Immunology of Transplant Rejection

Transplantation is the act of transferring cells, tissues, or organs from one site to another, typically between different individuals. The malfunction of an organ system can be corrected with transplantation of an organ (eg, kidney, liver, heart, lung, or pancreas) from a donor. However, the immune system remains the most formidable barrier to transplantation as a routine medical treatment. The immune system has developed elaborate and effective mechanisms to combat foreign agents. These mechanisms are also involved in the rejection of transplanted organs, which are recognized as foreign by the recipient's immune system.

Understanding these mechanisms is important, as it aids in understanding the clinical features of rejection and, hence, in making an early diagnosis and delivering appropriate treatment. Knowledge of these mechanisms is also critical in developing strategies to minimize rejection and in developing new drugs and treatments that blunt the effects of the immune system on transplanted organs, thereby ensuring longer survival of these organs.

In 1944, Medawar showed that skin allograft rejection is a host versus graft response. Mitchison later demonstrated the cell-mediated features of this response. The first successful identical twin transplant of a human kidney was performed by Joseph E. Murray in 1954 in Boston, followed by the first successful liver transplant by Dr. Thomas E. Starzl in 1967, the first heart transplant by Christian Barnard in 1967, and the first successful bone marrow transplant by E. Donnall Thomas in 1968.

Schwartz and Dameshek, in 1959, showed that 6-mercaptopurine was immunosuppressive in rats, ushering in the era of immunosuppressive drug treatment. Since then, many new and progressively more selective immunosuppressive agents have been developed. ^[1] These therapies have improved the survival of transplanted organs.

The degree of immune response to a graft depends partly on the degree of genetic disparity between the grafted organ and the host. Xenografts, which are grafts between members of different species, have the most disparity and elicit the maximal immune response, undergoing rapid rejection. Autografts, which are grafts from one part of the body to another (eg, skin grafts), are not foreign tissue and, therefore, do not elicit rejection. Isografts, which are grafts between genetically identical individuals (eg, monozygotic twins), also undergo no rejection.

Allografts are grafts between members of the same species that differ genetically. This is the most common form of transplantation. The degree to which allografts undergo rejection depends partly on the degree of similarity or histocompatibility between the donor and the recipient. ^{[2, 3, 4]}

The degree and type of response also vary with the type of the transplant. Some sites, such as the eye and the brain, are immunologically privileged (ie, they have minimal or no immune system cells and can tolerate even mismatched grafts). Skin grafts are not initially vascularized and so do not manifest rejection until the blood supply develops. The heart, kidneys, and liver are highly vascular organs and transplantation leads to a vigorous cell-mediated response in the host.

Genetic background

The antigens responsible for rejection of genetically disparate tissues are called histocompatibility antigens; they are products of histocompatibility genes. Histocompatibility antigens are encoded on more than 40 loci, but the loci responsible for the most vigorous allograft rejection reactions are on the major histocompatibility complex (MHC).

In humans, the MHC is called the human leukocyte antigen (HLA) system and is located on the short arm of chromosome 6, near the complement genes. Other antigens cause only weaker reactions, but combinations of several minor antigens can elicit strong rejection responses. The MHC genes are codominantly expressed, which means that each individual expresses these genes from both the alleles on the cell surface. Furthermore, they are inherited as haplotypes or 2 half sets (one from each parent). This makes a person half identical to each of his or her parents with respect to the MHC complex. This also leads to a 25% chance that an individual might have a sibling who is HLA identical.

Human HLA genes code for 3 major class I alleles (HLA-A, -B, -C), and 3 major class II alleles (HLA-DR, -DQ, -DP). Polymorphisms in HLA, especially HLA-A, -B, and -DR loci, are important biological barriers to a successful transplantation. As a closely HLA-matched graft is less likely to be recognized and rejected, HLA mismatching has a substantial impact on graft survival. ^[5]  

The MHC molecules are divided into 2 classes. The class I molecules are normally expressed on all nucleated cells, whereas the class II molecules are expressed only on the professional antigen-presenting cells (APCs), such as dendritic cells, activated macrophages, and B cells. The physiologic function of the MHC molecules is to present antigenic peptides to T cells, since the T lymphocytes only recognize an antigen when it is presented in a complex with an MHC molecule. The class I molecules are responsible for presenting antigenic peptides from within the cell (eg, antigens from intracellular viruses, tumor antigens, self-antigens) to CD8 T cells. The class II molecules present extracellular antigens such as extracellular bacteria to CD4 T cells.

Mechanisms of rejection

The immune response to a transplanted organ consists of both cellular (lymphocyte mediated) and humoral (antibody mediated) mechanisms. Although other cell types are also involved, the T cells are central in the rejection of grafts. The rejection reaction consists of the sensitization stage and the effector stage.

Sensitization stage

In this stage, the CD4 and CD8 T cells, via their T-cell receptors, recognize the alloantigens expressed on the cells of the foreign graft. Two signals are needed for recognition of an antigen; the first is provided by the interaction of the T cell receptor with the antigen presented by MHC molecules; the second, by a costimulatory receptor/ligand interaction on the T cell/APC surface. Of the numerous costimulatory pathways, the interaction of CD28 on the T-cell surface with its APC surface ligands, B7-1 or B7-2 (commonly known as CD80 or CD86, respectively), has been studied the most. ^[6] In addition, cytotoxic T lymphocyte–associated antigen-4 (CTLA4) also binds to these ligands and provides an inhibitory signal. Other costimulatory molecules include the CD40 and its ligand CD40L (CD154).

Typically, helices of the MHC molecules form the peptide-binding groove and are occupied by peptides derived from normal cellular proteins. Thymic or central tolerance mechanisms (clonal deletion) and peripheral tolerance mechanisms (eg, anergy) ensure that these self-peptide MHC complexes are not recognized by the T cells, thereby preventing autoimmune responses.

At least 2 distinct, but not necessarily mutually exclusive, pathways of allorecognition exist: the direct and indirect pathways. Each leads to the generation of different sets of allospecific T cell clones.

Direct pathway

In the direct pathway, host T cells recognize intact allo-MHC molecules on the surface of the donor or stimulator cell. Mechanistically, host T cells see allo-MHC molecule + allo-peptide as being equivalent in shape to self-MHC + foreign peptide and, hence, recognize the donor tissue as foreign. This pathway is presumably the dominant pathway involved in the early alloimmune response.

The transplanted organ carries a variable number of passenger APCs in the form of interstitial dendritic cells. Such APCs have a high density of allo-MHC molecules, and are capable of directly stimulating the recipient's T cells. The relative number of T cells that proliferate on contact with allogeneic or donor cells is extraordinarily high as compared with the number of clones that target antigen presented by self-APC. Thus, this pathway is important in acute allorejection.

Indirect pathway

In the indirect pathway, T cells recognize processed alloantigen presented as peptides by self-APCs. Secondary responses such as those that occur in chronic or late acute rejection are associated with T-cell proliferative responses to a more variable repertoire, including peptides that were previously immunologically silent. Such a change in the pattern of T-cell responses has been termed epitope switching or spreading.

A link between self-MHC + allopeptide-primed T cells and the development of acute vascular-type rejection has been demonstrated to be mediated in part by accelerated alloantibody production. In addition, chronic allograft vasculopathy may be mediated by T cells primed by the indirect pathway.

Molecular mechanisms of T-cell activation

During T-cell activation, membrane-bound inositol phospholipid is hydrolyzed into diacylglycerol (DAG) and IP3. This increases the cytoplasmic calcium concentration. The elevation in calcium promotes the formation of calcium-calmodulin complexes that activate a number of kinases as well as protein phosphatase IIB or calcineurin. Calcineurin dephosphorylates cytoplasmic nuclear factor of activated T cells (NFAT), permitting its translocation to the nucleus, where it binds to the interleukin-2 (IL-2) promoter sequence and then stimulates transcription of IL-2 messenger RNA (mRNA). Numerous other intracellular events, including protein kinase C (PKC) activation by DAG and activation of nuclear factor kappa B (NFkB) also occur at the molecular level.

Effector stage

Alloantigen-dependent and independent factors contribute to the effector mechanisms. Initially, nonimmunologic "injury responses" (ischemia) induce a nonspecific inflammatory response. Because of this, antigen presentation to T cells is increased as the expression of adhesion molecules, class II MHC, chemokines, and cytokines is upregulated. It also promotes the shedding of intact, soluble MHC molecules that may activate the indirect allorecognition pathway. After activation, CD4-positive T cells initiate macrophage-mediated delayed type hypersensitivity (DTH) responses and provide help to B cells for antibody production.

Various T cells and T-cell–derived cytokines such as IL-2 and interferon-γ (IFN-γ) are upregulated early after transplantation. Later, ß-chemokines such as RANTES (regulated upon activation, normal T cell expressed and secreted), interferon-inducible protein–10 (IP-10), and monocyte chemoattractant protein–1 (MCP-1) are expressed, and this promotes intense macrophage infiltration of the allograft. IL-6, tumor necrosis factor–α (TNF-α), inducible nitric oxide synthase (iNOS), and growth factors also play a role in this process. The growth factors, including transforming growth factor–ß (TGF-ß) and endothelin, cause smooth muscle proliferation; intimal thickening; interstitial fibrosis; and, in the case of the kidney, glomerulosclerosis.

Endothelial cells activated by T-cell–derived cytokines and macrophages express class II MHC, adhesion molecules, and costimulatory molecules. These can present antigen and thereby recruit more T cells, amplifying the rejection process. CD8-positive T cells mediate cell-mediated cytotoxicity reactions either by delivering a "lethal hit" or, alternatively, by inducing apoptosis.

Apoptosis

The final common pathway for the cytolytic processes is triggering of apoptosis in the target cell. ^[7] After activation of the CTLs, they form cytotoxic granules that contain perforin and granzymes. ^[7] At the time of target cell identification and engagement, these granules fuse with the effector cell membrane and extrude the content into the immunological synapse. By a yet unknown mechanism, the granzymes are inserted into the target cell cytoplasm where granzyme B can trigger apoptosis through several different mechanisms, including direct cleavage of procaspase-3 and indirect activation of procaspase-9. This has been shown to play the dominant role in apoptosis induction in allograft rejection.

Alternatively, CD8-positive CTLs can also use the Fas-dependent pathway to induce cytolysis and apoptosis. The Fas pathway is also important in limiting T cell proliferation in response to antigenic stimulation; this is known as fratricide between activated cytotoxic T lymphocytes. Cell-mediated cytotoxicity has been shown to play an important role in acute, although not chronic, allograft rejection.

Role of natural killer cells

The natural killer (NK) cells are important in transplantation because of their ability to distinguish allogeneic cells from self and their potent cytolytic effector mechanisms. ^[8] These cells can mount a maximal effector response without any prior immune sensitization. Unlike T and B cells, NK cells are activated by the absence of MHC molecules on the surface of target cells (“missing self” hypothesis). The recognition is mediated by various NK inhibitory receptors triggered by specific alleles of MHC class I antigens on cell surfaces.

NK cells also possess stimulatory receptors, which are triggered by antigens on nonself cells. The resulting effector responses include both cytokine release and direct toxicity mediated through perforin, granzymes, Fas ligand (FasL), and TNF-related apoptosis-inducing ligand (TRAIL).

In addition, NK cells provide help to CD28-positive host T cells, thereby promoting allograft rejection. ^[9] Their graft-versus-host alloresponse has been used for its potent graft-versus-leukemia effect and has contributed to an increase in the rate of sustained remission in patients with acute myeloid leukemia.

The importance of NK cells in rejection was first noted with bone marrow transplants. NK cells were subsequently recognized as active participants in the acute and chronic rejection of solid tissue grafts. ^[8] Donor NK cells in the allograft also play a role, particularly with liver transplants, as the liver has the most significant population of tissue-resident NK cells, but in other solid organ transplants, as well. In addition to exacerbating rejection, donor NK cells resident in graft tissue can mediate tolerance through the direct killing of recipient alloreactive T cells that might otherwise promote graft rejection. ^{[8, 9, 10, 11]}

Role of innate immunity

Although T cells have a critical role in acute rejection, the up-regulation of proinflammatory mediators in the allograft is now recognized to occur before the T cell response; this early inflammation following engraftment is due to the innate response to tissue injury independent of the adaptive immune system. Several studies have examined the role of Toll-like receptor (TLR) agonists and TLR signals in allorecognition and rejection.

These innate mechanisms alone do not appear sufficient to lead to graft rejection itself. However, they are important for optimal adaptive immune responses to the graft and may play a major role in resistance to tolerance induction. The development of methods to blunt innate immune responses, which has potential implications for a wide variety of diseases, is likely to have a significant impact on transplantation, as well.

Hyperacute rejection

In hyperacute rejection, the transplanted tissue is rejected within minutes to hours because vascularization is rapidly destroyed. Hyperacute rejection is humorally mediated and occurs because the recipient has preexisting antibodies against the graft, which can be induced by prior blood transfusions, multiple pregnancies, prior transplantation, or xenografts against which humans already have antibodies. The antigen-antibody complexes activate the complement system, causing massive thrombosis in the capillaries, which prevents the vascularization of the graft. The kidney is most susceptible to hyperacute rejection; the liver is relatively resistant, possibly because of its dual blood supply, but more likely because of incompletely understood immunologic properties.

Acute rejection

Acute rejection manifests commonly in the first 6 months after transplantation. It may be cellular or humoral.

Acute cellular rejection

Acute cellular rejection is mediated by lymphocytes that have been activated against donor antigens, primarily in the lymphoid tissues of the recipient. The donor dendritic cells (also called passenger leukocytes) enter the circulation and function as antigen-presenting cells (APCs).

Humoral rejection

Humoral rejection is form of allograft injury and subsequent dysfunction, primarily mediated by antibody and complement. It can occur immediately posttransplantation (hyperacute) or during the first week. The antibodies are either preformed antibodies or represent antidonor antibodies that develop after transplantation. Proteinuria is associated with donor-specific antibody detection and is likely to be an important factor that determines rapid glomerular filtration rate decline and earlier graft failure in patients developing de novo HLA antibodies. ^[12]

The presence of even low levels of donor-specific antibodies that may not be detected by complement-dependent cytotoxic and flow cytometry crossmatches have been shown to be associated with inferior kidney allograft outcomes. ^[13] These patients may require augmented immunosuppression.

The classic pathway inactive product C4d has been shown to be deposited in the peritubular capillaries (PTC), and immune detection of this product in kidney allograft biopsies is used in diagnosis of antibody-mediated rejection. However, one study has demonstrated substantial fluctuation in the C4d Banoff scores in the first year posttransplant, and this may reflect the dynamic and indolent nature of the humoral process. ^[14] Thus, C4d by itself may not be a sufficiently sensitive indicator, and microvascular inflammation with detection of donor-specific antibodies may be more useful in diagnosing humoral rejection.

Chronic rejection

Chronic rejection develops months to years after acute rejection episodes have subsided. Chronic rejections are both antibody- and cell-mediated. The use of immunosuppressive drugs and tissue-typing methods has increased the survival of allografts in the first year, but chronic rejection is not prevented in most cases.

Chronic rejection appears as fibrosis and scarring in all transplanted organs, but the specific histopathologic picture depends on the organ transplanted. In heart transplants, chronic rejection manifests as accelerated coronary artery atherosclerosis. In transplanted lungs, it manifests as bronchiolitis obliterans. In liver transplants, chronic rejection is characterized by the vanishing bile duct syndrome. In kidney recipients, chronic rejection (called chronic allograft nephropathy) manifests as fibrosis and glomerulopathy. The following factors increase the risk of chronic rejection:

• Previous episode of acute rejection
• Inadequate immunosuppression
• Initial delayed graft function
• Donor-related factors (eg, old age, hypertension)
• Reperfusion injury to organ
• Long cold-ischemia time
• Recipient-related factors (eg, diabetes, hypertension, hyperlipidemia)
• Posttransplant infection (eg, cytomegalovirus [CMV])

Rejection cannot be completely prevented; however, a degree of immune tolerance to the transplant does develop. Several concepts have been postulated to explain the development of partial tolerance. They include clonal deletion and the development of anergy in donor specific lymphocytes, development of suppressor lymphocytes, or factors that down-regulate the immune response against the graft. Other hypotheses include the persistence of donor-derived dendritic cells in the recipient that promote an immunologically mediated chimeric state between the recipient and the transplanted organ.

Tissue typing or crossmatching is performed prior to transplantation to assess donor-recipient compatibility for human leukocyte antigen (HLA) and ABO blood group. These tests include the following:

• The ABO blood group compatibility is tested first, because incompatibility between the blood groups leads to rapid rejection.

• In the lymphocytotoxicity assay, patient sera are tested for reactivity with donor lymphocytes. A positive crossmatch is a contraindication to transplantation because of the risk of hyperacute rejection. This is used mainly in kidney transplantation.

• Panel-reactive antibody (PRA) screens the serum of a patient for lymphocytic antibodies against a random cell panel. Patients with prior transfusions, transplants, or pregnancies may have a high degree of sensitization and are less likely to have a negative crossmatch with a donor. A reduced risk of sensitization at the time of second transplant has been observed when using more potent immunosuppression with rabbit antithymocyte globulin, tacrolimus, and mycophenolate mofetil/sodium for nonsensitized primary kidney or kidney/pancreas transplant patients. ^{[15, 16, 17]}

• Mixed lymphocyte reaction (MLR) can be used to assess the degree of major histocompatibility complex (MHC) class I and class II compatibility. However, it is not a rapid test and can be used only in cases involving living related donors. It is rarely used at present.

Initially, radiation and chemicals were used as nonselective immunosuppressive agents. In the late 1950s and 1960s, the agents 6-mercaptopurine and azathioprine were used in conjunction with steroids. Newer immunosuppressive agents have since been developed; they are more effective, more selective, and less toxic and have made possible the advances in the field of transplantation.

Immunosuppressive drugs are used in 2 phases: the initial induction phase, which requires much higher doses of these drugs, and the later maintenance phase. Immunosuppressive agents in current use include the following:

• Calcineurin inhibitors
• Mechanistic (mammalian) target of rapamycin (mTOR) inhibitor
• Antiproliferative agents
• Antibodies
• Corticosteroids

Calcineurin inhibitors

The calcineurin inhibitors used in transplantation are cyclosporine and tacrolimus. These agents are immunophilin-binding agents; they primarily suppress the activation of T lymphocytes by inhibiting the production of cytokines, specifically IL-2. They are associated with numerous toxicities that are often dose-dependent. Nephrotoxicity occurs with both the drugs. Hirsutism, gingival hypertrophy, hypertension, and hyperlipidemia develop more often with cyclosporine than tacrolimus. Potential drug interactions are also important to recognize.

Tacrolimus is a macrolide lactone antibiotic produced by the soil fungus Streptomyces tsukubaensis. It binds to a different intracellular protein (FK Binding Protein–12 [FKBP-12]) than cyclosporine but has the same mechanism of action. Neurotoxicity, alopecia, and posttransplant diabetes mellitus develop more frequently with tacrolimus than with cyclosporine.

Conversion from brand name to generic tacrolimus is routinely feasible, but it requires close monitoring of tacrolimus levels. ^{[18, 17]}

mTOR inhibitor

Sirolimus is a macrocyclic antibiotic produced by fermentation of Streptomyces hygroscopicus. It inhibits T-cell activation and proliferation that occurs in response to antigenic and cytokine (IL-2, IL-4, and IL-15) stimulation. Sirolimus binds to the immunophilin, FKBP-12, and the resulting complex inhibits mTOR; in turn, this inhibits IL-2–mediated signal transduction, which blocks the progression from the G1 to the S phase of the cell cycle. ^[19]

Sirolimus is associated with numerous adverse effects, such as leukopenia, thrombocytopenia, anemia, hypercholesterolemia, and hypertriglyceridemia. It has also been associated with mucositis, delayed wound healing, lymphocele formation, pneumonitis, and prolonged delayed graft function.

Although calcineurin inhibitors reduce short-term kidney transplant failure, chronic nephrotoxicity may contribute to late transplant loss. However, in the 3C Study, which compared maintenance therapy with tacrolimus versus sirolimus in 394 kidney transplant recipients, follow-up at 18 months showed that sirolimus-based maintenance therapy did not improve transplant function and was associated with significant hazards of rejection and infection. ^[20]

Antiproliferative agents

Azathioprine and mycophenolate mofetil (MMF) are the agents commonly used in this category. Other antiproliferative agents, such as cyclophosphamide and, more recently, leflunomide, have also been used.

Antiproliferative agents inhibit DNA replication and suppress B- and T-cell proliferation. MMF is an organic synthetic derivative of the natural fermentation product mycophenolic acid (MPA) that causes noncompetitive reversible inhibition of inosine monophosphate dehydrogenase. This interferes with purine synthesis. Adverse effects of MMF are nausea, diarrhea, leukopenia, and thrombocytopenia. Invasive CMV infection has been sometimes associated with MMF. The introduction of MMF has been shown to be associated with improvement or stabilization of kidney function, even several years after transplantation. ^{[21, 16]}

Antibodies

Basiliximab, an IL-2 receptor antagonist, is a monoclonal antibody approved by the US Food and Drug Administration (FDA) for preventing kidney transplant rejection, and has unlabeled uses for preventing acute heart and liver transplant rejection. Another IL-2

Antilymphocyte globulin, such as the monoclonal antibody muromonab-CD3, and the polyclonal antibodies, antithymocyte globulins derived from either equine or rabbit sources, are approved for the treatment of rejection. They also have been used as induction agents at some transplantation centers.

Antibodies interact with lymphocyte surface antigens, depleting circulating thymus-derived lymphocytes and interfering with cell-mediated and humoral immune responses. Lymphocyte depletion also occurs either by complement-dependent lysis in the intravascular space or by opsonization and subsequent phagocytosis by macrophages. Adverse effects such as fever, chills, thrombocytopenia, leukopenia, and headache typically occur with the first few doses.

Belatacept, a fusion protein composed of the Fc fragment of human IgG1 linked to the extracellular domain of cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4), selectively inhibits T-cell activation through costimulation blockade. It has been approved by the FDA and the European Medicines Agency for use in combination with basiliximab induction, MMF, and corticosteroids in the prevention of acute rejection of kidney transplants in adults. ^[22]

Belatacept was also associated with a small but increased risk for posttransplant lymphoproliferative disorder (PTLD). The risk for PTLD is higher for transplant patients who have not been exposed to Epstein-Barr virus (EBV). Transplant patients who have not been exposed to EBV have more difficulty mounting an effective immune response to the virus if they get infected after transplant.

Corticosteroids

Steroids have historically been the cornerstone of immunosuppression and are still used. However, the newer regimens are trying to minimize the use of steroids and thereby avoid the adverse effects that are associated with them. Steroids are still important in treating episodes of acute rejection.

Investigational agents

Alemtuzumab, a recombinant monoclonal antibody against CD52 (lymphocyte antigen), is used off-label as part of various induction regimens in patients undergoing kidney transplantation, and has achieved steroid-sparing effects in high-risk transplant recipients. ^[23] In the 3C Study, which compared with induction therapy with alemtuzumab versus standard basiliximab-based treatment in 852 adult recipients of kidney transplants, follow-up at six months showed that alemtuzumab-based induction therapy followed by reduced calcineurin inhibitor and mycophenolate exposure and steroid avoidance, reduced the risk of biopsy-proven acute rejection in a broad range of patients. ^[24]

Rituximab is a chimeric monoclonal antibody aginst CD20 that can deplete CD20-positive B-cell populations. B cells play an important role in antibody-mediated rejection, and small studies in both adults and pediatric kidney transplant recipients have shown improved outcomes with rituximab treatment of acute antibody-mediated rejection. ^[25]