch was used in patients who had a minimum of 6 months negative antibody screening and who had had no recent sensitisation events.  The authors concluded that a virtual crossmatch could be safely performed in patients who meet these criteria, reduced CIT and may reduce delayed graft function Taylor. Regardless of whether the performed crossmatch is laboratory based or virtual it is still a crucial element in preventing allograft rejection. Plasmapheresis and Immunoadsorption: An estimated 25% of patients awaiting a solid organ transplant are highly sensitised against HLA. The presence of HLA antibodies may be contra-indicative for a transplant to go ahead. A highly sensitised individual may have an extended wait of the transplant list awaiting a suitable organ compared with a non-sensitised individual. One potential technique that may reduce their waiting time for a matching organ is antibody depletion via plasmapheresis or immunoadsorption. Plasmapheresis is a process that removes all blood proteins and is therefore non-specific for removal of HLA antibodies cozzi. Immunoadsorption is a more tailored method that gives broad HLA antibody removal using specific adsorbers Cozzi. Both plasmapheresis and immunadsorption are used to directly prevent antibody-mediated rejection.  Best results for plasmapheresis and immunoadsorption are noted in cases with living donors as recipients can be treated prior to transplantation, reducing the chances of AMR occurring. Immunosuppression: Immunosuppressive therapies utilise one of three mechanisms; 1) lymphocyte depletion, 2) blocking lymphocyte response and 3) diversion of lymphocytes. The majority of currently available immunosuppressive agents are designed to either block lymphocyte response or disrupt the cell cycle resulting in lymphocyte depletion. There are a wide variety of immunosuppressive drugs including small-molecule drugs, monoclonal and polyclonal antibodies, intravenous immunoglobulin (IVIG) and corticosteroids. Use of immunosuppression that causes lymphocyte depletion reduces the risk of acute rejection, but can increase the risk of infection and development of post-transplant lymphoproliferative disorder. Immune tolerance: Immunosuppression is a significant burden both for recipients of a transplant and for the healthcare provider. For the transplant patient they face a lifetime drug regime that increases their risk of infection and developing cancer. For healthcare providers the burden is financial. Immune tolerance, defined as a lack of immune response to specific donor tissues is therefore a future aim for solid organ transplants (6). Currently the aim is to achieve operational tolerance, where recipients have long-term (more than 1 year) allograft survival following transplant with no immunosuppression (6). The immune tolerance network (ITN) have been involved in several clinical trials   Chimerism as a method for preventing allograft rejection was first demonstrated in 1953 in mice Billingham.    Despite use of these methods rejection is still a significant risk for any solid organ transplant. Therefore, there must be effective treatment options available.     Treatment options for allograft rejection With an increased understanding of how cellular and antibody rejection occurs there has been development of several therapeutic agents that can target specific facets of the immune system. As stated previously, due to the rapid onset, there is no treatment available for hyperacute rejection. While there is no standard anti-rejection therapy for accelerated rejection, some successes in preventing allograft rejection have been achieved with plasmapheresis, IVIG, anti-thymoglobulin (ATG) and eculizumab therapies. Immunosuppressive therapies currently focus on disrupting the adaptive immune response involved in acute and chronic rejection. The following section discusses available treatment options and how they exert their immunosuppressive effect. Targeting cellular rejection: T-cell mediated, or cellular rejection is heavily involved in early onset acute rejection. The majority of immunosuppressants currently used target one of the three signals required for T-cell activation.   ACR (acute cellular rejection) has been shown to respond to treatment with corticosteroids Kasiske. In cases where corticosteroid treatment is ineffective, or ACR is recurrent, then treatment with an anti-T-cell antibody such as OKT3 (muromonab), ATG or ALG may prevent allograft loss Kasiske. Targeting antibody-mediated rejection: There are several treatment options available to treat acute AMR. However, there is, as yet, no successful treatment available for chronic AMR. Treatment options can include suppression of the T-cell response, elimination of circulating antibodies, suppression or deletion of B cells or blockage of the complement cascade. Table X summarises the potential therapies that can mediate these effects. Strategies for 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