Allograft rejection is initiated by an immune response to donor MHC proteins. We recently reported that this response can result in breakdown of immune tolerance to a recipient self Ag. However, the contribution of this autoimmune response to graft rejection has yet to be determined. Here, we found that after mouse allogeneic heart transplantation, de novo CD4+ T cell and B cell autoimmune response to cardiac myosin (CM), a major contractile protein of cardiac muscle, is elicited in recipients. Importantly, CM is the autoantigen that causes autoimmune myocarditis, a heart autoimmune disease whose histopathological features resemble those observed in rejected cardiac transplants. Furthermore, T cell responses directed to CM peptide myhcα 334–352, a known myocarditogenic determinant, were detected in heart-transplanted mice. No responses to CM were observed in mice that had received an allogeneic skin graft or a syngeneic heart transplant, demonstrating that this response is tissue specific and that allogeneic response is necessary to break tolerance to CM. Next, we showed that sensitization of recipient mice with CM markedly accelerates the rejection of allogeneic heart. Therefore, posttransplant autoimmune response to CM is relevant to the rejection process. We conclude that transplantation-induced autoimmune response to CM represents a new mechanism that may play a significant role in cardiac transplant rejection.

Heart transplantation is a life-saving procedure for patients with incurable chronic cardiac diseases. However, immunological rejection represents a major obstacle to long-term survival of transplanted hearts. This is underscored by the fact that, 3 yr after transplantation, nearly 30% of the cardiac grafts are rejected (1). Despite recent advances in immunosuppressive therapy, such treatment is nonspecific and often associated with increased risk of infections and cancer in transplanted patients. In addition, it is generally poorly effective in preventing chronic rejection (2, 3). Hence, there is an obvious need to design selective Ag-specific therapies that achieve long-lasting immune tolerance to donor organs, a task that requires elucidation of the cellular and molecular mechanisms underlying allograft rejection process.

T cell responses to intact or processed allogeneic MHC molecules displayed on the donor tissues initiate allograft rejection (4, 5, 6). We recently reported that, following injection of recipient mice with allogeneic cells, in vivo T cell response to donor MHC molecule resulted in the breakdown of immune T cell tolerance to a recipient self Ag (7). In this model, the self MHC class I peptide Dd 61–80 was dominant in syngeneic BALB/c mice (H-2d). T cell tolerance to Dd 61–80 in this mouse strain resulted in the absence of T cell proliferation following in vivo priming with Dd 61–80 peptide. However, we showed that transplantation of BALB/c mice with allogeneic B10.A (H-2a) splenocytes led to an autoimmune T cell response toward the dominant self peptide Dd 61–80. This phenomenon was Ag specific, since no T cell responses to Dd 61–80 peptide were observed after transplantation of “third party” C57BL/6 (H-2b) splenocytes into BALB/c recipients (7). In addition, we provided evidence that indicated that the breakdown of tolerance to Dd 61–80 self peptide resulted from the presentation of the donor cross-reactive peptide Kk 61–80 at the surface of recipient APCs. This previous study demonstrated that T cell-mediated alloresponse can induce autoimmune responses to a previously tolerogenic autoantigen. However, whether this type of immune response occurs following organ transplantation and the relevance of this phenomenon to the rejection process remained open to question.

In the present article, to investigate whether heart autoimmunity is induced during the rejection of cardiac allografts, we tested T and B cell responses to cardiac myosin (CM)4 in transplanted mice. CM was chosen because it is a well characterized cardiac tissue-specific protein that has been identified as the target autoantigen in autoimmune myocarditis. This cardiac autoimmune disease causes severe cardiac malfunction and ultimate failure in patients. Experimental autoimmune myocarditis (EAM) in mice represents the best characterized animal model for human myocarditis. In EAM, chronic inflammation and myocyte damage are associated with activation of CD4+ T lymphocytes specific to CM and with local deposition of anti-CM autoantibodies. Furthermore, immunization of susceptible mice with CM is sufficient to elicit EAM in rodents (8, 9, 10, 11). In the present article, we observed that, after transplantation of allogeneic heart in mice, B and T cell immune tolerance to CM self protein was broken, as demonstrated by the presence of activated anti-CM autoreactive lymphocytes in graft recipients. Tansplantation of mice with allogeneic hearts triggered a vigorous T cell autoimmune response to CM and its dominant determinant myhcα 334–352. This phenomenon was associated with a histopathology that is typical of EAM (12). Importantly, we then provided direct evidence showing that this postheart transplant autoimmune response to CM can enhance antigraft immunity, thus contributing to the rejection process. The implications of this finding for understanding the immunological mechanisms underlying allotransplant rejection are discussed.

The A/J (Kk Ak Ek Dd) and A.TL (Ks Ak Ek Dd) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The care of all animals involved in this study was in accordance with institutional guidelines. Vascularized heterotopic cardiac transplantation was performed as described by Corry et al. (13). Briefly, mice were anesthetized with pentobarbital injected i.p. (2 mg/mouse) and used either as donors or recipients. Donor hearts were anastomosed to recipient aorta and vena cava using microsurgical procedures. The function of transplanted hearts was monitored daily by palpation through the abdominal wall. Heart beat intensity was graded on a scale of 0 (no palpable impulse) to 4 (strong impulse). Rejection was determined to be the time when heart impulse declined to 1–0 for two consecutive days. Statistical analysis was performed using paired t test.

Peptides were synthesized utilizing F-moc chemistry by Research Genetics (Huntsville, AL) and purified by HPLC (purity >95%). The amino acid sequences of the peptides were as follows: myhcα 334–352: DSAFDVLSFTAEEKAGVYK; P12–26: LEDARRLKAIYEKKK.

At the time of rejection, mice were sacrificed; then donor hearts were removed, fixed in neutral buffered formalin (Sigma, St. Louis, MO), and embedded in paraffin. Several cross-sections of each heart in the atrial-apical axis were then prepared. Sections were stained with hematoxylin and eosin.

To induce EAM, mice were injected into rear footpads and i.p. with 80 μg of murine CM emulsified in CFA on days 0 and 7. Mice also received a single i.p. injection of 500 ng of pertussis toxin (List Biologicals, Detroit, MI) on day 0 (10). Murine CM was purified as described by Schiverick et al. (14). The purity of preparations (>95%) was determined by SDS-PAGE. The myosin concentration was assessed spectrophotometrically using BCA Protein Assay kit (Pierce, Rockford, IL). Myosin was dissolved in 50 mM sodium pyrophosphate and stored at −80°C.

At the time of rejection (10–13 days posttransplant), spleens were harvested from recipient A/J mice transplanted with either syngeneic (A/J) or allogeneic A.TL hearts. Suspensions of 106 spleen cells were plated in 96-well dishes in AIM-V medium (Life Technologies, Grand Island, NY) supplemented with 1% of FCS (Gemini Bioproducts, Calabases, CA). IFN-γ production was measured using ELISA assay. Briefly, 96-well microtiter ELISA plates (Corning, Corning, NY) were coated with capturing rat anti-mouse IFN-γ mAb R4-6A2 (PharMingen, San Diego, CA) at 1 μg/ml in bicarbonate coating buffer (pH 8.2) and incubated overnight at 4°C. After blocking with PBS containing 2% BSA (Sigma), supernatants from cell cultures were added to the wells and incubated overnight at 4°C. For detection of bound IFN-γ, biotinylated rat anti-mouse IFN-γ mAb XMG 1.2 (PharMingen) was used, followed by incubation with avidin D-coupled HRP (Vector, Burlingame, CA). Peroxidase activity was revealed with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) tablets (Sigma) dissolved in phosphate-citrate substrate buffer (pH 5.0) containing H2O2. Absorbance was measured at 405 nm. Concentration of detected IFN-γ (pg/ml) was calculated using recombinant murine IFN-γ (PharMingen) as a standard.

Mouse sera were tested for Abs to CM by ELISA as described elsewhere (15).

We have previously reported that allotransplantation can disrupt immune tolerance to some self proteins (7). Here, we investigated whether this phenomenon occurs after cardiac transplantation and whether it is associated with the rejection process. To address this, T cell response to purified mouse CM was investigated in the spleen of mice transplanted with allogeneic hearts. CM was chosen because it represents a major contractile protein of heart tissue and because it is a well-characterized cardiac tissue-specific Ag that has been implicated in heart autoimmune pathology. In our study, single MHC class I allele-mismatched A/J (Kk) and A.TL (Ks) mice were used either as donors or recipients in vascularized heterotopic cardiac transplant model (13). Allogeneic hearts were consistently rejected at day 9.4 ± 0.3 (A/J → A.TL) and at day 8.6 ± 0.5 (A.TL → A/J) after transplantation. In contrast, syngeneic grafts survived indefinitely (>100 days). As shown in Fig. 1,A, vigorous anti-CM T cell response, as determined by IFN-γ production, was observed in mice tested 10–13 days after transplant, and it was found to be mediated by CD4+, MHC class II (Ak)-restricted T cells (data not shown). No IL-4 and IL-5 production was detected, indicating that anti-CM response was mediated by Th1 cells. No response was detected in the absence of Ag or in the presence of the irrelevant hen eggwhite lysozyme (HEL) control Ag (Fig. 1). No anti-myosin T cell autoimmunity was observed in normal mice and in those grafted with syngeneic hearts (Fig. 1) as well as in mice that received an allogeneic skin transplant (data not shown). These control experiments demonstrated that this phenomenon was Ag and tissue specific. Most importantly, the absence of anti-CM response after syngeneic heart graft showed that donor MHC-mediated allogeneic response was necessary to break tolerance to CM. We conclude that, following cardiac allograft, T cell tolerance to CM had been disrupted, a phenomenon that resulted in de novo activation of anti-CM CD4+ autoreactive T cells displaying Th1 phenotype.

FIGURE 1.

T cell and B cell responses to CM in transplanted mice. A, Spleen cells were cultured in either medium alone (open bars) or with the following Ags: purified murine CM (solid bars) or control Ag, HEL (hatched bars). Data are expressed as concentration of IFN-γ (pg/ml). The data are representative of three to eight mice tested individually in each group. B, Ab responses to CM in mice transplanted with allogeneic heart (solid bars), syngeneic heart (hatched bars), and in nontransplanted mice (open bars) are shown. Data are presented as mean absorbance at OD 405 nm ± SE after subtraction of absorbance values obtained with plates coated with control Ag. Each bar shows an individual mouse, representative of three to five mice tested in each group.

FIGURE 1.

T cell and B cell responses to CM in transplanted mice. A, Spleen cells were cultured in either medium alone (open bars) or with the following Ags: purified murine CM (solid bars) or control Ag, HEL (hatched bars). Data are expressed as concentration of IFN-γ (pg/ml). The data are representative of three to eight mice tested individually in each group. B, Ab responses to CM in mice transplanted with allogeneic heart (solid bars), syngeneic heart (hatched bars), and in nontransplanted mice (open bars) are shown. Data are presented as mean absorbance at OD 405 nm ± SE after subtraction of absorbance values obtained with plates coated with control Ag. Each bar shows an individual mouse, representative of three to five mice tested in each group.

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Abs are known to play a critical role in cardiac immune-mediated pathology. It was therefore important to determine whether T cell tolerance breakdown to self CM in heart-transplanted mice was accompanied by anti-CM autoantibody production by B cells. As shown in Fig. 1,B, high titers of CM-specific autoantibodies were detected in transplanted mice sera. Similar to EAM, the vast majority of these anti-myosin Abs were of IgG1 isotype (15). No anti-myosin B cell responses were observed in normal mice, or in mice grafted with syngeneic hearts (Fig. 1 B). The divergence between the prevalent Th1-type cell response to CM and IgG1 isotype Ab production may reflect a preexisting IgG1-type B cell memory to CM. Taken together, our results demonstrate that, during acute cardiac allograft rejection in mice, alloresponses to donor MHC Ags lead to the induction of both humoral and cellular autoimmunity to CM.

CM has been shown to induce EAM in mice (12). Based upon the data presented in Fig. 1, we investigated whether similar immune response to CM in both EAM and allotransplantation would be associated with comparable histopathology between hearts with EAM and cardiac allografts. It has been reported that, following induction of EAM, heart infiltration by anti-CM T cells is associated with adjacent myocyte damage. To perform histologic examination of rejected hearts and hearts with EAM, A/J mice were either transplanted with A.TL heart allografts or injected with mouse CM along with CFA and pertussis toxin to cause EAM (12). Hearts were removed following graft rejection (10 days posttransplant) or EAM induction (21 days postimmunization), fixed, and embedded in paraffin. Cross-sections were stained with hematoxylin and eosin. Histologic analysis revealed epicardial and endocardial interstitial inflammatory cell infiltrates, myocyte dropout, and necrosis in both transplanted and myocarditic hearts (Fig. 2, B and C). We therefore conclude that, following cardiac allotransplantation, allogeneic immune response leads to breakdown of tolerance to CM and the subsequent induction of an autoimmune histopathology that is characteristic of autoimmune myocarditis.

FIGURE 2.

Histopathology of cardiac allografts and hearts from mice with EAM. Photomicrograph of normal mouse heart (A) shows representative view of epicardium and myocardium with several blood vessels in the absence of inflammation, ×200. Both rejected donor heart (B) and a heart from a mouse with EAM (C) display a subepicardial and myocardial interstitial lymphocytic inflammatory cell infiltrate and adjacent myocyte damage.

FIGURE 2.

Histopathology of cardiac allografts and hearts from mice with EAM. Photomicrograph of normal mouse heart (A) shows representative view of epicardium and myocardium with several blood vessels in the absence of inflammation, ×200. Both rejected donor heart (B) and a heart from a mouse with EAM (C) display a subepicardial and myocardial interstitial lymphocytic inflammatory cell infiltrate and adjacent myocyte damage.

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Next, it was crucial to investigate whether the anti-CM autoimmune response does actually influence the allograft rejection process. To address this, we tested the effect of pretransplant sensitization of recipients with CM on the course of cardiac transplant rejection. Recipient mice were immunized i.p. with CM and then transplanted 21 days later with allogeneic hearts. As shown in Fig. 3, CM-sensitized mice rejected donor grafts in an accelerated fashion (5.2 ± 0.6 days, p < 0.001; n = 5). Sensitization with a control Ag, OVA, had no effect on transplant survival (9.5 ± 0.6; n = 4).

FIGURE 3.

Accelerated rejection of cardiac allografts in mice sensitized with CM. Allogeneic (A/J) hearts were transplanted to untreated A.TL mice (filled squares) or to A.TL mice sensitized with either CM (filled circles) or OVA, control Ag (Boehringer Manheim) (open circles) 21 days before transplantation. A.TL mice transplanted with A.TL-syngeneic hearts are also shown (open squares). In sensitization experiments, mice were immunized with murine CM as described for EAM induction but in the absence of pertussis toxin. Data represent the median score of heart function (0–4) determined for three to eight mice in each experimental group. Heart beat intensity was graded on a scale of 0 (no palpable impulse) to 4 (strong impulse).

FIGURE 3.

Accelerated rejection of cardiac allografts in mice sensitized with CM. Allogeneic (A/J) hearts were transplanted to untreated A.TL mice (filled squares) or to A.TL mice sensitized with either CM (filled circles) or OVA, control Ag (Boehringer Manheim) (open circles) 21 days before transplantation. A.TL mice transplanted with A.TL-syngeneic hearts are also shown (open squares). In sensitization experiments, mice were immunized with murine CM as described for EAM induction but in the absence of pertussis toxin. Data represent the median score of heart function (0–4) determined for three to eight mice in each experimental group. Heart beat intensity was graded on a scale of 0 (no palpable impulse) to 4 (strong impulse).

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To further demonstrate the relevance of anti-CM response in heart transplantation, we tested whether, in the absence of an alloresponse, induction of anti-CM autoimmune response alone could affect cardiac graft rejection. Recipient A.TL mice were sensitized to CM and then transplanted with a syngeneic heart. Strikingly, in contrast to untreated recipients that retained syngeneic grafts indefinitely, CM-sensitized mice rejected syngeneic transplants after 40 days (Table I). In addition, rejected syngeneic hearts displayed histopathological features that were similar to those of allogeneic transplants undergoing rejection (Fig. 4). This result demonstrated that, in the absence of allogeneic stimulation, anti-myosin autoimmune response per se is sufficient to elicit graft rejection.

Table I.

Rejection of syngeneic cardiac transplants following presensitization of recipient mice with cardiac myosina

TreatmentTransplantDay of Graft RejectionMean Day of Graft Rejection ± SE
None Syngeneic >100 (n = 3) >100 
None Allogeneic 8, 9, 9, 9, 9, 10, 10, 11 (n = 8) 9.4 ± 0.3 
OVA sensitization Allogeneic 8, 9, 10, 11 (n = 4) 9.5 ± 0.6 
CM sensitization Allogeneic 4, 4, 5, 6, 7 (n = 5) 5.2 ± 0.6 
CM sensitization Syngeneic 32, 32, 33, 50, 56, 56 (n = 6) 43.2 ± 4.9 
TreatmentTransplantDay of Graft RejectionMean Day of Graft Rejection ± SE
None Syngeneic >100 (n = 3) >100 
None Allogeneic 8, 9, 9, 9, 9, 10, 10, 11 (n = 8) 9.4 ± 0.3 
OVA sensitization Allogeneic 8, 9, 10, 11 (n = 4) 9.5 ± 0.6 
CM sensitization Allogeneic 4, 4, 5, 6, 7 (n = 5) 5.2 ± 0.6 
CM sensitization Syngeneic 32, 32, 33, 50, 56, 56 (n = 6) 43.2 ± 4.9 
a

In sensitization experiments, recipient mice were immunized s.c. in their rear footpads and i.p. with 80 μg of murine cardiac myosin or control OVA protein emulsified in CFA on days 0 and 7. After 21 days, sensitized mice received either syngeneic or allogeneic heterotopic heart transplants. Rejection was monitored daily by palpation analysis. Heart beat intensity was graded on a scale of 0 (no palpable impulse) to 4 (strong impulse). Rejection was determined to be the time when heart impulse declined to 1-0 for 2 consecutive days.

FIGURE 4.

Histopathology of syngeneic grafts from mice sensitized with CM pretransplant. Photomicrograph of a syngeneic heart from a CM-sensitized recipient mouse displays a subepicardial and myocardial interstitial lymphocytic inflammatory cell infiltrate and adjacent myocyte damage.

FIGURE 4.

Histopathology of syngeneic grafts from mice sensitized with CM pretransplant. Photomicrograph of a syngeneic heart from a CM-sensitized recipient mouse displays a subepicardial and myocardial interstitial lymphocytic inflammatory cell infiltrate and adjacent myocyte damage.

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It is important to note that, in all CM sensitization experiments, mice were injected with murine CM in the absence of pertussis toxin, an adjuvant that is required for EAM induction. These mice were primed to CM as shown by their T cell response to CM (data not shown). However, there was no inflammatory cell infiltration in the heart of these mice, and they did not develop any signs of EAM in the absence of transplantation. Therefore, the rejection of the graft, as well as the EAM-like pathology in CM-sensitized recipient mice, was not due to EAM induction.

Taken together, our results show that 1) initial response to donor MHC molecule is a prerequisite for breaking tolerance to CM and, 2) once it has been elicited, anti-CM response alone can cause the rejection of transplanted heart. Therefore, posttransplant autoimmune response to CM is likely to represent an essential element of the rejection of allogeneic cardiac transplants.

Next, we investigated the nature of the determinant(s) on CM presented to T cells during heart allograft rejection. A previous study by Donermeyer et al. has shown that immunization of A/J mice with the peptide 334–352 of CM H chain α (myhcα 334–352) induces EAM (16). It was possible that the same determinant could be involved in anti-CM autoimmune responses occurring after heart transplant. As shown in Fig. 5, when tested 10–13 days after allogeneic cardiac transplantation, no T cell response to myhcα 334–352 peptide was detected in recipient A.TL mice. However, at 50 days posttransplant, vigorous T cell proliferative response (Fig. 5,A) and IFN-γ production (Fig. 5 B) to myhcα 334–352 were observed in recipient spleens. Moreover, immunization of mice with myhcα 334–352 before transplantation resulted in modest but statistically significant accelerated allograft rejection (7.3 ± 0.4 days, p < 0.03). Therefore, in contrast to EAM, initial anti-CM T cell response (day 10) is apparently not directed to myhcα 334–352 peptide but to another yet unidentified CM determinant. However, at day 50 after grafting, secondary T cell response to myhcα 334–352 peptide occurs. This result shows that 1) initial posttransplant T cell response to CM is directed to a CM determinant that is distinct from the myhcα 334–352 EAM-inducing peptide and, 2) after transplant rejection has occurred, autoimmunity to CM persists and spreads to other determinants on CM. This suggests that anti-CM autoimmune responses may play an important role in long-term or chronic rejection of allotransplanted hearts.

FIGURE 5.

T cell response to myocarditogenic CM peptide myhcα 334–352 in heart-transplanted mice. A, T cell proliferative response to myhcα 334–352 peptide in A.TL mice transplanted with allogeneic A/J hearts is shown. Recipient spleens were removed at either 10–13 days (open symbols) or 50 days (filled symbols) following transplantation. Suspensions of spleen cells were cultured in the presence of myhcα 334–352 peptide (triangles), control λ repressor peptide P12–26 (circles), or medium alone for 4 days. Data are expressed as Δcpm (experimental counts − medium counts). Data for an individual mouse are shown, representative of three to eight mice tested. B, myhcα 334–352 peptide-mediated recipient spleen T cell release of IFN-γ was measured at 10–13 days and 50 days following heart transplantation using ELISA assay. Splenocytes were cultured for 48 h either with myhcα 334–352 (solid bars) or control P12–26 peptide at 50 μM (open bars). Data are expressed as pg/ml of IFN-γ following substraction of control values obtained with cells cultured with medium alone. The data shown are representative of three to five mice tested individually in each group.

FIGURE 5.

T cell response to myocarditogenic CM peptide myhcα 334–352 in heart-transplanted mice. A, T cell proliferative response to myhcα 334–352 peptide in A.TL mice transplanted with allogeneic A/J hearts is shown. Recipient spleens were removed at either 10–13 days (open symbols) or 50 days (filled symbols) following transplantation. Suspensions of spleen cells were cultured in the presence of myhcα 334–352 peptide (triangles), control λ repressor peptide P12–26 (circles), or medium alone for 4 days. Data are expressed as Δcpm (experimental counts − medium counts). Data for an individual mouse are shown, representative of three to eight mice tested. B, myhcα 334–352 peptide-mediated recipient spleen T cell release of IFN-γ was measured at 10–13 days and 50 days following heart transplantation using ELISA assay. Splenocytes were cultured for 48 h either with myhcα 334–352 (solid bars) or control P12–26 peptide at 50 μM (open bars). Data are expressed as pg/ml of IFN-γ following substraction of control values obtained with cells cultured with medium alone. The data shown are representative of three to five mice tested individually in each group.

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In this article, we showed that transplantation of allogeneic hearts in mice induced immune tolerance breakdown to CM, the autoantigen that causes the heart autoimmune disease, autoimmune myocarditis. In addition, cardiac tissues from transplanted and autoimmune diseased mice displayed histological similarities. Finally, pretransplant sensitization of recipient mice with CM caused marked acceleration of allogeneic heart grafts. Taken together, these results strongly suggest that allotransplantation-induced autoimmunity to CM contributes to cardiac graft rejection. A/J and A.TL mice have identical background genes and differ only at the K class I locus in the MHC. Since, autografts are not rejected and show no signs of autoimmunity to CM, we conclude that anti-MHC class I alloresponse is a prerequisite to tolerance breakdown to CM after cardiac allotransplantation. However, while initial alloresponse to donor MHC is necessary to disrupt tolerance to CM, once anti-CM autoimmunity has been induced, we provided evidence showing that it is sufficient alone to mediate the rejection of the cardiac transplant. This was confirmed by the fact that CM-sensitized mice reject autografts in the absence of alloresponse.

Two non-mutually exclusive mechanisms described in Fig. 6 may explain how the autoimmune process is triggered by the alloresponse. 1) Inflammatory cytokines produced by activated alloreactive T cells at the site of the graft may up-regulate the presentation of existing CM/MHC class II complexes and induce the expression of costimulatory molecules on cardiac APCs. Efficient presentation of myocarditogenic CM determinants in the transplanted heart may lower the threshold required for activation of formerly silent CM-specific autoreactive T lymphocytes. 2) Alternatively, myocardial damage by alloreactive T cells may cause the release of circulating CM. De novo exogenous processing of extracellular autoantigen by recipient APCs in peripheral lymphoid organs and/or in the grafted heart might result in the presentation of new CM determinants, thus sensitizing myocarditogenic T cells.

FIGURE 6.

Mechanisms involved in breakdown of T cell tolerance to CM following heart transplantation. The infiltration of the transplanted heart by anti-donor MHC-activated alloreactive T cells leads to local inflammation and cytokine release. Model 1: Local inflammation and cytokine release at the site of the graft associated with up-regulation of MHC class II expression and induction of costimulatory receptors on cardiac APCs of donor origin may lead to efficient presentation of CM/donor MHC class II complexes to anti-CM T cells. Model 2: Tissue damage at the site of the graft may induce the release of CM and its processing and presentation in peptide form by infiltrating macrophages and dendritic cells of recipient origin. Such de novo presentation of CM determinants in recipient MHC class II context could induce the activation of anti-CM autoreactive T cells of recipient origin.

FIGURE 6.

Mechanisms involved in breakdown of T cell tolerance to CM following heart transplantation. The infiltration of the transplanted heart by anti-donor MHC-activated alloreactive T cells leads to local inflammation and cytokine release. Model 1: Local inflammation and cytokine release at the site of the graft associated with up-regulation of MHC class II expression and induction of costimulatory receptors on cardiac APCs of donor origin may lead to efficient presentation of CM/donor MHC class II complexes to anti-CM T cells. Model 2: Tissue damage at the site of the graft may induce the release of CM and its processing and presentation in peptide form by infiltrating macrophages and dendritic cells of recipient origin. Such de novo presentation of CM determinants in recipient MHC class II context could induce the activation of anti-CM autoreactive T cells of recipient origin.

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Donermeyer et al. previously reported that injection with cyanogen bromide-derived CM fragments in mice induces a T cell response to myhcα 334–352 peptide on CM (16). In the same study, immunization with this peptide was shown to be sufficient to mediate EAM. In our model, at day 10, no T cell response to myhcα 334–352 peptide could be detected in transplanted mice while strong autoimmune response to CM was measured. This indicates that, in heart grafted mice, initial anti-CM immune response is directed to determinants other than the myhcα 334–352 peptide. However, diversification of T cell response to myhcα 334–352 self peptide occurs at a later time point (50 days) after transplantation. Therefore, Ag spreading, a phenomenon previously described in autoimmune diseases such as multiple sclerosis and insulin-dependent diabetes (17, 18, 19), is also a feature of T cell response to CM determinants during cardiac graft rejection.

It is noteworthy that allotransplanted mice bear two vascularized hearts, their original heart and the transplanted hearts located in the thoracic and abdominal cavities, respectively. Interestingly, we observed only marginal focal inflammatory cell infiltration in the mouse’s own original heart, and, in contrast to the transplanted heart, no obvious signs of myocyte necrosis were observed. It may seem surprising that autoimmunity had apparently not spread from the transplanted to the native mouse’s cardiac tissue. However, the absence of trauma and of local inflammation in the nontransplanted heart may have resulted in the lack of chemokine and inflammatory cytocine production and of adhesion molecule up-regulation, thereby accounting for the absence of infiltration by immunocompetent cells. It is also important to note that initiation of autoimmunity to the unmanipulated heart in EAM model requires coinjection of the Ag and pertussis toxin. By activating APCs, pertussin toxin may elicit or exacerbate the presentation of self determinants to autoreactive T cells, thereby promoting local inflammation and cellular infiltration in the otherwise unmanipulated heart tissue.

Previous studies have provided some evidence for the involvement of tissue-specific Ags in allotransplant rejection (20, 21, 22). These responses were directed to minor histocompatibility Ags, i.e., tissue-specific proteins differing between donors and recipients. In addition, little information exists about their precise nature and the degree of their involvement in the rejection process. In contrast, in our model, CM is identical between donor and recipient mice, and it therefore does not represent a minor histocompatibility Ag. For the first time, to our knowledge, our study clearly identifies a tissue-specific Ag shared by donor and recipient that is involved in cardiac transplantation, and it establishes its contribution to the rejection process.

It is possible that anti-CM autoreactive responses account for the following clinical observations in heart transplant patients: 1) patients originally diagnosed with chronic myocarditis experience more frequent and severe rejection episodes than patients with other heart diseases (23); 2) an increase in the amounts of circulating CM following transplantation is associated with poor prognosis for cardiac transplant survival (24). Thus, heart transplantation-induced anti-CM autoimmunity is clinically relevant. Together with our observations, this suggests the need for monitoring this response as a diagnostic indicator of rejection.

Induction of autoimmunity may represent a general phenomenon in allotransplantation. Tissue Ags known to induce autoimmune disease, such as glutamic acid decarboxylase (insulin dependent diabetes) and type IV collagen (Goodpasture’s syndrome), could also be involved in the rejection of donor islets and kidney transplants, respectively (17, 25, 26, 27, 28, 29). Therefore, pathologies observed in transplanted patients and often diagnosed as recurrent autoimmune diseases may instead be the result of de novo autoimmunity caused by the transplantation itself (30). While T cell response to donor MHC alloantigens undoubtedly initiates graft rejection, secondary autoreactive responses to some organ-specific Ags may perpetuate and amplify the immune destruction of transplanted tissues. This implies that, in addition to the inhibition of T cell responses to donor MHC molecules, blocking of autoimmune responses to key tissue Ags, such as CM in heart transplantation, may represent a necessary therapeutic approach to achieve immune tolerance to donor cells and subsequent long-term transplant survival.

We thank E. McLashina and V. Ossovskaya for help in purificaton of murine CM; B. Diamond for providing anti-myosin mAb-producing hybridoma cells (11C6-E3) and for helpful comments on the manuscript; E. Sercarz for encouragement and valuable discussion; J. Sprent, R. C. Tam, and J. Kanellopoulos for critical review of the manuscript; and J. Pete for technical assistance.

1

This work was supported by National Institutes of Health Grant AI-33704 (to G.B.) and by a fellowship from the National Kidney Foundation (to E.V.F.).

4

Abbreviations used in this paper: CM, cardiac myosin; EAM, Experimental autoimmune myocarditis; HEL, hen eggwhite lysozyme.

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