Rejection After Transplantation

Models Used to Define Rejection

Our current understanding of rejection is derived from human and animal data. In vitro study of cells, direct histological examination of the allograft, and animal models of acute rejection are the main methods used to understand the mechanisms of acute liver transplant rejection. Because current concepts of rejection are based on these models, it is important to be aware of the limitations of a given model relative to our total understanding of the transplantation process. In vitro coculture systems, such as cell-mediated cytotoxicity, define potential cellular interactions and have proved invaluable in the definition and characterization of helper T cells, cytotoxic T cells, and antigen-presenting cells (APCs). The in vitro system is limited, however, because the coculture environment allows for optimization of culture conditions and the relevant cytokines. As such, it may not reflect the in vivo response, in which cytokines may be absent or at low levels and the precursor cells may be compartmentalized rather than in direct proximity with each other or with host antigen.

As an alternative to isolated in vitro models, one may remove cells from the graft and examine their functional capacity in vitro. In this way the cells can be activated and undergo the immunological events occurring in the graft and then be examined in vitro. The cells of interest could be recovered from peripheral blood or biopsy specimens or by using other techniques, such as an allogeneic-coated sponge matrix allograft. For example, the ability of the cells found in the peripheral blood after transplantation to damage target cells sharing major histocompatibility antigens with the donor can be studied. The cells may be used directly in a cytotoxicity assay if enough cells can be removed. Using this approach, one is dependent on the adequacy of the in vitro assay to infer the in vivo function of the cell. If only a few cells can be directly removed, they may be cultured in vitro to expand the population. This approach has been criticized because the in vitro culturing and expansion are done in the presence of donor antigen and therefore may select for recipient cells with reactivity against donor antigen. The use of peripheral blood cells and cell products to study the process in the graft depends upon the material in the peripheral blood reflecting what is occurring in the graft or other immune compartments.

Histological examination of acute cellular rejection allows recording of the rejection response, with the presence of a specific cell type being used to infer the importance of that cell in the rejection process. However, the histological picture does not help us understand the cell-cell interaction or the specific mechanisms of the immune response. As methods for determining the state of activation of a cell improve, the inferences regarding the role of a given cell type may become more accurate. The use of immunohistochemistry is expanding rapidly with the ability to examine tissue for a wide number of proteins. This field may make important contributions to our understanding of the rejection process.

Animal models may be limited because of different responses to alloantigen in different species, different sensitivity to immunosuppression, variation in the toxicity profile of agents in different species, tolerance to portal revascularization, and the ease with which tolerance is induced in some models. Perhaps the models that have the greatest potential to define specific cellular mechanisms are those in which the host animal is depleted of responding cells and is subsequently repopulated with one or more specific cell types or clones (i.e., adoptive transfer experiments). Although this approach does not address the issue of the compartmentalization of cells or cytokines within the host, it is useful for screening of cell types that can injure allografts. Use of mouse models reconstituted with the human immune system may allow expansion of the understanding of rejection.

The mouse is frequently used in models to define allograft response. Although the ability to perform liver transplantation in the mouse is limited, some investigators have achieved success in this area and found that success after liver transplantation did not follow major histocompatibility complex (MHC) disparity between donor and recipient as would be predicted by in vitro coculture studies. Because of the technical difficulties involved in the mouse liver transplantation procedure, the rat is the model most frequently used to study liver allograft response. The rat, which has been a common model for the testing of factors related to liver preservation and transplantation, does not require rearterialization for successful liver transplantation. Rat donor-recipient combinations vary widely in the kinetics of the rejection response and the ability to induce tolerance with short courses of immunosuppression ; the explanation of this variability is unclear but must be borne in mind by investigators planning to study a specific treatment modality.

Rejection has been described extensively in rat liver allografts; the histological differences in the high versus the low responder have been mainly in the kinetics of the rejection response and in different patterns of infiltrating cells. Rejection has also been described in larger-animal models, and these findings offer the advantage of being more clinically relevant. Unlike most rat liver allografts, canine and porcine allograft tolerance with a short course of immunosuppression is not to be expected.

Another factor that separates animal studies from the experience in humans is the historical exposure to infectious agents. Exposure to infectious agents in specific pathogen-free laboratory rodents can be controlled, unlike in large nonhuman primates and human patients. This acquired immune history in humans may result in a heterologous immune response—specifically, virally induced alloreactive memory—that is a potent barrier to tolerance induction.

Thus an understanding of the human liver allograft response depends on a composite of animal models and observations and of the histological patterns and response to treatment seen in liver transplant patients.

Cellular Basis for Liver Allograft Rejection

Acute rejection is the most common type of rejection after liver transplantation. The histological characteristics of acute rejection are defined by lymphocyte infiltration of the portal tracts as well as bile duct injury and inflammation of the venous endothelium. If acute rejection is untreated, hepatocytes may demonstrate signs of injury, either from direct immune attack or as an effect of vascular injury and subsequent ischemia.

The lymphocyte is believed to be the dominant cell type in acute cellular rejection based on infiltrates. The lymphocyte population contains both CD4 ⁺ and CD8 ⁺ , and once activated, these cells proliferate, differentiate, and secrete cytokines. The appearance of CD4 ⁺ lymphocytes within the portal tracts predicts rejection even before biochemical evidence is present. At the time of the rejection response (as defined by injury to the small bile ducts and elevated serum alkaline phosphatase level), the predominant lymphocyte within the portal tracts displays the CD8 ⁺ phenotype. Acute rejection and chronic rejection have been correlated with the presence of CD8 ⁺ within portal tracts. Investigators at the Mayo Clinic have found an association between the patterns of the inflammatory response and the response to treatment of rejection. These investigations indicate that CD4 ⁺ cells are important in initiating and amplifying the immune response and that CD8 ⁺ cells have an important effector role in the rejection process.

The other cells present within the portal tracts are considered to be part of a nonspecific inflammatory response. Their appearance likely reflects the influence of local cytokines in the inflammatory response. Polymorphonuclear neutrophils appear in the setting of late or partially treated rejection but also appear with acute cholangitis and cytomegalovirus infection, making the diagnosis of rejection versus biliary disease particularly difficult. Eosinophils are a frequent feature of liver allograft rejection, although the relative amount of these cells is generally less than 5%. Peripheral eosinophilia has also been identified in kidney and liver allograft recipients with acute rejection. CD4 ⁺ helper T cells type 2 (T _H 2) secrete interleukin (IL)-5, which has been identified in rejecting liver allografts and is known to attract eosinophils to the portal triads, causing further inflammation and injury. The identification of message for IL-5 in rejecting liver allografts supports the role of eosinophils in liver graft rejection. The predictive value of the blood eosinophil count in the diagnosis of acute cellular rejection and the value as a marker of response to treatment have been examined. An elevated eosinophil count has a positive predictive value for acute cellular rejection of the liver, whereas a normal count generally excludes moderate or severe rejection. Improvement in peripheral eosinophilia may also be useful in predicting histological improvement after treatment of rejection.

Macrophages and plasma cells make up the remainder of the cells present at the graft site. The macrophages are presumed to be part of a nonspecific inflammatory response. Multiple studies have attempted to identify a pattern of peripheral blood lymphocytes that can be associated with specific types of rejection. Although differences in peripheral blood lymphocyte subsets have been identified in transplant recipients, research has failed to reliably establish peripheral lymphocytes as a clinically significant marker of rejection.

Class I MHC molecules (human leukocyte antigen [HLA]-A, HLA-B, HLA-C) are expressed on all nucleated cells. They consist of a 44-kD heavy chain noncovalently linked to β ₂ -microglobulin. Class II MHC molecules (HLA-DR, HLA-DP, HLA-DQ) contain a 34-kD α chain and a 29-kD β chain. These molecules are normally expressed on APCs (macrophages and dendritic cells), activated T cells and B cells, and Kupffer cells. According to classic models, CD4 ⁺ cells recognize antigens (i.e., exogenous peptides) in the context of MHC class II antigen, and CD8 ⁺ cells recognize antigens (i.e., endogenous or viral antigens) in the context of class I antigens. Essential to the initiation of acute cellular rejection is recipient T-cell recognition of allo-MHC-peptide complexes on donor APCs. Transplanted livers contain large numbers of donor APCs, and these serve as the primary stimulation of the recipient’s alloreactive T-cell receptors.

Ibrahim et al and Dollinger et al analyzed rejecting versus nonrejecting specimens according to cell phenotype and location using markers of cell activation. Proliferation of mononuclear leukocytes inside the liver allograft was a prominent feature of acute rejection; they were located predominantly in the portal tracts at the site of the inflammatory infiltrate and were found to decrease in response to treatment with corticosteroids. Increased numbers of portal CD3 ⁺ T cells were found in rejecting compared with nonrejecting liver grafts. The increased number of CD3 ⁺ cells could be accounted for mainly by an increased number of CD8 ⁺ cells. Examination of the CD45 marker revealed an increase in memory cells (CD45 RO ⁺ ) but not in “naive cells” (CD45 RA) ; these were located periportally. CD8 ⁺ T lymphocytes, CD57 ⁺ natural killer (NK) cells, and CD68 ⁺ macrophages, however, were located intraparenchymally throughout the lobules, whereas CD20 ⁺ B lymphocytes were present only in some of the portal tracts. It is believed that the CD8 ⁺ cells that are present cause graft injury via cytolytic activity directed against donor alloantigen. This scheme is consistent with classic models of cytotoxic effector cells that are dependent on helper T cells for differentiation and maturation.

Other investigations have found that early acute rejection is also characterized by a higher expression of CD4 ⁺ CD7 ⁺ and CD8 ⁺ CD38 ⁺ T lymphocytes in the liver than in peripheral blood. Moreover, a preferential proinflammatory (T _H 1; see CD4 ⁺ Cells and Rejection) cytokine profile was related to liver-resident T lymphocytes in comparison with corresponding plasma. This change was accompanied by a decrease in regulatory T cells (Tregs) in patients with acute rejection. These studies suggest that a T _H 1 immune mechanism works in a local fashion and may be involved in acute rejection as indicated by the reduction of Tregs in the liver and blood.

Investigations at Duke support an effector role of memory CD8 ⁺ cells. These cells may be independent of the requirement of CD4 ⁺ cell-based help and capable of maturation and cytolytic function in the presence of inflammatory mediators and cytokines. The central role of the lymphocyte in acute liver allograft rejection is also supported by the Pittsburgh group led by Zeevi and Duquesnoy. These investigators were able to culture clones of antidonor reactive cells from small liver biopsy specimens in the setting of acute rejection, but they were unsuccessful when rejection was not present. Although lymphocytes infiltrated the biopsy specimens of patients with hepatitis, the lymphocytes were much more resistant to in vitro propagation compared with cells in the biopsy specimens of patients with rejection. This work has been confirmed by Kolbeck et al in liver transplant recipients.

Other investigators have shown similar antidonor activity of cells cultured from rejecting kidney allografts and that lymphocyte propagation correlates with rejection. The reactive clones identified in this work demonstrated antidonor cytolytic activity and were directed against either class II or class I HLA of the donor. The fact that lymphocytes from biopsy specimens showing no rejection could also be propagated with alloreactivity demonstrates the particular conditions used: donor alloantigen in the form of the liver tissue and exogenous IL-2 favor alloreactive lymphocyte proliferation. The work from the Pittsburgh group in liver and cardiac allograft recipients indicates that the presence of antidonor reactive cells within biopsy specimens may precede evidence of injury to specific liver or heart targets.

The induction of tolerance in liver transplantation is associated with an increased rate of apoptosis of T lymphocytes in the portal inflammatory infiltrate and the presence of an intragraft T _H 2-like T-cell population. Kupffer cells reside in the hepatic sinusoids and are believed to be able to directly interact with circulating T lymphocytes. As such, Kupffer cells may play a unique role in immunomodulation. Recent investigation has demonstrated the Kupffer cells can suppress T-cell proliferation in vitro in mixed leukocyte reactions. In addition, Kupffer cells express functional Fas ligand and can induce apoptosis of Fas-positive cells. This process can be blocked by the addition of neutralizing anti-Fas ligand antibody. Using an allogeneic liver transplant model, Sun et al have demonstrated that Kupffer cells recovered from chronically accepted hepatic allografts have increased Fas ligand messenger RNA (mRNA) and protein expression. In addition, they have a greater ability to induce apoptosis of alloreactive T cells compared with Kupffer cells obtained from animals with acute rejection. Furthermore, the authors have demonstrated that Kupffer cells not only induce apoptosis of T cells, but also regulate cytokine production and the T _H 2/T _H 3-like cytokine mRNA expression in allogeneic mixed lymphocyte reaction. Finally, they were able to demonstrate that administration of Kupffer cells derived from chronically accepted liver allografts actually prolongs the survival of hepatic allografts in animals with acute rejection.

CD4 ⁺ Cells and Rejection

The concept that CD4 ⁺ cells have a central role in liver allograft rejection by acting through CD8 ⁺ effector cells is supported by clinical observation. Agents that act through suppression of IL-2 production and release, such as cyclosporine and tacrolimus, play a major role in inhibition of the rejection response. Helper T cells have been categorized as helper T cells type 1 (T _H 1) or helper T cells type 2 (T _H 2) based on a function and cytokine profile. These cytokines result in the activation, proliferation, and differentiation of other lymphocytes that have been implicated in tolerance induction and acute cellular rejection. When CD4 ⁺ T _H 0 cells are activated, IL-2, IL-4, and interferon-γ are secreted. This activation results in two phenotypes: (1) T _H 1 cells producing IL-2, interferon-α, and supporting cellular responses, including acute allograft rejection (generating cytotoxic T lymphocytes and activating macrophages); and (2) T _H 2 cells producing IL-4, IL-5, IL-6, IL-10, and IL-13 and supporting humoral (antibody-mediated [immunoglobulin (Ig)G1 and IgE]) responses. In addition, these cytokines may counteract acute cellular rejection by suppressing delayed-type hypersensitivity and inhibiting activation of macrophages induced by T _H 1 cells.

Recently a new subset of CD4 ⁺ T cells, T _H 17 cells, characterized by their section of IL-17, has also been implicated in acute cellular rejection. Studies show that these cells may be stimulated by IL-6 and transforming growth factor secreted by Kupffer cells. Both infiltrates of T _H 17 cells and their secreted IL-17 are increased in allografts compared to isografts in rat models of liver transplantation.

In long-surviving hepatic allografts in animals, a T _H 2 cytokine profile appears to predominate and T _H 1 cytokines are absent. Conversely, a T _H 1 cytokine profile predominates in rejection. IL-10 may be instrumental in the shift in phenotype from T _H 1- to T _H 2-dominant profile necessary to achieve a state of tolerance. IL-10 downregulates the expression of the costimulatory molecule B7, diminishing T-cell activation and potentially inducing T-cell anergy. In rat models of liver transplantation, rats undergoing isograft transplantation have higher IL-10 levels, as well as less rejection and longer survival, compared rats undergoing allograft transplantation. Similarly, rats undergoing allograft transplantation have higher serum interferon-γ (T _H 1 cytokine) levels. Furthermore, transplanted rats treated with 1,25-(OH) ₂ vitamin D3 had lower interferon-γ levels, higher IL-10 levels, lower rejection activity index, and prolonged survival, which also supports the concept that a shift to a T _H 2 type cytokine profile may prevent rejection.

In vitro models of immune response using human or murine cells also support a central role for CD4 ⁺ cells and strong supporting roles for macrophages (or APCs) and cytotoxic lymphocytes. Lafferty’s two-signal hypothesis is well accepted : donor antigen provides one signal, and APCs provide a second signal to effectively stimulate responder CD4 ⁺ cells. These cells elaborate cytokines, the most important of which is IL-2. IL-2 in turn stimulates the proliferation of additional CD4 ⁺ cells and the proliferation and maturation of CD8 ⁺ cells via newly expressed IL-2 receptors. Both CD4 ⁺ T cells and CD8 ⁺ T cells can act as cytotoxic T lymphocytes. However, analysis indicates the CD8 ⁺ cells are the primary effector cells that infiltrate the bile ducts and cause apoptosis (CD8-to-CD4 ratio of 5:1). Mature cytolytic CD8 ⁺ cells can damage donor tissue through contact and the release of active enzymes.

The in vitro models of immunoactivity have generally used lymphoid cells such as splenocytes for all three components of the immune response: responder cells, stimulator cells, and target cells. However, the applicability of in vitro studies to organ transplantation may be served better by substituting parenchymal cells for the stimulator cells and target cells. Lymphoid cells from the graft may serve as the sensitizing antigen at the graft site, or they may migrate out of the graft and elicit host sensitization at another site. Graft parenchymal cells may provide sensitization of the host by having their antigens indirectly presented through host APCs. Because the graft is damaged as a result of the rejection response, one would assume that the donor parenchymal cells represent an appropriate target for the antidonor response seen in rejection. Using the mixed lymphocyte-hepatocyte coculture system, purified murine hepatocytes or nonparenchymal cells (Kupffer cells, epithelial cells, and endothelial cells) can stimulate the development of specific antidonor cytotoxic cells. Because hepatocytes express only class I antigens on their surfaces, using purified hepatocytes as simulators requires the presence of APCs within the responder cell population; removal of these APCs abrogates the cytolytic response. Work by Bumgardner et al has demonstrated that the immune response to murine parenchymal and nonparenchymal cells involves both direct and indirect antigen presentation. The cytotoxic response of lymphocytes that develop in mixed lymphocyte-hepatocyte coculture is generally tested with blast targets sharing MHC antigens with the stimulating hepatocytes in a chromium release assay. Hepatocytes may also be used as targets, but instead of using chromium labeling, transaminase release from injured hepatocytes has been monitored.

Transplant Tolerance and Regulatory T Cells

Tolerance, or the survival of a liver allograft without the need for ongoing immunosuppression, has been described for decades in both animal models and small patient populations. A short course of immunosuppression therapy in the rat can lead to long-term graft acceptance without the need for further therapy. In human transplantation, multiple institutional reports have documented successful withdrawal of immunosuppression without injury to the allograft. In fact, in HCV patients there may actually be a benefit to immunosuppression withdrawal. Recently Feng et al conducted a prospective study of complete immunosuppression withdrawal in a pediatric population of living donor liver transplant recipients. This study found that 60% of recipients were able to tolerate complete withdrawal without allograft dysfunction for at least 1 year. This finding has tremendous implications for future trials because certain recipients or recipient/donor combinations clearly allow for operational tolerance. Even more appealing would be the identification of these individuals with the goal of understanding their tolerance and using this information to help all transplant recipients.

In the discussion of transplant tolerance, Tregs have also moved to the forefront. Tregs can be classified into naturally occurring and induced. Although multiple subsets of Tregs have been described, it is the CD4 ⁺ CD25 ⁺ FOXP3 Tregs that have generated the most interest in relation to liver transplant tolerance. Initially discovered in the 1970s, CD4 ⁺ CD25 ⁺ T cells were shown to be critical in self-tolerance based on the development of autoimmune diseases in CD4 ⁺ CD25 ⁺ –depleted mice. Graca et al went on to demonstrate that induction of tolerance in skin grafts involved the presence of Tregs. Finally, the transcription factor forkhead box P3 (FOXP3) was found to be the master regulator of CD4 ⁺ CD25 ⁺ Tregs, with deletion of FOXP3 leading to a loss of Treg suppressive abilities and development of autoimmune diseases.

Tregs themselves suppress immune responses through multiple mechanisms. These include secretion of inhibitory molecules such as IL-10, IL-35, and TGF-β. Furthermore, Tregs can suppress the immune system by inducing cell death through the creation of molecules such as granzyme and perforin.

At this point, multiple studies have demonstrated the role of Tregs in animal models of transplant tolerance. Tregs have been shown to be increased in the periphery of a spontaneous mouse model of liver transplant tolerance and CD4 ⁺ CD25 ⁺ FOXP3 cells are present in human liver transplant recipients who have achieved tolerance. Similarly, the expression of Treg effector molecules is predictive of tolerance. However, the use of Tregs in the clinic is just beginning. Both the stimulation of native Tregs and the transfusion of ex vivo expanded Tregs are being proposed as possible treatments. The use of Treg effector molecules may also prove useful in the promotion of tolerance or prevention of rejection. Key to progress in this field was the finding that Tregs can be isolated and expanded ex vivo, all while retaining their immune function. Under this tenant, small trials of ex vivo expanded Tregs in patients with hematological malignancy undergoing bone marrow and stem cell transplant have been completed, all with an improvement in graft-versus-host disease. This success has spurred greater interest in the use of Tregs in solid organ transplantation, and multiple studies are on the horizon with the goal of achieving tolerance or at least minimizing immunosuppression.