Pathogenesis Of Chronic Allograft Rejection Research Articles

Combined abdominal heterotopic heart and aorta transplant model in mice.

Allograft vasculopathy (AV) remains a major obstacle to long-term allograft survival. While the mouse aortic transplantation model has been proven as a useful tool for study of the pathogenesis of AV, simultaneous transplantation of the aorta alongside the transplantation of another organ may reveal more clinically relevant mechanisms that contribute to the pathogenesis of chronic allograft rejection. Therefore, we developed a combined abdominal heart and aorta transplantation model in mice which benefits from reducing animal and drug utilization, while providing an improved model to study the progressive nature of AV. The middle of the infrarenal aorta of the recipient mouse was ligatured between the renal artery and its bifurcation. Proximal and distal aortotomies were performed at this site above and below the ligature, respectively, for the subsequent anastomoses of the donor aorta and heart grafts to the recipient infrarenal aorta in an end-to-side fashion. The distal anastomotic site of the recipient infrarenal aorta was connected with the outlet of the donor aorta. Uniquely, the proximal anastomotic site on the recipient infrarenal aorta was shared to connect with both the inlet of the donor aorta and the inflow tract to the donor heart. The outflow tract from the donor heart was connected to the recipient inferior vena cava (IVC). The median times for harvesting the heart graft, aorta graft, recipient preparation and anastomosis were 11.5, 8.0, 9.0 and 40.5 min, respectively, resulting in a total median ischemic time of 70 min. The surgery survival rate was more than 96% (29/30). Both the syngeneic C57Bl/6 aorta and heart grafts survived more than 90 days in 29 C57Bl/6 recipients. Further, Balb/c to C57Bl/6 allografts treated with anti-CD40L and CTLA4.Ig survived more than 90 days with a 100% (3/3) survival rate. (3/3). This model is presented as a new tool for researchers to investigate transplant immunology and assess immunosuppressive strategies. It is possible to share a common anastomotic stoma on the recipient abdominal aorta to reconstruct both the aorta graft entrance and heart graft inflow tract. This allows for the study of allogeneic effects on both the aorta and heart from the same animal in a single survival surgery.

Role of antibodies to self-antigens in chronic allograft rejection: Potential mechanism and therapeutic implications

Significant progress has been made in preventing acute allograft rejection following solid organ transplantation resulting in improved allograft survival. However, long term function still remains disappointing primarily due to chronic allograft rejection. Alloimmune responses primarily defined by the development of antibodies (Abs) to donor mismatched major histocompatibility antigens during the post-transplantation period have been strongly correlated to the development of chronic rejection. In addition, recent studies have demonstrated an important role for autoimmunity including the development of Abs to organ specific self-antigens in the pathogenesis of chronic allograft rejection. Based on this, a new paradigm has evolved indicating a possible cross-talk between the alloimmune responses and autoimmunity leading to chronic rejection. In this review, we will discuss the emerging concept for the role of cellular and humoral immune responses to self-antigens in the immunopathogenesis of chronic allograft rejection which has the potential to develop new strategies for the prevention and/or treatment of chronic rejection.

Human Cytomegalovirus Induces Systemic Immune Activation Characterized by a Type 1 Cytokine Signature

Mechanisms underlying the onset and perpetuation of chronic immune activation in individuals without overt infectious or autoimmune diseases are unclear. Cytomegalovirus (CMV) is a persistent virus that induces a permanent increase of highly differentiated, interferon-gamma-secreting effector T cells. We hypothesized that, because of this increase, CMV also induces a systemic inflammatory response. We measured acute phase proteins, cytokines, and chemokines in serum samples from renal transplant recipients who developed a primary CMV infection and healthy CMV serum-positive or -negative individuals. Primary CMV infection induced a clear proinflammatory response that was maintained during latency. This response was characterized by increased levels of acute phase proteins, such as serum amyloid-A and C-reactive protein, and type 1 cytokines, such as interleukin-18, interferon-inducible protein-10, and interferon-gamma. This continuous activation of the immune system may play a role in the pathogenesis of chronic allograft rejection and potentially contribute to the acceleration of chronic diseases.

IL-17 Contributes to the Development of Chronic Rejection in a Murine Heart Transplant Model

Although interleukin-17 (IL-17) has been reported to participate in the pathogenesis of infectious, autoimmune and allergic disorders, the precise role in allograft rejection remains uncertain. This study illustrates that IL-17 contributes to the pathogenesis of chronic allograft rejection. Utilizing a murine heterotopic heart transplant model system, IL-17-deficient recipient mice had decreased allograft inflammatory cell recruitment, decreased IL-6, MCP-1, and KC production, and reduced graft coronary artery disease (GCAD). Intragraft gamma delta (gammadelta) T cells appear to be the predominant source of IL-17 production. Therefore, IL-17 neutralization may provide a potential target for novel therapeutic treatment for cardiac allograft rejection.

Does transforming growth factor beta 1 play a role in the pathogenesis of chronic allograft rejection?

To investigate the potential role of Transforming Growth Factor beta 1 (TGF beta 1) in the pathogenesis of chronic allograft rejection, we studied TGF beta 1 expression in a rat aortic allograft model. mRNA and protein expression of total and endogenously active TGF beta 1 were analysed in infra-renal orthotopic aortic syngeneic and allogeneic grafts and matched with the histological appearances of the grafts, 2, 4 and 12 weeks post-transplantation. Serum levels of TGF beta 1 were also measured. The level of TGF beta 1 m RNA and protein expression appeared highest 2 and 4 weeks following transplantation in both syngeneic and allogeneic grafts, with significantly elevated levels of mRNA expression in the 2 week allograft specimens. These time-points correlate histologically with maximal inflammatory cell infiltration of the grafts. By 12 weeks post-transplantation, TGF beta 1 mRNA expression is reduced in allogeneic grafts compared to syngeneic grafts. However, detectable levels of total and endogenously active TGF beta 1 protein levels in the allografts exceed those measured in the syngeneic grafts at this time point. These results demonstrate the complex expression pattern of this growth factor during the progression of chronic rejection and suggest an aetiological link between TGF beta 1 and the process of accelerated graft atherosclerosis.

Does transforming growth factor β1 play a role in the pathogenesis of chronic allograft rejection?

Does transforming growth factor β1 play a role in the pathogenesis of chronic 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.

Antibody-induced transplant arteriosclerosis is prevented by graft expression of anti-oxidant and anti-apoptotic genes.

We investigated the pathogenesis of chronic allograft rejection in mouse cardiac allografts. Long-term survival occurred after administration of monoclonal antibody to CD4 or CD40-ligand (CD40L) plus donor cells. Both treatments induced permanent graft survival, but, in contrast to transplants in mice treated with CD4 monoclonal antibody, grafts in mice treated with CD40L monoclonal antibody lacked evidence of chronic rejection, including transplant arteriosclerosis. Freedom from chronic rejection in the group treated with CD40L monoclonal antibody correlated with vascular expression of the 'protective' genes heme oxygenase-1 (HO-1), Bcl-xL and A20. Moreover, arteriosclerosis was induced in allografts in immunoglobulin-deficient mice by antibody transfer only when the transfer was done before expression of protective genes. A direct role for protective gene expression in endothelial cells was demonstrated by in vitro experiments in which induction of HO-1 or Bcl-xL suppressed alloantibody-stimulated endothelial activation. Finally, induction of HO-1 in vivo protected allografts against chronic injury. These data show a role for protective genes in the prevention of chronic rejection, and indicate new approaches to protect grafts against development of transplant arteriosclerosis.

Role of donor and recipient antigen-presenting cells in priming and maintaining T cells with indirect allospecificity.

It has been suggested that the sensitization of recipient T lymphocytes against peptides derived from allogeneic major histocompatibility complex (MHC) antigens in the context of self-MHC molecules may contribute to the pathogenesis of chronic allograft rejection. The purpose of this study was to quantitate and characterize the indirect alloresponse in renal transplantation. An HLA-A2-negative patient whose A2-positive kidney transplant failed as a result of chronic rejection was selected for this study. T-cell clones were raised using a cocktail of peptides corresponding to polymorphic regions of the A2 sequence and studied by measuring their proliferation using [3H]thymidine incorporation. The presence in vivo of HLA-A2-specific T cells was assessed using limiting dilution analysis. T-cell clones were specific for a single peptide of HLA-A2, residues 92-120, and restricted by HLA-DRB1*1502. The frequency of interleukin-2-secreting T cells specific for this A2 peptide was 1:86,000, only 2-fold lower than that measured against the recall antigen tetanus toxoid. Capitalizing on the similarity of the donor and recipient DR15 alleles (DRB1*1501 and 1502), the question was addressed as to how these T cells had been primed in vivo. Although the large majority of clones responded to A2 synthetic peptide presented by both DR15 alleles, only 3 of 10 clones responded to cells co-expressing DRB1*1501 and A2. These data suggest that antigen presentation by recipient APCs is responsible for maintaining T cells with indirect allospecificity in vivo and that, in the context of partial DR matching, indirect presentation by the parenchymal cells of the graft may serve to induce tolerance in T cells with indirect allospecificity.

LATE BLOCKADE OF B7-1 COSTIMULATION INTERRUPTS THE DEVELOPMENT OF CHRONIC REJECTION IN A RAT MODEL OF CARDIAC TRANSPLANTATION.

720 We have previously shown that CTLA4Ig prevents the development of chronic allograft rejection in the Lew to F344 rat heterotopic cardiac transplant model (JCI, 1996; 97: 833). CTLA4Ig blocks both B7-1 and B7-2: two costimulatory molecules which are known to exhibit differing temporal expression and functional characteristics in vitro and in vivo. To dissect the individual role of B7-1 in the pathogenesis of chronic allograft rejection we used a mutant form of CTLA4Ig, Y100F, which blocks B7-1 but not B7-2. Early administration of Y100F, either as a single dose (0.5mg i.p.) (group A, n=5) or multiple doses (0.5mg i.p. 2x/wk) (group B, n=4) starting day 2 post transplantation failed to prolong graft survival as compared to untreated control (Group C, n=6; all animals rejected their grafts within 3 weeks). This is in sharp contrast to the effects of CTLA4Ig given at this time point which results in prolonged graft survival (> 100 days) in more than 60% of animals. We then studied the effects of late blockade of B7-1. Animals received 30 days of low dose CsA (1 mg1day s.c.) to prevent acute rejection followed by either Y100F (group D; n=8), or control fusion protein (group E, n=19) at 0.5mg 2x/wk for 4 wks starting 1 month post transplantation, or continuous CsA (1 mg1day s.c.) (group F, n=5). Both selective late blockade of B7-1 by Y100F (D) and continuous CsA treatment (F) significantly prolonged allograft survival but only Y100F treatment interupted development of graft arteriosclerosis, the sine qua non of chronic allograft rejection. Mean arteriosclerotic score in grafts surviving >100days was 0.91±0.5* in the Y100F group (D), compared to 1.76±1.53 in the continuous CsA treated group (F) and 1.72±0.53 in the control fusion protein group (E). Furthermore, histological analysis of graft specimens revealed significantly less graft fibrosis in the Y100F treated groups (D) compared to control (E) and continuous CsA (F) groups. Consistent with these findings, semi-quantitative PCR revealed significantly reduced tissue expression of TGF-β mRNA in grafts from the Y100F treated group. These data show that blocking B7-1 late but not early posttransplantation is effective at interrupting progression of chronic rejection. These findings are consistent with the later temporal expression of B7-1 during the alloimmune response. *p<0.05 compared to control group (D).Figure

CHARACTERIZATION OF MHC ALLOPEPTIDE REACTIVE T CELL CLONES FROM HUMAN RENAL ALLOGRAFT RECIPIENTS

382 We have recently demonstrated that T cells obtained from patients with chronic renal allograft dysfunction are primed to recognize and respond to specific donor-derived class II MHC allopeptides in vitro. In experimental animal models, class II MHC allopeptide-specific Th1 clones generated from rejecting animals express restricted TCR Vß repertoire and transfer delayed type hypersensitivity responses in vivo. In this study we generated T cell clones from PBMCs of two human renal transplant recipients; the first patient has chronic allograft dysfunction and the second control patient has stable graft function. Patient 1 received a HLA-DR*0101 mismatched living donor renal allograft and currently has chronic allograft dysfunction (creatinine: 2.9mg/dl) with typical histopathological features of chronic rejection. We have previously demonstrated T cell reactivity to peptide (42-62) of HLA-DR*0101 in this patient. Patient 2 received a HLA-DR*1501 mismatched cadaver renal allograft and currently has stable renal function (creatinine: 1.6mg/dl). Allopeptide reactivity was not detectable to any of 5 HLA-DR*1501 allopeptides on each of three separate occasions in this patient. T cell lines were generated from PBMCs of both patients by repeated stimulation with self APC pulsed allopeptide; peptide (42-62) of HLA-DR*0101 was used for patient 1 and peptide(1-21) of HLA-DR*1501 was used for patient 2. T cell clones were then generated by limiting dilution and were phenotyped using flow cytometric analysis. Culture supernatants were analyzed for cytokine production by ELISA, and the TCR Vß expression was characterized by RT-PCR. T cell clones from patient 1 were found to be CD4+ cells which specifically proliferated to peptide (42-62) of HLA-DR*0101 and which produced IL-2 and IFN-γ after re-stimulation with the peptide. Clones from patient 2 proliferated weakly, yet specifically to peptide (1-21) of HLA-DR*1501 and were found to be CD4+ cells which produced IL-4 and IL-10 after re-stimulation with the peptide. RT-PCR transcript analysis with specific human TCR Vß primers showed that the clones from both patients expressed a restricted TCR Vβ repertoire: 5.2, 8, 9, 13.1 and 13.2 for patient 1, and Vß 4, 6, 13.1, 13.1 and 13.1 for patient 2. The is the first demonstration, at the clonal level, that chronic allograft rejection in humans is associated with the presence of CD4+ MHC allopeptide-specific T cell clones that express a Th1 phenotype. In contrast, MHC allopeptide-specific T cell clones generated in the absence of rejection are associated with a (? protective) Th2 phenotype. Expanding these studies and understanding the mechanisms of immune deviation in humans should yield relevant information on the pathogenesis of chronic allograft rejection, the major clinical problem in organ transplantation.

936-85 Intrathymic Injection of Donor Splenocytes Prolongs Rat Cardiac Allograft Survival but Does not Inhibit Graft Arteriosclerosis

Chronic rejection , manifested by the development of graft arteriosclerosis, remains the leading cause of late death in cardiac transplant recipients. Immunologic mechanisms have been implicated in both experimental and clinical studies, yet the pathogenesis of chronic allograft rejection remains poorly understood. Recent studies have shown that intrathymic (i.t.) injection of donor cells or processed allo-MHC antigens with or without transient immunosuppression induces specific systemic T cell tolerance and prevents acute allograft rejection in several experimental transplantation models. In this study we examined the effects of i.t. injection of donor cells with or without systemic anti-lymphocyte serum (ALS) on the development of chronic rejection in the Lewis-to-F344 rat cardiac allograft model. Recipients were divided into 4 groups: control group A was pre-treated with saline i.t., group B was pre-treated with a one-time dose of ALS (1 mI) via intraperitoneal injection (i.p.). group C received donor (Lewis) splenocytes i.t. (2 × 10 6 ), and group D received both the splenocytes i.t. and a one-time dose of ALS i.p. two weeks prior to transplantation. Allografts were followed by daily palpation and graded from 0–4 (grade 0 = rejection, grade 4 = normal heartbeat). Graft survival on post-op day 90 for all three experimental groups was 100% vs. 33% for the control (n = 6/group). Moreover, mean heartbeat grade for the three experimental groups (B = 2.83 ± 0.17, C = 2.0 ± 0.41, D = 2.67 ± 0.21) was significantly higher (p &lt; 0.01) than that of the control group (A = 0.4 ± 0.24). Histologic and immunocytochemical analysis of 90-day old grafts revealed the following results: GROUPS Inflammation % Vessels Diseased % Luminal Occlusion Intima:Media Ratio Control(A) severe 89 ± 1 64 ± 5 0.97 ± 0.33 Splenocyte only (C) moderate-severe 79 ± 8 57 ± 7 068 ± 0.16 ALS only (B) minimal 25 ± 7 ** 8 ± 3 ** 0.09 ± 0.04 * Splenocyte + ALS (D) minimal 27 ± 4 ** 8 ± 3 ** 0.07 ± 0.03 * * p &lt; 0.02. ** p &lt; 0.003 vs. Control (1) The induction of systemic unresponsiveness with donor splenocyte i.t. plus ALS i.p. or ALS i.p. alone markedly reduced inflammation and inhibited graft arteriosclerosis indicating that the immune response and graft inflammation playa critical role in the pathogenesis of chronic allograft rejection. (2) The donor cells i.t. alone significantly prolonged graft survival and prevented rejection as measured by functional criteria but did not significantly inhibit graft inflammation or graft arteriosclerosis. These results suggest that partial suppression of the immune response without significant inhibition of graft inflammation prolongs rat cardiac allograft survival but does not prevent graft arteriosclerosis.