Introduction to Transplantation Immunology

During the past few decades, the field of transplantation has undergone a dramatic expansion. Sophisticated surgical procedures have enabled the transplantation of almost any type of vital organ or tissue as an effective therapeutic modality. Since most transplants are done from genetically different donors, we must consider the immunological response of the recipient to the transplantation antigens presented by the donor graft. In general, this immune response is designed to cause rejection of the transplanted tissue and both cellular (or lymphocyte-mediated) and humoral (antibody-mediated) mechanisms are involved. However, under certain circumstances, a state of immunological tolerance develops which permits long-term normal function of a transplanted organ. This overview is intended as an introduction to the field of transplantation immunology. Since most transplants are performed between different members of the same species, we will focus on the basic mechanisms of allogeneic immune responses in transplant immunity.

Transplant immunity results after exposure of a recipient to different histocompatibility antigens. These transplantation antigens have been grouped as major (i.e. HLA and ABO blood groups) and minor histocompatibility antigens (although the latter have been documented in mice and other animals, little is known about them in humans). HLA antigens are controlled by a series of genes on chromosome 6, referred to as the human Major Histocompatibility Complex MHC). These genes have been classified into major categories. HLA-A and HLA-B are examples of Class I genes and HLA-DR and HLA-DQ represent Class II genes. These genes are highly polymorphic. For instance, HLA-A has more than 20 alleles and HLA-B has more than 50 alleles. Therefore, it is highly unlikely that two unrelated persons have the same HLA type. Both HLA class I and class II alloantigens can induce transplant immunity at humoral (antibody) and cellular (T lymphocyte) immune levels. ABO incompatibility is important only in antibody-mediated injury of the graft.

Since transplant immunity leads to rejection, it seems best that the patient and donor are matched for histocompatibility antigens. Although this is possible for genetically identical twins (syngeneic grafts) and to a lesser extent between HLA- and ABO-identical siblings, the vast majority of transplants come from incompatible unrelated (cadaveric) donors. Immunosuppressive drugs are given to control transplant immunity so that rejection can be prevented or controlled and that the recipient develops a long-term acceptance (or tolerance) of the transplant. Rejection can be mediated by antibodies, lymphocytes or both and can manifest itself in different ways: hyperacute rejection (during the early post-transplant period), acute rejection (may occur at any time) and chronic rejection (a slowly developing process causing a progressive decline in graft function).

Hyperacute Rejection

Vascularized organs (especially kidney and heart) are at risk for hyperacute rejection if the patient has preformed donor-specific alloantibodies. This humoral presensitization (or alloimmunization) may be caused by a previous transplant, blood transfusions and pregnancy. The primary target of the donor-specific antibodies is the vascular endothelium of the transplanted organ. The most common target antigens are HLA class I, ABO but other less-well defined antigens have also been considered. HLA class II antigens seem less relevant in hyperacute rejection because they are not stongly expressed on the vascular endothelium.

Antibodies mediating hyperacute rejection are almost always Complement-fixing and they could be IgG or IgM type or both. Alloreactive T lymphocytes are occasionally involved. A critical step in alloantibody-mediated rejection is the initiation of the Complement cascade. This involves binding and activation of C1q, which requires at least two Complement-fixing sites on the antigen/antibody complex. This requires a close proximity between antigens and no Complement activation will occur if the antigen density is too low on the cell surface.

Activation of the Complement cascade leads to the release of various inflammatory mediators and the initiation of the coagulation and fibrinolytic systems. Hyperacute rejection in manifested by rapid vascular constriction, edema and thrombotic occlusion. Endothelium stimulation and exposure to subendothelial basement membrane proteins activate platelets which then adhere and aggregate in the vasculature. Soon afterward, polymorphonuclear leukocytes (PMN) adhere to the vascular endothelium.

The histopathology of hyperacute rejection is manifested by thrombosis and edematous changes but no pronounced mononuclear cell infiltration. Immunostaining shows deposits of immunoglobulin, Complement component C3 and fibrinogen.

Antibody-mediated rejection may also occur without thrombotic occlusion. In this case the histological pattern is a vasculitis which may lead to necrosis of individual cells of graft blood vessels. Because lymphocytes may be also involved, this process is referred to as acute vascular rejection.

Cellular Rejection

Acute cellular rejection is mediated by lymphocytes which have become alloactivated against donor transplantation antigens. In vivo stimulation of alloreactive T cells takes places primarily in the peripheral lymphoid tissues of the recipient although intragraft sensitization may occur. The strongest stimulus is provided by donor dendritic cells (also referred to as passenger leukocytes) in the allograft which will enter the circulation and end up on the lymphoid tissues of the recipient. These dendritic cells function as antigen-presenting cells and they provide a strong stimulus of HLA class II reactive CD4 cells, which can stimulate the growth and differentiation of HLA class I reactive CD8+ cytolytic lymphocytes. Alloreactive lymphocytes enter the circulation and they will react with allograft vascular endothelium, the primary target of the initial stage of cellular rejection.

The lymphocyte-endothelial interactions depend on the expression of appropriate target antigens on the endothelium. HLA class I antigens are constitutively expressed at the cell surface whereas HLA class II antigens expression must be induced by various cytokines, especially gamma interferon. Furthermore, adhesion molecules (ICAM, VCAM, integrins, selectins) play an important role in the cell surface interactions between lymphocytes and endothelium cells and their activation and release of cytokines and inflammatory mediators. These processes lead to migration of lymphocytes through the vascular wall.

Graft-infiltrating lymphocytes can mediate various effector mechanisms of allograft immunity. Besides a direct cytotoxic effect on graft parenchymal cells, lymphocytes may mediate a delayed type hypersensitivity mechanism of graft rejection. The latter involves the recruitment of macrophages and the release of various cytokines. Other inflammatory cells may also be seen during rejection, including neutrophils, eosinophils, B lymphocytes and NK cells. Varying proportions of CD4 and CD8 lymphocytes are found in cellular infiltrates of rejecting allografts. Many of them express T cell activation markers, like IL-2 receptors and probably through release of gamma-interferon and other cytokines, there is an increased expression of HLA antigens (especially class II) on the vascular endothelium and other target cells in the graft parenchyma.

Consideration must be given to additional immunological mechanisms of graft injury secondary to infection, especially by viruses. This means that other types of lymphocytes may infiltrate the graft and they could mediate immune effector responses against virus infected cells in the graft. Another possibility is that graft infiltrating lymphocytes mediate recurrent autoimmune disease processes.

Chronic Rejection

This major complication, affecting most long-term transplant survivors, is not well understood. Chronic rejection is characterized by vasculopathy, fibrosis and a progressive loss of organ function. Its pathogenesis probably involves both humoral and cellular immune mechanisms. Chronic rejection may be mediated by a low-grade, persistent delayed type hypersensitivity response in which activated macrophages secrete mesenchymal cell growth factors. Of potential importance are persistent viral infections which induce cellular immune responses which may synergize with donor-specific alloreactive T cells within the allograft. Chronic rejection may also reflect chronic ischemia secondary to injury of blood vessels by antibody or cell-mediated mechanisms. Vascular occlusion may occur as a result of smooth muscle cell proliferation in the intima of arterial walls.

Transplant Tolerance

Although the goal of transplantation is to achieve a long-term acceptance of the allograft, little is known about the mechanisms responsible for this successful outcome. Several concepts have been forwarded to explain the immunological tolerance to the transplant. One deals with the clonal deletion of donor-specific lymphocytes in long-term transplant survivors. This means that rejection mediating donor-specific alloreactive T cells have selectively been eliminated in these patients. Another possibility is that donor-specific T lymphocytes have become anergic (or non-reactive) so that they cannot function anymore as effector cells mediating graft rejection. A third concept is that long-term survivors have developed suppressor lymphocytes or soluble suppressor factors which down regulate the donor-specific immune effector responses against the allograft. Relevant to this is the idiotypic network concept whereby the immune response triggers anti-antibodies and anti-T cells as regulatory mechanisms of the immune response. The most recent hypothesis to explain long-term graft acceptance is the persistence in the recipient of donor-derived dendritic cells which promote an immunologically-mediated chimeric state between the recipient and the transplanted organ.

Regardless of the mechanism of transplant tolerance, it must be noted that most long-term transplant patients require continuous immunosuppressive treatment for maintaining graft function although some notable exceptions have been documented.

Immunosuppressive Drugs

Virtually every transplant recipient requires treatment with immunosuppressive drugs to control graft rejection. During the past decade, improved immunosuppressive strategies have increased the success of organ transplantation. Although potent immunosuppressive drugs may prevent graft rejection, we must consider adverse side effects on the patient. Immunosuppression was first done with metabolic toxins that inhibit DNA replication of lymphocytes. Some drugs are still in clinical use especially azathioprine (or Imuran) and to a lesser extent, cyclophosphamide. During the early 1980's, Cyclosporine was introduced as a potent immunosuppressive drug. This drug is a cyclic peptide produced by a fungus. Its major site of action is the T cell in which cyclosporine blocks transcription of IL-2 and other genes, thereby inhibiting the production of lymphokines which induce the activation and differentiation of alloreactive T cells. Cyclosporine binds to a small protein (12kD) called cyclophilin, which functions as a peptidyl-prolyl-isomerase enzyme involved in correct folding of the transcription protein for the regulatory sequence of the IL-2 gene. During recent years a new drug tacrolimus (FK506) has been successfully used to treat transplant patients, especially those with liver allografts. Although tacrolimus is structurally unrelated to cyclosporine, it also inhibits IL-2 synthesis by T lymphocytes through binding to the FK506 binding protein (which is different from cyclophilin) thereby blocking lymphokine gene transcription.

Corticosteroids are usually administered in combination with Cyclosporine or FK506. These steroids exert their immunosuppressive effects through lysis of certain T cell subsets and especially by blocking cytokine gene transcription in macrophages, especially for IL-1, IL-6 and Tumor Necrosis Factor (TNF) which are important costimulators of T cell activation.

During recent years, other immunosuppressive drugs are being evaluated in clinical trials with organ transplant patients. These include mycophenylate mofetil, rapamycin and deoxyspergulin.

Besides chemical immunosuppression, transplant patients are often treated with specific antibodies against T cell surface structures. These antibodies have been produced as polyclonal antisera in horses and rabbits immunized with human lymphocytes or thymocytes. Mouse monoclonal antibodies have been generated against specific T cell molecules like CD3, CD4, IL-2 receptor, T cell receptor, etc. Anti-CD3 antibodies (OKT3) are most widely used and their immunosuppressive effect is likely mediated through a modulating effect on the expression of CD3 through a series of events involving binding, aggregation, capping and endocytosis of the CD3 molecule. A major limitation of monoclonal antibodies is their immunogenicity which triggers anti-mouse Ig antibodies in the recipient.

Any type of immunosuppressive drug has adverse side effects which compromise the transplant recipient. Immunosuppression is not limited to the transplant response but also effects the immune system as a whole and this increases the patient's susceptibility to infection. Indeed, opportunistic infections by a variety of microorganisms (including viruses) are the most frequent complications in transplant patients. Decreased immunosurveillance also increases the risk for malignancy, in particular post-transplant lymphoproliferative disease (PTLD). A major problem is drug toxicity. The adverse side effects of cyclosporine and tacrolimus include nephrotoxicity, liver toxicity, hypertension (less for tacrolimus) and neurological disorders. Metabolic complications are associated with steroid treatment.

Most transplant patients receive a combination of immunosuppressive drugs. Optimal drug treatments require a careful clinical management of the patient which includes proper dosage strategies, measurement of drug levels in the blood, monitoring of graft function including biopsy histology and assessment of potential side effects of the drug.

Tissue Typing for Clinical Transplantation

The purpose of the tissue typing laboratory is to assess donor-recipient compatibility for HLA and ABO to analyze patient serum for lymphocytotoxic antibodies which may be specific for the potential transplant donor. Most relevant is the crossmatch assay whereby patient sera are tested for their reactivity with donor lymphocytes. This is usually done by lymphocytotoxicity testing whereby donor lymphocytes are first incubated with patient serum, then with rabbit Complement and lysis of lymphocytes is assessed by the uptake of an extravital dye like trypan blue or eosin red. A positive crossmatch is a contraindication for organ transplantation because of the risk for antibody-mediated (hyperacute) rejection. This applies particularly for kidney transplants whereas the liver allograft shows a relative resistance to antibody-mediated injury. In kidney transplantation, several modifications of the crossmatch assay have been used to increase its sensitivity including antiglobulin augmentation and flow cytometry. Serum treatment with dithiothreitol (DTT) is used to distinguish clinically irrelevant IgM type antibodies.

Transplant candidates may become sensitized following a prior transplant, blood transfusion and previous pregnancies. Serum screening for lymphocytotoxic antibodies against a random cell panel will provide an assessment of the degree of sensitization expressed as the percentage Panel Reactive Antibody (PRA). The PRA can vary between 0% (non-sensitized) to 80-100% indicating a high degree of sensitization. Patients with high PRA values are less likely to have crossmatch negative donors and they must wait much longer for a transplant and some may never receive a transplant. Recent studies have provided insight about the HLA specificities of antibodies in highly sensitized patients. This is based on the concept that each HLA molecule has multiple antigenic determinants (epitopes) which can be grouped as private (e.g. HLA-A1, HLA-B7, etc.) and public determinants (sometimes referred to as crossreactive groups, CREGs). A public determinant is an epitope shared by molecules with different private specificities (e.g. A1+A3+A11, A2+A9+A28, B5+15+35, B7+22+27+40, etc.). Most highly sensitized patients show a persistence of the same pattern of antibody specificity against one or a few public epitopes. A better understanding of the antibody specificity improves the selection of transplant donors with acceptable HLA mismatches.

Considerable evidence has been obtained that matching for HLA reduces allograft rejection thereby promoting survival of kidney and heart transplant patients. Although the HLA system comprises multiple class I and especially class II genes, most matching strategies consider only HLA-A, HLA-B and HLA-DR. Because of the codominant expression of HLA genes, the degree of compatibility ranges from 0 to 6 matches (two for each locus). Survival statistics from kidney and heart transplant registries have shown the best survival rates of cadaveric transplants with 6 matches, followed by 5 matches, etc. Although it make sense to find a perfect match for each transplant patient, the reality of practice dictates the selection of less well-matched donors because of the limitations caused by extensive polymorphisms of HLA, the limited availability of donors and the urgency status of the patient. However, recent experience has shown that transplanted kidneys with "permissible" HLA mismatches have excellent graft survivals.

Post-Transplant Monitoring of Organ Transplants

Different methodologies have been developed to monitor various organ transplants. In kidney transplant patients, the easiest way to assess renal function is by measuring serum creatinine levels. Elevations suggest rejection although cyclosporine induced nephrotoxicity may also be responsible. Histopathological examination of a renal biopsy may enable a differential diagnosis between rejection and cyclosporine toxicity. Immunostaining of renal tubular cells, a primary target of infiltrating T cells, shows increased expression of HLA class II antigens during rejection. Heart transplant patients are monitored by histopathological analysis of endomyocardial biopsies at regular intervals. These biopsies are obtained through a catheter passed into the right ventricle and the histological rejection is assessed by the degree of cellular infiltration and myocyte damage. In liver transplants, histological analysis of biopsies shows that the biliary epithelium is the primary target of cellular rejection. Liver function is monitored by measuring serum levels of bilirubin and liver enzymes like alkaline phosphatase. Lung transplant patients are monitored for rejection by X-ray analysis, pulmonary function tests, transbronchial biopsy histology and bronchoalveolar lavage analysis. Since the transplanted lung is susceptible to infection, a differential diagnosis between rejection and infection is very important for the proper management of these patients. Acute rejection episodes are treated with augmented immunosuppression mostly in the form of a bolus treatment with steroids or administration of OKT3 or other anti-lymphocyte antibodies.

The major complication of any organ transplant is chronic rejection which is frequently associated with an obstructive vasculopathy, e.g. graft coronary disease in heart transplant patients. Chronic liver transplant rejection also leads to a progressive loss of biliary epithelium, refeered to as vanishing bile duct syndrome. In lung transplant patients, chronic rejection is manifested by a progressive obstructive disease of the airways, referred to as obliterative bronchiolitis. An early detection of chronic rejection is neccessary for effective immunotherapeutic intervention.

Submitted by:

Rene J. Duquesnoy, Ph.D.
Professor of Pathology and Surgery
Division of Transplant Pathology
Thomas E. Starzl Transplant Institute
University of Pittsburgh Medical Center
June 18, 2009

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