Mechanisms of rejection: current perspectives

Rejection is the major barrier to successful transplantation. The immune response to an allograft is an ongoing dialogue between the innate and adaptive immune system that if left unchecked will lead to the rejection of transplanted cells, tissues, or organs. Activation of elements of the innate immune system, triggered as a consequence of tissue injury sustained during cell isolation or organ retrieval and ischemia reperfusion, will initiate and amplify the adaptive response. T cells require a minimum of two signals for activation, antigen recognition, and costimulation. The activation requirements of naive T cells are more stringent than those of memory T cells. Memory T cells are present in the majority of transplant recipients as a result of heterologous immunity. The majority of B cells require help from T cells to initiate antibody production. Antibodies reactive to donor human leukocyte antigen molecules, minor histocompatibility antigens, endothelial cells, RBCs, or autoantigens can trigger or contribute to rejection early and late after transplantation. Antibody-mediated rejection triggered by alloantibody binding and complement activation is recognized increasingly as a significant contribution to graft loss. Even though one component of the immune system may dominate and lead to rejection being described in short hand as T cell or antibody mediated, it is usually multifactorial resulting from the integration of multiple mechanisms. Identifying the molecular pathways that trigger tissue injury, signal transduction and rejection facilitates the identification of targets for the development of immunosuppressive drugs.     

Graft produced interleukin-6 functions as a danger signal and promotes rejection after transplantation

BACKGROUND: Interleukin (IL)-6 is a pleiotropic cytokine that functions in both the innate and adaptive immune responses. However, the role of IL-6 in allograft rejection remains poorly understood. METHODS: In this study, we demonstrate a critical role for graft-produced IL-6 in allograft rejection in a murine model of cardiac allograft transplantation. RESULTS: The results show that IL-6-deficient grafts transplanted into allogeneic wild-type recipients have significantly prolonged survival, approximately three times the survival time of wild-type controls. In contrast, allogeneic cardiac transplants into IL-6-deficient recipients do not have prolonged graft survival, indicating that donor graft cells are the relevant source of IL-6. Our investigation of potential mechanisms shows that graft-produced IL-6 promotes the activation of peripheral CD4 and CD8 T cells. Furthermore, we show that IL-6 deficiency prolongs graft survival only in the presence of CD25+ T cells that have a phenotype consistent with regulatory T cells. Interestingly, IL-6 production by the graft is triggered by antigen-independent innate immune mechanisms. CONCLUSIONS: Thus, our results suggest the paradigm that graft rejection versus tolerance is determined by a balance between the activation of effector T cells versus immune suppression by regulatory T cells, and that after transplantation, IL-6 functions as a systemic danger signal that overcomes constitutive immune suppression mediated by regulatory T cells and promotes the activation of effector T cells.     

Late CD8+ T cell-dependent xenoantibody production in innate tolerant nude rats after hamster islet grafting but not after hamster heart grafting

BACKGROUND: Xenograft rejection can be provoked by both the innate and adaptive immune compartments and close reciprocal interactions exist between these two systems. We investigated the interdependent roles of T and B lymphocytes in vascularized (heart) and cellular (islet) xenograft rejection in a model with established xeno-nonreactivity of the innate immune system. METHODS: Specific innate xenotolerance was induced in nude rats bearing either a hamster heart or a hamster pancreatic islet graft by a tolerizing regimen consisting of donor antigen infusion, temporary natural killer cell depletion and a 4-week administration of leflunomide. One month after transplantation, syngeneic CD4 and CD8 T cells were adoptively transferred. RESULTS: Both vascular and cellular xenografts were rejected after CD4 T cell reconstitution, corresponding with production of high IgM and IgG xenoantibody titers. Deposition of xenoantibodies and complement was seen in the heart but not in the islet xenografts. After infusion of CD8 T cells, xenohearts underwent a delayed type of rejection without xenoantibody production and xenoislets were not rejected. In xenoislet recipients, CD8 dependent B cells were not tolerized, resulting in the production of IgG xenoantibodies belonging to Th2-dependent isotypes, known not to cause graft rejection, and deposited at the graft implantation site. CONCLUSIONS: We conclude that distinct mechanisms of immune activation underlie xenogeneic reactions against vascular and cellular grafts.     

Regulatory functions of alloreactive Th2 clones in human renal transplant recipients

BACKGROUND: Chronic allograft rejection is the major clinical problem in organ transplantation. There is evidence that indirect T cell recognition of donor-specific HLA peptides may play an important role in the immunopathogenesis of chronic allograft rejection. We have recently shown that HLA allopeptide-specific T cell clones generated from renal transplant recipients with chronic allograft nephropathy are of the Th1 phenotype, while those from stable patients are Th2. There is evidence in experimental animal models of autoimmunity and transplantation that Th2 cells may function to regulate immune responses, but the biological relevance of these observations in humans has not been reported. METHODS: The purpose of this study was to investigate the putative regulatory functions of alloreactive human Th2 clones. HLA-DR allopeptide-specific Th1 and Th2 cell clones were generated from peripheral blood lymphocytes of human renal allograft recipients with chronic allograft nephropathy (CAN) or with stable renal function (SRF), respectively. RESULTS: An in vitro co-culture system showed that the proliferative responses of Th1 clones from patients with CAN were significantly inhibited by the Th2 clones in response to the donor-derived HLA allopeptides. In addition, co-culture of the Th2 clones inhibited cytokine production (IFN-gamma) by the Th1 clones in response to the donor-specific peptides. The regulatory functions of Th2 clones were antigen-specific since they only occurred when both the Th1 and Th2 clones were reactive to the same HLA-DR allopeptide, and were mediated by IL-4 and IL-10. CONCLUSIONS: This is the first demonstration, to our knowledge, indicating that Th2 cells may function to regulate indirect Th1 alloimmune responses that are critical for the progression of CAN in humans.     

The role of macrophages in allograft rejection

Macrophage accumulation has long been recognized as a feature of allograft rejection, yet the role of macrophages in rejection remains underappreciated. Macrophages contribute to both the innate and acquired arms of the alloimmune response and thus may be involved in all aspects of acute and chronic allograft rejection. Recent advances in macrophage biology have allowed a better understanding of the mechanisms of macrophage accumulation, their state of activation and the pleuripotent roles they play in allograft rejection. Therapeutic attention to macrophages, in addition to T lymphocytes, may lead to improved outcomes in organ transplantation.     

Principles of Transplantation

The immunological barriers that provide protection from pathogens also provide a major hurdle to successful organ transplantation. Immune responses to infectious pathogens can be divided into innate and acquired responses. Innate immune mechanisms are non-specific and include physical barriers, complement-mediated phagocytosis, and the acute inflammatory reaction. In contrast, the acquired immune response shows specificity, memory, and improves with repeated exposure. Sir Peter Medawar showed in the 1940s that graft rejection exhibits all the characteristics of an acquired immune response.This article provides an overview of the cells and antigens involved in recognition of an allograft and describes the cellular pathways and molecular mechanisms by which a graft may be rejected. The emphasis is on acute, rather than chronic, rejection.     

Acute allograft rejection occurs independently of inducible heat shock protein-70

Dendritic cells (DCs) are key mediators of the innate response to transplantation. Yet, the substances that activate these cells during acute allograft rejection remain elusive. Previous work has suggested that heat shock protein (HSP)-70 is associated with acute allograft rejection. Hence, the goal of this study was to determine whether HSP-70 activates DCs and plays a critical role in acute allograft rejection in an experimental model that is dependent on innate MyD88 signaling. Our in vitro data indicate that HSP-70 does not activate DCs. In vivo transplant studies demonstrate that HSP-70 levels are not increased during acute allograft rejection and that an absence of the inducible form of HSP-70 neither delays acute allograft rejection, impairs DCs maturation, nor alters Th1 immune responses during acute allograft rejection. In conclusion, our results indicate that HSP-70 in our experimental models does not play an essential role in acute allograft rejection.     

Chronic allograft rejection. Do the Th2 cells preferentially induced by indirect alloantigen recognition play a dominant role?

Chronic rejection has been the major obstacle to the long-term allograft survival in the clinic. Although the etiology of this rejection reaction is multifactorial, alloantigen-specific immune activation plays the most critical role. We herein hypothesize that CD4+ Th2 cells that are preferentially induced by the indirect recognition of allogeneic histocompatibility antigens late in transplantation may play the most critical role in the initiation and/or maintenance of chronic allograft rejection. Immunosuppression used to prevent acute rejection and the nature of antigen-presenting cells and alloligands in the graft may all contribute to immune deviation to the Th2 response. This response may be further perpetuated by type 2 cytokines conceivably produced by activated macrophages, NK cells, and CD8+ T cells in the graft. Cytokines and growth factors induced by this type 2 response, in turn, allow for activation of B, endothelial, and smooth muscle cells that collectively contribute to the pathogenesis of chronic allograft rejection by producing alloantibodies and growth hormones required for interstitial fibrosis, extracellular matrix deposition, and vascular neointimal hyperplasia.     

The T cell as a bridge between innate and adaptive immune systems: implications for the kidney

The T cell as a bridge between innate and adaptive immune systems: Implications for the kidney.The immune system is classically divided into innate and adaptive components with distinct roles and functions. T cells are major components of the adaptive immune system. T cells are firmly established to mediate various immune-mediated kidney diseases and are current targets for therapy. Ischemic acute renal failure, a major cause of native kidney and allograft dysfunction, is mediated in part by inflammatory components of the innate immune system. However, recent data from experimental models in kidney as well as liver, intestine, brain and heart implicate T cells as important mediators of ischemia reperfusion injury. These data reveal new insights into the pathogenesis of ischemic acute renal failure, as well as identify novel and feasible therapeutic approaches. Furthermore, the identification of T cells as a mediator of early alloantigen-independent tissue injury demonstrates that the functional capacity of T cells spreads beyond adaptive immunity into the realm of the innate immune response.     

Innate immunity alone is not sufficient for chronic rejection but predisposes healed allografts to T cell-mediated pathology

BackgroundAcute allograft rejection is dependent on adaptive immunity, but it is unclear whether the same is true for chronic rejection. Here we asked whether innate immunity alone is sufficient for causing chronic rejection of mouse cardiac allografts.MethodsWe transplanted primarily vascularized cardiac grafts to recombinase activating gene-knockout (RAG^?/?) mice that lack T and B cells but have an intact innate immune system. Recipients were left unmanipulated, received adjuvants that stimulate innate immunity, or were reconstituted with B-1 lymphocytes to generate natural IgM antibodies. In a second model, we transplanted cardiac allografts to mice that lack secondary lymphoid tissues (splenectomized aly/aly recipients) and studied the effect of NK cell inactivation on T cell-mediated chronic rejection.ResultsAcute cardiac allograft rejection was not observed in any of the recipients. Histological analysis of allografts harvested 50 to 90 days after transplantation to RAG^?/? mice failed to identify chronic vascular or parenchymal changes beyond those observed in control syngeneic grafts. Chronic rejection of cardiac allografts parked in splenectomized aly/aly mice was observed only after the transfer of exogenously activated T cells. NK inactivation throughout the experiment, or during the parking period alone, reduced the severity of T cell-dependent chronic rejection.ConclusionsThe innate immune system alone is not sufficient for causing chronic rejection. NK cells predispose healed allografts to T cell-dependent chronic rejection and may contribute to chronic allograft pathology.     

Innate immunity and cardiac allograft rejection

The development of immunosuppressive drugs to control adaptive immune responses has led to the success of heart transplantation as a therapy for end-stage heart failure. However, these agents are largely ineffective in suppressing components of the innate immune system. This distinction has gained clinical significance as mounting evidence now indicates that innate immune responses have important roles in the acute and chronic rejection of cardiac allografts including cardiac allograft vasculopathy (CAV). Whereas clinical interest in natural killer (NK) cells was once largely confined to the field of bone marrow transplantation, recent findings suggest that these cells can also participate in the acute rejection of cardiac allografts and in the development of CAV. Stimulation of Toll-like receptors (TLRs), another important component of innate immunity, by endogenous ligands released in response to ischemia/reperfusion is now known to cause an inflammatory milieu favorable to graft rejection. Finally, new data indicate that activation of complement is linked to acute rejection and CAV. In summary, the conventional wisdom that the innate immune system is of little importance in whole-organ transplantation is no longer tenable. The addition of strategies that target TLRs, NK cells, and complement will be necessary to prevent CAV completely and to eventually achieve long-term tolerance to cardiac allografts.     

Effects of immunosuppressive agents on Th17 cells involved in transplantation

The lymphocyte‐derived helper T (Th) cells are critical regulators of the adaptive immune response and are associated with inflammatory disease. The most recently recognized Th‐cell lineage, Th17, plays an important role in host defense against extracellular pathogens by secreting the proinflammatory cytokine, interleukin 17, and recruiting reactive oxygen species (ROS)‐producing monocytes to the site of infection. However, accumulating evidence has implicated Th17‐cell dysregulation as an underlying cause for some immune‐related pathogenic conditions, including allograft rejection. Recent studies of human transplant patients have indicated that Th17 cells exhibit resistance to current immunosuppressive therapies that would otherwise prevent allograft rejection. In this review, we will discuss the most current research findings related to Th17‐cell function in various kinds of allografts.     

Role of secondary lymphoid tissues in primary and memory T-cell responses to a transplanted organ

Secondary lymphoid tissues are the hub of adaptive immune responses wherein rare cognate lymphocytes encounter dendritic cells bearing antigen from peripheral tissues and differentiate into effector and memory cells that eliminate antigen. It is accepted that immune responses against microbial and tumor antigens are initiated within secondary lymphoid tissues. There is less agreement on whether the same principle applies to immune responses to a transplanted organ because an allograft expresses foreign major histocompatibility complex and contains donor antigen presenting cells that could activate T cells directly in situ leading to rejection. Recent studies confirm that although naïve T cells can be primed within the allograft, their differentiation to effect rejection is dependent on secondary lymphoid tissues. Antigen-experienced memory T cells, unlike Naïve T cells, function largely independent of secondary lymphoid tissues to cause allograft rejection. In an alloimmune response, secondary lymphoid tissues support not only immune activation but also immune regulation essential for allograft survival. Here, we will review recent findings and discuss the role of secondary lymphoid tissues in primary and memory alloimmune responses.     

T-cell alloimmunity and chronic allograft dysfunction

Solid organ transplantation is the standard treatment to improve both the quality of life and survival in patients with various end-stage organ diseases. The primary barrier against successful transplantation is recipient alloimmunity and the need to be maintained on immunosuppressive therapies with associated side effects. Despite such treatments in renal transplantation, after death with a functioning graft, chronic allograft dysfunction (CAD) is the most common cause of late allograft loss. Recipient recognition of donor histocompatibility antigens, via direct, indirect, and semidirect pathways, is critically dependent on the antigen-presenting cell (APC) and elicits effector responses dominated by recipient T cells. In allograft rejection, the engagement of recipient and donor cells results in recruitment of T-helper (Th) cells of the Th1 and Th17 lineage to the graft. In cases in which the alloresponse is dominated by regulatory T cells (Tregs), rejection can be prevented and the allograft tolerated with minimum or no immunosuppression. Here, we review the pathways of allorecognition that underlie CAD and the T-cell effector phenotypes elicited as part of the alloresponse. Future therapies including depletion of donor-reactive lymphocytes, costimulation blockade, negative vaccination using dendritic cell subtypes, and Treg therapy are inferred from an understanding of these mechanisms of allograft rejection.     

Innate immunity and organ transplantation: focus on lung transplantation

Ischemia reperfusion injury (IRI) that occurs with solid organ transplantation activates the innate immune system to induce inflammation. This leads to enhanced acute allograft rejection, impaired transplant tolerance and accelerated progression of chronic rejection. In this review, we discuss the innate immune signaling pathways that have been shown to play a role in organ transplantation. In particular, we focus on Toll‐like receptor signaling pathways and how they have influenced outcomes after organ transplantation both experimentally and from clinical studies. Furthermore, we describe the substances that trigger the innate immune system after transplantation and several of the key cellular mediators of inflammation. We specifically point out unique aspects of activation of the innate immune system after lung transplantation. Finally, we discuss the areas that should be investigated in the future to more clearly understand the influence of the innate immune system after organ transplantation.     

NK cells and transplantation

The requirement for cytotoxic T lymphocytes during allograft rejection is controversial. We have demonstrated that CD8+ T cells are not essential for allograft rejection or for the induction of apoptosis in two experimental models of transplantation. To determine candidate cells types which may play a role in the events leading to graft rejection, the cellular composition of rejecting allografts was determined. We demonstrate that substantial numbers of NK cells, of recipient origin, infiltrate allografts as early as 12 h after transplantation. These NK cells produce cytokines and express cytotoxic mediators such as granzyme B and FasL. It is unknown which NK cell receptors are expressed and activated during transplantation. NK cells express multiple cell surface receptors, including MHC class I binding inhibitory receptors, which deliver a negative signal, and activation receptors, which stimulate cytokine secretion and cytotoxicity of NK cells. To begin to understand NK cell activation in the context of transplantation, we have recently cloned a novel rat immunoglobulin-like surface receptor from a rejecting liver allograft. Sequence analysis demonstrates that this putative activation receptor contains 71% identity to human NKp30 at the DNA level, suggesting that it is the rat homologue (rNKp30). Characterization of NK activation receptors may lead to better understanding of the interactions between the innate and adaptive immune responses in transplantation.     

Bronchoalveolar immunologic profile of acute human lung transplant allograft rejection

Bronchoalveolar lavage fluid (BALF) offers a potential means to diagnose acute rejection and could provide insight into the immune mechanisms responsible for lung allograft rejection. Transbronchial biopsies from 29 bronchoscopic procedures were assessed for rejection. Concurrent BALF lymphocyte subsets were examined by flow cytometry, including CD4 and CD8 T cells and their activation status by CD38 expression, natural killer (NK), NK-like T (NT), B, regulatory T, and invariant receptor NK-T cells. Percentages of CD4 were reduced, and CD8 and activation of CD4 T cells correlated with rejection. There were trends for increased NT, reduced NK, and increased B cell percentages with rejection, suggesting potential roles of these cells. Among regulatory cells, the percentages of regulatory T cells decreased and CD4/CD8 invariant NK-T cells increased during rejection, suggesting a proinflammatory profile. A unique BALF lymphocyte profile was associated with rejection and may provide insight into the pathogenesis of allograft rejection.     

Adoptive Transfer of Tracer‐Alloreactive CD4+ T Cell Receptor Transgenic T Cells Alters the Endogenous Immune Response to an Allograft

T cell receptor transgenic (TCR‐Tg) T cells are often used as tracer populations of antigen‐specific responses to extrapolate findings to endogenous T cells. The extent to which TCR‐Tg T cells behave purely as tracer cells or modify the endogenous immune response is not clear. To test the impact of TCR‐Tg T cell transfer on endogenous alloimmunity, recipient mice were seeded with CD4⁺ or CD8⁺ TCR‐Tg or polyclonal T cells at the time of cardiac allograft transplantation. Only CD4⁺ TCR‐Tg T cells accelerated rejection and, unexpectedly, led to a dose‐dependent decrease in both transferred and endogenous T cells infiltrating the graft. In contrast, recipients of CD4⁺ TCR‐Tg T cells exhibited enhanced endogenous donor‐specific CD8⁺ T cell activation in the spleen and accelerated alloantibody production. Introduction of CD4⁺ TCR‐Tg T cells also perturbed the intragraft accumulation of innate cell populations. Transferred CD4⁺ TCR‐Tg T cells alter many aspects of endogenous alloimmunity, suggesting that caution should be used when interpreting experiments using these adoptively transferred cells because the overall nature of allograft rejection may be altered. These results also may have implications for adoptive CD4⁺ T cell immunotherapy in tumor and infectious clinical settings because cell infusion may have additional effects on natural immune responses.     

Non‐Classical Pathways of Cell‐Mediated Allograft Rejection: New Challenges for Tolerance Induction?

Allograft rejection results from separate pathways primarily controlled by CD4+ T cells. Refinement of transplantation models together with investigations on rejection occurring despite co-stimulation blockade revealed unexpected pathways involving CD8+ T cells, NK cells and Th2 cytokines. In this minireview, we discuss these non-classical pathways of allograft rejection and their relevance for the induction of tolerance in the clinics.