The transplantation of allogeneic organs triggers a massive inflammatory immune response mediated by recipient T lymphocytes recognizing donor major histocompatibility complex (MHC) molecules. Allorecognition takes place primarily in the recipient's secondary lymphoid organs where T cells interact with intact allo-MHC molecules present on donor dendritic cells (direct allorecognition) and with donor MHC peptides processed and presented by recipient antigen presenting cells (indirect allorecognition) (1). In addition, minor histocompatibility antigens, defined as peptides derived from non-MHC donor polymorphic proteins, are presented directly or indirectly, and can trigger an alloimmune response (2). The activation of alloreactive CD4+ and CD8+ T cells triggers a cascade of events including cytotoxic T-cell differentiation, delayed type hypersensitivity (DTH) reactions, and anti-donor antibody production that lead to the rejection of allografts. On the other hand, the involvement of tissue-specific antigens in anti-graft immunity has long been postulated (3). Although some of these antigens have been identified, little is known regarding their contribution to the rejection of allografts.
Recent studies in our laboratory and others' have demonstrated that rodents receiving allogeneic heart or lung transplants mount an autoimmune response to graft tissue-specific antigens (4–6). In a mouse heart transplant model, we have reported that the T-cell response to donor MHC class I molecules is followed by an autoimmune response to cardiac myosin (CM), a contractile protein expressed by cardiomyocytes (4). CM also represents the target autoantigen in experimental autoimmune myocarditis (EAM), a mouse model of autoimmune heart disease (7). These findings suggest that anti-CM immunity may cause damage to the transplanted heart, thereby contributing to its rejection by the host. There is a report supporting this view that induction of an autoimmune response to CM in mouse recipients before transplantation accelerates the rejection of allogeneic heart transplants (4). In this case, the anti-CM autoimmune response was mediated by activated MHC class II-restricted CM-specific CD4+ TH1 cells (4). In addition, high titers of IgG1 anti-CM autoantibodies were detected in the serum of transplanted mice (4). Most importantly, tolerance induction to CM and collagen type V has been shown to significantly prolong the survival of heart and lung transplants, respectively (8, 9). Finally, several studies have provided evidence suggesting the relevance of CM autoimmunity in clinical cardiac transplantation (10–13). Collectively, these studies suggest that secondary immunity to tissue-specific antigens may represent an essential component of allograft rejection. However, the actual relevance of this phenomenon to the rejection of minor antigen-mismatched allografts in a large-animal model remains to be established.
In this study, we investigated the influence of CM autoimmunity on the rejection of minor antigen-mismatched cardiac allografts performed in a swine model. We observed that the induction of an anti-CM response led to the rejection of minor antigen-mismatched cardiac allografts. The implications of these findings for the design of novel diagnostic tools and therapies in transplantation are discussed.
In this study, we investigated the effect of CM sensitization on the rejection of minor antigen-mismatched cardiac allografts in miniature swine. To address this question, we compared DTH, T-cell proliferation and antibody responses with CM and transplant rejection in swine immunized with CM emulsified in Freund's adjuvant and control nontreated or adjuvant-immunized recipients.
None of the five control (nonimmunized or immunized with adjuvant alone) swine (C1–C5) acutely rejected its graft, an expected result given the poor immunogenicity of minor antigens (Figs. 1 and 2). Although histological examination of these grafts at posttransplant day 60, 120, and 180 revealed some inflammatory cell infiltrates, no signs of cardiac allograft vasculopathy were observed (Fig. 1). No DTH response (induration<1 mm) to CM was detected in the control animals (Table 1). In contrast, experimental swine P1, P2, and P3 mounted potent DTH responses to CM (>10 mm) (Table 1) and rejected their allografts in an acute fashion (grade 3R, Figs. 1 and 2) (P=0.02). The fourth swine (P4) mounted a poor DTH response after immunization with CM (8 mm) and was diagnosed with a low grade (1R at day 62) acute rejection characterized by focal perivascular or interstitial infiltrate without myocyte damage. The correlation between CM-specific DTH response and acute rejection was statistically significant (two-tailed t test assuming equal variance P=0.0003).
As shown in Figure 3(A), CM-immunized P1 to P3 pigs exhibited high and sustained serum levels of anti-CM antibodies. These antibodies displayed an IgG isotype. In contrast, no CM-specific antibodies were detected in control nonimmunized swine and swine immunized with adjuvant alone (Fig. 3A). The swine P4, characterized by a poor DTH and low grade acute cellular rejection, displayed an early antibody response to CM which subsided rapidly after transplantation as anti-CM antibodies could no longer be detected by day 27 posttransplantation (Fig. 3A).
Next, we measured T-cell proliferative responses in two CM-immunized swine (P1 and P2) and two control swine (C1 and C2) at different time points pre- and postcardiac transplantation. As shown in Figure 3(B), T-cell proliferation was detected in both immunized pigs, 21 and 42 days after immunization (day −21 and d0). This proliferative response was no longer detectable at day 27. However, interestingly, it reappeared at day 62 posttransplantation and remains high at day 125. This suggests that, during the course of transplant rejection, endogenous CM determinants derived from cardiac myocytes are processed and presented to T cells thereby reactivating some anti-CM memory T cells. No T-cell proliferation was observed in control nonimmunized swine (Fig. 3B).
De novo induction of autoimmunity to tissue-specific antigens has now been observed in heart, lung, kidney, skin, and liver transplant models (4, 5, 14–16). We have previously shown the involvement of CM-specific immunity in the rejection of MHC disparate cardiac allografts studied in an experimental mouse model (4, 9). It is noteworthy that patients originally diagnosed with chronic myocarditis experience more frequent and severe rejection episodes than patients with other heart diseases (17). In addition, Latif et al. (11) have shown an association between the presence of anti-heart autoantibodies and clinical course after heart transplantation in patients. Actually, increases in the amounts of circulating CM after transplantation have been correlated with poor prognosis for cardiac transplant survival (18). Most importantly, Warraich et al. (13) recently reported the presence of anti-CM autoantibodies during acute rejection of cardiac allografts in patients with dilated cardiomyopathy. Recently, the presence of autoimmune responses to CM has been observed in human cardiac allograft recipients with antibody-mediated rejection and cardiac allograft vasculopathy (10, 12). Therefore, autoimmunity to CM represents a general phenomenon in cardiac allotransplantation. The present study shows for the first time that CM-specific autoimmunity can trigger the acute rejection of minor antigen-mismatched cardiac allografts in a large-animal model.
Apparently, the nature of the rejection was dependent on the strength of the response to CM because high responder swine (P1–P3) rejected their allografts acutely whereas the swine displaying low and transient DTH and antibody responses to CM (P4) did not. The suboptimal immune response to CM detected in the fourth experimental animal may simply be due to a poor immunization. Alternatively, it may reflect genetic differences in susceptibility to CM autoimmunity among these swine. This result is reminiscent of the observation that tolerance to CM can be broken only in certain mouse strains that are also susceptible to EAM whereas other strains do not mount autoimmune responses to CM on immunization and are resistant to EAM (7, 19). Finally, this observation may reflect differences in the T- and B-cell repertoires acquired through the pig's “immunological history.”
Similar to previous observations reported in rodent lung and heart allotransplant models, no signs of tissue injury were detected in the native heart of the swine injected with CM. It is important to note that while CM autoimmunity is known to trigger EAM, CM immunization is insufficient on its own to cause cardiac tissue injury. Serial coinjections with Pertussis toxin are necessary to induce EAM in mice (7, 20, 21). It is likely that Pertussis toxin activates antigen-presenting cells and exacerbates the processing and presentation of CM peptides to autoimmune T cells and thereby promotes local inflammation and cellular infiltration in the otherwise unmanipulated mouse heart tissue. Likewise, it is firmly established that, under normal conditions, individuals do not develop autoimmune diseases, despite the presence of high numbers of potentially pathogenic autoreactive T cell in the periphery. Altogether, this suggests that, in our and other transplant models, the absence of trauma and of local inflammation in the native organ accounts for lack of infiltration by activated lymphocytes and subsequent tissue injury.
Anti-CM autoantibodies have been shown to be pathogenic and play a key role in the initiation of EAM in rodent models (22–24). The mechanisms by which antibodies directed against an intracellular protein can cause tissue damage are still unclear. It is possible that T cells recognizing CM peptides presented by MHC molecules on cardiac antigen-presenting cells cause initial tissue injury and release of soluble CM, thereby inducing an autoantibody response. Subsequently, these antibodies can form immune complexes and opsonize cardiac target cells thus exacerbating the autoimmune process. In addition, it is known that apoptosis is known to reorient intracellular proteins and allow their expression on cell surface blebs (25, 26). This phenomenon may contribute to reveal some CM epitopes for recognition by CM-specific B cells and antibodies.
In summary, our findings further support the view that autoimmune reactivity to tissue-specific antigens expressed by the transplanted graft represents an important component of the process of allograft rejection. Therefore, monitoring the level of CM-specific immunity in the blood of heart-transplanted patients may help to predict the rejection of cardiac allografts.
MATERIALS AND METHODS
The Massachusetts General Hospital miniature swine preclinical model has been previously described (27). The genotyping was controlled by strict pedigree breeding and confirmed with flow cytometric analysis using indirect allospecific antibodies. All donor-recipient pairs were matched for MHC and mismatched for minor histocompatibility antigens. The animals were 3 to 6 months of age at the time of transplantation. Animal care and procedures were performed in compliance with both the Principles of Laboratory Animal Care formulated by the National Society of Medical Research and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.
Purification of CM
Swine CM was purified as described by Shiverick et al. (28). The purity of preparations (>95%) was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The myosin concentration was assessed spectrophotometrically using the BCA Protein Assay kit (Pierce, Rockford, IL). Myosin was dissolved in 50 mM sodium pyrophosphate and was stored at −80°C.
Heterotopic heart transplantation was performed as previously described (29). Cyclosporine A, generously provided by Novartis (Hanover, NJ), was administered intravenously at 10 to 13 mg/kg per day beginning on the day of surgery (postoperative day 0) and continuing until posttransplant day 11 (29). Cardiac function was monitored by daily palpation and serial electrocardiograms and echocardiograms. The survival of cardiac allografts was analyzed using the Kaplan-Meier method, and survival curves were compared using the log-rank test.
Four control swine (C1–C4) received minor antigen-mismatched heart transplants. Four experimental swine (P1–P4) were injected subcutaneously with 2 mg purified CM emulsified in complete Freund's adjuvant and in incomplete Freund's adjuvant 42 days and 21 days, respectively, before receiving a minor antigen-mismatched heart transplant. A fifth control swine (C5) was injected with adjuvant alone (complete Freund's adjuvant and incomplete Freund's adjuvant given 42 and 21 days, respectively, before transplantation) before transplantation of a minor histocompatibility-mismatched heart. All animals were treated with cyclosporine A for 12 days after transplantation and monitored for acute and chronic rejection. The animals were tested for DTH and antibody responses against CM before and after transplantation.
Measurement of DTH Responses
DTH responses were evaluated 14 days after CM immunization by re-challenging recipients with 200 μg of purified CM in 0.1 mL phosphate-buffered saline (PBS) injected intradermally. Width of induration was measured at 48 hr after injection by blinded observers using calipers. A response of more than 10 mm of induration was scored as positive, whereas negative responses were less than 10 mm.
Histology and Immunohistochemistry
Formalin-fixed tissue was stained with hematoxylin-eosin. Acute interstitial rejection of heart allografts was scored from 1R to 3R based on the International Society for Heart and Lung Transplantation system (30).
Detection of anti-CM Antibodies by Enzyme-Linked Immunosorbent Assay
Plates were coated with 50 μL of purified CM (2 μg/mL) or PBS and incubated overnight at 4°C. The plates were washed twice with 200 μL of PBS+0.1% Tween20 and then blocked by dispensing 200 μL of PBS+0.05% Tween20 and 1% bovine serum albumin with a 1-hr incubation at room temperature. The plates were then washed three times, and swine serum at 1:10 dilution in PBS+0.1% Tween20 was serially diluted. After a 2-hr incubation at room temperature, plates were washed five more times. Rabbit anti-pig IgG (1:250) and IgM (1:500) in PBS+0.05% Tween20 and 1% bovine serum albumin were added to each well and incubated for 2 hr at room temperature. After five more washes, 50 μL streptavidin horseradish peroxidase developing solution (1:1000) was added to each well and allowed to incubate for 1 hr at room temperature and in the dark. Another five washes were performed, and hydrolysis was measured after adding 2,2′-azino-bis-[3-ethylbenzthiazoline-6-sulfonic acid] peroxidase solution to each well. Product absorbances were measured using an enzyme-linked immunosorbent assay plate reader at 405 nm (BioRad; Hercules, CA).
A total of 4×105 peripheral blood mononuclear cells were cultured for 5 days along with purified CM protein (10 μg/mL). In all proliferation assays, 1 uCi of [3H]thymidine was added to each well and incubated for 5 hr to allow for incorporation. [3H]-incorporation was determined in triplicate samples by β-scintillation counting. Results were expressed as stimulation indices (SI), calculated as stimulation index (SI)=average count per million (cpm)±standard deviation for a responder stimulated with CM/cpm of the same responder stimulated by medium, alone. Average maximum SI of naïve responses was 1.2.
The authors thank Ms. K. Stenger for her assistance in the preparation of the manuscript. They also thank Drs. G. Tocco and H. Winn for helpful discussions and Drs. T. Millington and A. Aoyama for expert review of the manuscript.
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