Organ Transplantation - The Rejection Response

In the late 1800s and early 1900s, surgeons gained the technical ability to transplant organs and tissues from one animal to another. It soon became evident that, following transplantation, the organ would rapidly become ischemic and necrotic. In 1923, Dr. Carl Williamson, at the Mayo Clinic, demonstrated that cells of the immune system were responsible for the death of transplanted tissues and organs. This discovery set the stage for the study of immune-mediated rejection and the development of effective immunosuppressive strategies [1].

Transplantation of organs and tissues in veterinary medicine is becoming more common. Corneal transplantation is performed to replace diseased or scarred corneas. Corneoscleral transplantation is performed for the treatment of canine epibulbar melanomas. Allogeneic bone marrow transplantation has been performed in cats to aid in the treatment of lymphohematopoietic neoplasias, aplastic anemias, and feline retrovirus infections. In the dog and cat, for the treatment of acute and chronic renal failure, kidney transplantation is performed at university hospitals and private practices. Feline renal transplant patients have now survived for over 13 years with normal renal function.

Nomenclature

A graft is tissue or an organ used in a transplant procedure. An autograft is tissue or an organ that is removed from and then transplanted on or into the same individual. A common example is a cancellous bone graft used to speed fracture healing. Autografts do not incite a rejection response. An isograft is tissue or an organ transplanted between two genetically identical individuals; identical twins or closely inbred individuals. An allograft is tissue or an organ transplanted between genetically nonidentical members of the same species. Virtually all renal transplants performed in clinical veterinary medicine are allografts. A xenograft is tissue or an organ transplanted between members of different species.

Mechanism of the Immune Response to an Allograft [2-4].

Rejection of the transplanted tissue or organ is determined by T-cell (lymphocyte) recognition of differences in the composition of cell surface glycoproteins between graft and host tissues. These glycoproteins are termed histocompatibility antigens or histocompatibility molecules. The histocompatibility antigens that incite the most vigorous rejection response are encoded by genes of the major histocompatibility complex (MHC).

A MHC is found in all vertebrates. In dogs, this cluster of genes on a single chromosome is termed the dog leukocyte antigen (DLA); in cats it is termed the feline leukocyte antigen (FLA). Polymorphism, or the presence of many different variations of the same gene (alleles) at a single location, or locus, is characteristic of the MHC. Each individual will have two, one on each paired chromosome, of many possible alleles at each locus in his or her MHC. This variation in the genetic makeup of the MHC, results in the production of a tremendous variety of cell surface histocompatibility antigens. This variety ensures that the host T-cells will recognize virtually all tissue and organ allografts as foreign, resulting in a rejection response.

The genes of the MHC are closely linked, and the genetic information inherited from each parent on a single chromosome is transferred as a block. This group of genes is termed the MHC haplotype; each offspring receives one haplotype from each parent. The genes of each haplotype are expressed codominantly; therefore, cell surface histocompatibility derived from each parent will be present in the offspring. This basic understanding of the genetics of the MHC is important clinically, particularly in a species like the dog, in which it is difficult to immunosuppress or control the rejection response. Because each offspring inherits one haplotype from each parent and each haplotype is expressed codominantly, 25% of littermates have the possibility of being MHC identical, 50% might share one haplotype, and 25% will not share a haplotype. Without the administration of immunosuppressive agents, renal allografts from MHC-nonmatched dogs survive approximately 10 days, renal allografts from dogs matched for one haplotype survive approximately 24 days, and MHC-matched allografts survive for 150 days or more. In the latter group, the rejection response can be more readily controlled using immunosuppressive agents; therefore, selection of MHC-identical littermates as donor/recipient pairs can greatly enhance the chance of long-term graft survival.

The fact that allograft survival is not indefinite when a dog receives a kidney from an MHC-identical donor demonstrates that the MHC is not the only genetic region coding for histocompatibility antigens. Minor histocompatibility genes have been isolated in mice and human beings and probably code for changes in peptide structure in endogenous proteins that allow recognition by host T-cells, but do not affect their physiologic function. Fortunately, the rejection response produced by minor histocompatibility differences is relatively easy to prevent using immunosuppressive agents.

It is important to understand that the MHC-encoded cell surface glycoproteins, or transplantation antigens, did not evolve to prevent the transplantation of genetically dissimilar tissue and organs, but rather to protect the host from invasion by viruses, fungi, nematodes, and other parasites. To do so effectively, the immune system must distinguish between antigens against which an immune response would be beneficial (pathogenic or allogeneic) or harmful (host or self). In the thymus, during fetal development, T-cells are propagated or destroyed based on their ability to recognize MHC glycoproteins as "self" or "nonself". The interaction between MHC glycoproteins and T-cells, in the presence of antigen, results in a series of transmembrane and cytosolic chemical reactions that result in T-cell cytotoxic activity and the production and/or release of cytokines. Cytokines (interleukins [ILs], tumor necrosis factor [TNF], and others) result in the further activation of T-cells, B-cells, macrophages, and other immunoreactive cells.

The cell surface glycoproteins of the MHC are divided into two major classifications: Class I and II. Class I glycoproteins or molecules are expressed on all nucleated cells. Class I molecules have a folded region that holds and presents antigens from virus-infected, tumor, and allogeneic cells to antigen-responsive cytotoxic (CD8+) T-cells. The linkage of the T cell receptor -CD8 complex on the surface of cytotoxic T-cells with the class I molecule-antigen complex of allograft or antigen-presenting cells (monocytes, macrophages, Langerhans cells of the epidermis, and dendritic cells of lymphoid organs) results in the proliferation and differentiation of a clone of cytotoxic T cells specific for that class I-antigen combination. Thus, the cytotoxic activity of T-cells is both antigen specific and class I restricted.

Class II molecules are only constitutively expressed on the surface of cells that are essential for immune responses. These include B-cells (lymphocytes), thymic epithelial cells, and the cells listed above that present antigen to T-cells. T cells, vascular endothelial cells, smooth muscle cells, and others can express class II antigens when activated by cytokines such as interferon-γ (INF-γ). The function of class II molecules is similar to that of class I. Antigen-sensitive T helper cells (CD4+) recognize the class II-antigen complexes on allogeneic or antigen-presenting cells. The linkage of the CD4-T-cell receptor complex with the class II-antigen complex results in the proliferation and differentiation of a clone of T helper cells specific for that class II-antigen complex. Activated T helper cells begin the cascade of events responsible for acute allograft rejection by the release of cytokines (IL-2, IL-3, IL-4, IL-5, IL-6, INF-γ, TNFs, granulocyte macrophage colony stimulating factor [GM-CSF], and others) that propagate an inflammatory response, activate cytotoxic cells, and promote antibody formation.

In addition to their role as antigen-presenting molecules, it is believed that foreign class I and class II molecules, in the absence of antigen, may stimulate alloreactive T cells and serve as the stimuli for rejection reactions. Also, rejection may be initiated following T-cell recognition of class I or II - minor histocompatibility antigen complexes.

The allograft rejection response begins with the migration of antigen-presenting cells of the host or donor organ to T-cell areas of secondary lymphoid organs. These T cells circulate between lymphoid tissues, regulated by chemokine and sphingosine-1 phosphate receptors. T-cell activation by allogeneic or antigen-presenting cells requires cell contact and cell surface molecule interaction. Cell surface molecules that interact are termed "ligand-receptor pairs". A ligand is any molecule that forms a complex with another molecule. Antigen presentation through class I or class II molecules can be split into four stages that produce three signals needed to stimulate the rejection reaction: adhesion, antigen-specific activation, co-stimulation, and cytokine signaling (Fig. 10-1). Association of the antigen-presenting cells and T cells first involves nonspecific, reversible binding through adhesion molecules such as LFA-1 and ICAM. When the T-cell receptor complex is presented with an alloantigen, a conformational change occurs in the adhesion molecules that results in tighter binding and prolonged cell to cell contact. The first signal of the rejection response is provided by the class I/antigen-CD8/T-cell receptor ligand-receptor pair and the class II/antigen-CD4/T-cell receptor ligand-receptor pair (CD3 complex) (Fig. 10-2). The specific MHC/peptide -T-cell receptor interaction, although necessary, is not sufficient to fully activate the T cell. A second signal is required, otherwise the T cell becomes unresponsive. The second signal, also termed co-stimulation, is of critical importance (Fig. 10-3).

Figure 10-1. Association of APCs and T cells first involves non-specific, reversible binding through adhesion molecules, such as the LFA-1/ICAM interaction. Recognition of the peptide antigen in the MHC molecule by the TCR, that provides the specificity of the interaction, results in prolonged cell-cell contact. A second signal (co-stimulation) is necessary for the T cell to respond efficiently, otherwise tolerance may result. Activation results in upregulation of cytokines and their receptors, which boost the activatory signals and help to decide the cell fate.

Figure 10-2. T-cell activation through three signals.

Figure 10-3. A T cell requires signals from both the T-cell receptor and CD28 for activation.

(a) In the absence of co-stimulatory molecules inactivation or anergy results. This situation would prevail in order to tolerize T cells not removed by central tolerance to self anti96ns expressed on peripheral tissues.
(b) In the absence of an antigen-specific signal (wrong peptide for example) there is no effect on the T cell.
(c) Co-reception of both signals, from the surface of a professional APC, activates the T cell to produce IL-2 and its receptor. The cell divides and differentiates into an effector T cell, which no longer requires signal 2 for its effector function. (d) At the termination of the immune response, CTLA-4 replaces CD28 and downregulates T-cell function.

Two of the most potent co-stimulatory molecules expressed on antigen-presenting cells are B7-1 (CD80) and B7-2(CD86). They are ligands for the T-cell molecules CD28 and CTLA-4. CD28 is the main co-stimulatory ligand expressed on naïve T cells. CD28 stimulation has been shown to prolong and augment the production of IL-2 and other cytokines and is probably important in preventing the induction of tolerance. CTLA-4, the alternative ligand for B-7 is an inhibitory receptor, limiting T-cell activation, and resulting in less IL-2 production. CD28, which is constitutively expressed, initially interacts with B7, producing signal 2 that leads to T-cell activation. The upregulation of CTLA-4, which has a higher affinity to B7 than CD28, limits the degree of activation. Signals 1 and 2 activate 3 signal transduction pathways: the calcium-calcineurin pathway, the RAS-mitogen-activated pathway (MAP), and the nuclear factor-κB pathway. These pathways activate transcription factors that bind to regulatory proteins in enhancer regions of specific genes involved in proliferation and differentiation, including cytokines such as IL-2, cytokine receptors such as IL-2R (CD25), and receptors involved in co-stimulation such as CD40L (CD154). Interleukin and other cytokines produce signal 3 via activation of the mammalian target of rapamycin (mTOR) pathway. This pathway is the trigger for cell proliferation (see Fig. 10-2).

Proliferation and differentiation lead to a large number of effector T cells. The primary mechanism of the destruction of an allograft is by generation of T cells that leave the lymph nodes, migrate to the allograft, and are cytotoxic for the cells of the graft. Graft-cell lysis is accomplished through the direct action of T cytotoxic cells, including release of secretory granules containing granzyme B and perforin and by the induction of apoptosis, and by the activation of cascading enzyme systems, including the complement, clotting, and the kinin pathways. Other cellular mediators such as plasma cells, macrophages, platelets, and polymorphonuclear leukocytes also migrate into the allograft and have both a direct and indirect role in allograft rejection. B cells are activated when antigen engages their antigen receptors, usually in lymphoid follicles or in extrafollicular sites, such as the red pulp of the spleen or in the transplant, producing alloantibody against donor MHC antigens. Alloantibody targets capillary endothelium and fixes complement that results in cell lysis, thrombosis, and ischemia.

The severity of the immune response to allografts differs greatly between dogs and cats. Dogs mount a virulent rejection response, and without immunosuppression, a MHC-mismatched renal allograft is destroyed in 6 to 8 days. In cats, a MHC-mismatched renal allograft will function for approximately 23 days.

The Tempo of Rejection

Three overlapping types of organ rejection are recognized clinically. Hyperacute rejection is an accelerated form of rejection that is associated with naturally occurring or preformed circulating antibody in the serum of the recipient that reacts with donor cells, particularly the endothelium of blood vessel walls as described above. In hyperacute allograft rejection, the recipient has been sensitized to the allograft MHC antigens by previous blood transfusions, pregnancy, or transplantation. Preexisting antibody can be identified before transplantation by lymphocyte cross-match, which involves testing leukocytes of the potential donor with serum of the recipient in the presence of complement. If preexisting antibody is present in the serum, the host leukocytes will be lysed. In addition, antibodies against the donor's blood type (erythroid cell surface antigens) can cause hyperacute rejection. The donor and recipient should be tested for blood type compatibility and a blood cross match should be performed prior to transplantation.

Acute allograft rejection typically occurs 7 to 21 days after transplantation or when effective immunosuppression is terminated. Pathologic studies of the rejected organ reveal a predominant pattern of mononuclear leukocyte infiltration in the tissue.

Chronic rejection is characterized by gradual loss of organ function over months to years, often without any clinically recognized rejection episode. Chronic rejection is a major cause of death of all human organ transplant recipients and the primary cause of death for heart transplant recipients. Kidneys undergoing chronic rejection show severe narrowing of numerous arteries and thickening of the glomerular capillary basement membrane. Heart transplants show progressive thickening of the coronary arteries caused mainly by smooth muscle cell proliferation and migration. Occlusion of coronary artery blood flow results in diminished function of the cardiac muscle, and, eventually, in myocardial infarction. The factors causing chronic rejection are multiple and appear to produce chronic inflammation and injury via both immune and nonimmune mechanisms [5]. Factors that have been implicated in the development of chronic rejection in human beings include donor age, donor race, HLA mismatches, ischemia time, reperfusion injury to the allograft, viral infections, hyperlipidemia, hypertension, acute and subacute rejection, and drug toxicity. Many peptide growth factors, including transforming growth factor β, platelet-derived growth factor, and the fibroblast growth factors, have been shown to be upregulated in chronically rejecting organs. Growth factors have been shown in vitro and in vivo to promote fibroplasia, collagen synthesis, and smooth muscle cell proliferation and migration. Currently, growth factor inhibitors, coenzyme A reductase inhibitors, antiviral strategies, and changes in organ procurement and preparation are being investigated to reduce the incidence and severity of chronic rejection.