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 myhcalpha 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.

Recent studies using synthetic altered peptide ligands (Analogues) have led to the fine dissection of TCR-mediated T cell functions elicited by Ag recognition. Certain Analogues behave as full agonists of the antigenic peptide while others are partial agonists in that they only trigger selected T cell functions. Additionally, peptide Analogues can behave as antagonists by inhibiting functions of T cell clones when coincubated with the wild-type peptide. In fetal thymic organ cultures, synthetic altered peptide ligands can impact T cell repertoire selection. However, the influence of naturally occurring peptide Analogues on T cell immunity in vivo remains hypothetical. We previously reported that, in B10.A mice, immunogenicity and tolerogenicity of the self-MHC class I peptide, Ld 61-80, were influenced by the presentation of a cross-reactive self-peptide, Kk 61-80. Here, we show that Kk 61-80 self-peptide represents a partial agonist of Ld 61-80 in that it induced the proliferation but not the lymphokine production of Ld 61-80-primed T cells. Next, we showed that presentation of Kk 61-80 Analogue peptide mediated T cell tolerance toward Ld 61-80 self-peptide. Alternatively, when Ld protein represented an alloantigen displayed on transplanted cells, immunization with Kk 61-80 Analogue sensitized recipient mice to Ld 61-80 peptide, thus inducing potent immune responses to donor cells. These results show that the presentation of natural Analogue peptides may represent an essential component of T cell responses involved in autoimmunity and transplant rejection.

72 Allograft rejection is initiated by immune response to either intact or processed donor major histocompatibility complex (MHC) proteins. We previously 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-antigen However, the contribution of this de novo autoimmune response to the graft rejection remains unknown. Here, we investigated whether de novo induction of heart autoimmunity could be detected during the rejection of allogeneic cardiac transplants in mice. 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. Allogeneic hearts were consistantly rejected at day 9.4± 0.3 (A/J→ A.TL) and at day 8.6 ± 0.5 (A.TL→A/J) after transplantation. Histologic examination of rejected donor hearts revealed an interstital inflammatory cell infiltrate, adjacent myocyte damage and extensive subepicardial calcifications. Interestingly, we found that these histopathological features were strikingly similar to those observed in the hearts of mice with experimental autoimmune myocarditis (EAM). This observation prompted us to determine whether following cardiac allograft the myocarditis-like histopathology of transplanted heart is caused by immune responses to the known myocarditic autoantigen, cardiac myosin. To test this, T cell response to CM was investigated in the spleens of mice transplanted with allogeneic hearts. Vigorous anti-CM T cell response was observed in transplanted mice, and it was found to be mediated by CD4+, MHC class II (Ak)-restricted T cells (data not shown). In addition, high titers of CM-specific autoantibodies were detected in the sera of transplanted mice. Next, to demonstrate the relevance of anti-CM autoimmune response to the allograft rejection process, recipient mice were sensitized with CM and then transplanted with either allogeneic or syngeneic hearts. This resulted in accelerated rejection of allogeneic cardiac grafts. Most importantly, CM-sensitized were found to reject syngeneic transplants. Therefore, in the absence of allogeneic stimulus, anti-CM alone was sufficient to ensure the rejection of transplanted cardiac grafts. We conclude that while T cell response to donor MHC alloantigen is the initiatory event in graft rejection, breakdown of tolerance associated withde novo autoimmunity to key organ-specific antigens is likely to perpetuate and amplify the immune destruction of transplanted tissues.

Breakdown of T cell tolerance to self-myelin basic protein induces an autoimmune process that leads to demyelination of the central nervous system (CNS) in multiple sclerosis (MS) patients. While the autoimmune disease is initiated by antigen-specific autoreactive T cells, there is accumulating evidence that CNS injury is essentially mediated by CNS-infiltrating inflammatory cells. In addition, it is established that activated macrophages and polymorphonuclear cells contribute to tissue damage in several inflammatory diseases by releasing highly reactive oxygen metabolites. It was therefore possible that demyelination associated with MS results from oxidative injury caused by a cascade of oxygen reactive metabolites produced by CNS-infiltrating activated macrophages and other inflammatory cells. To address this question, we tested the effect of a synthetic catalytic scavenger of oxygen radicals, EUK-8, on experimental allergic encephalomyelitis (EAE) in mice, the animal model for MS in humans. We observed that repeated injection of EUK-8 starting at the time of EAE induction delayed the onset and markedly reduced the severity of the disease. Strikingly, all EUK-8-treated mice completely recovered after 40 days. In addition, we showed that posttreatment with EUK-8 4 days after EAE induction also resulted in a significant amelioration of EAE disease. These results indicate that oxygen metabolites secreted by inflammatory cells at the site of tissue destruction play a major role in the induction and presumably the perpetuation of the autoimmune disease. This study also suggests that treatment with oxygen metabolites scavengers may represent a novel and promising strategy to prevent the onset and to block the course of ongoing autoimmune encephalomyelitis and other inflammatory autoimmune diseases. (C) 1997 Academic Press.

T cell tolerance to self-antigens is established through the recognition by immature T cells of dominant self-peptides presented in association with self-MHC molecules in the developing thymus (negative selection). The self-peptide D-d 61-80 is dominant in syngeneic BALB/c mice (H2(d)), T cell tolerance to D-d 61-80 in this mouse strain resulted in the absence of T cell proliferation following in vivo priming with D-d 61-80 peptide, Here, we show that transplantation of BALB/c mice with allogeneic B10.A (H2(a)) splenocytes led to an autoimmune T cell response toward the dominant self-peptide D-d 61-80, NO T cell responses to D-d 61-80 peptide were observed after transplantation of C57BL/6 (H2(b)) splenocytes into BALB/c recipients. In addition, we provide evidence indicating that the breakdown of tolerance to D-d 61-80 self-peptide resulted from the presentation of the donor crossreactive peptide K-k 61-80 at the surface of recipient antigen-presenting cells, Taken together, our results suggest that following allotransplantation, T cell responses to donor antigens could spread to crossreactive determinants on self-proteins, thus perpetuating and amplifying the rejection process and presumably initiating tissue-specific autoimmune disorders.

There is accumulating evidence indicating that the T cell response to donor major histocompatibility complex (MHC)* peptides plays a crucial role in graft rejection. We and others previously demonstrated the involvement of MHC class-II-restricted recognition of donor MHC class I and II peptides by alloreactive CD4(+) T helper cells in graft rejection. Here we studied the in vivo induction of CD8(+) cytotoxic T lymphocytes (CTL) directed to donor MHC class I peptides following allotransplantation in the mouse. To address this question, BALB/c irradiated splenocytes (H-2(d)) (K-d, A(d), E(d) L(d), D-d) were injected into L(d)-deficient BALB/c-dm2 (dm2) mutant mice (K-d, A(d), E(d) -, D-d). Nine days after allogeneic cell transplant, recipient lymph node T cells were tested for cytolytic activity using peritoneal macrophages as targets. We observed that in addition to BALB/c targets, dm2 macrophages could also be lysed but only when incubated with a dominant peptide on donor L(d) molecule, L(d) 61-80. This response was abolished by anti-CD8 but not anti CD4 monoclonal antibodies. In addition, after immunization of dm2 mice with the peptide L(d) 61-80, alloreactive CTL were generated in vivo and shown to destroy allogeneic donor BALB/c target cells in the absence of exogenously added peptide. We conclude that after allotransplantation, concomitant in vivo priming of alloreactive CD8(+) CTL by donor MHC class I peptides occurs through both direct and indirect pathways of allorecognition.

Self-proteins are regularly processed for presentation to autoreactive T cells in association with both class I and class II major histocompatibility complex (MHC) molecules. The presentation of self-peptides plays a crucial role in the acquisition of T cell repertoire during thymic selection. We previously reported that the self-MHC class I peptide Ld 61-80 was immunogenic in syngeneic B10.A mice (H-2a). We showed that despite its high affinity for self-MHC class II molecules, Ld 61-80 peptide failed to induce elimination of autoreactive CD4+ T cells, presumably due to incomplete processing and presentation in the B10.A's developing thymus (cryptic-self peptide). In this report, we showed that the cryptic phenotype was not an intrinsic property of the self-peptide Ld 61-80 since it was found to be naturally presented and subsequently tolerogenic in BALB/c mice (H-2d) (dominant self-peptide). In addition, the self-peptide Ld 61-80 was found to be immunogenic in different H-2a mice while it was invariably tolerogenic in H-2d mice regardless of their background genes. We observed that Ld 61-80 bound equally well to H-2d and H-2k MHC class II molecules. Also, no correlation was found between the quantity of self-Ld protein and the tolerogenicity of Ld 61-80. Surprisingly, Ld 61-80 was not naturally presented in (H-2d x H-2a) F1 mice, indicating that the H-2a MHC locus contained a gene that impaired the presentation of the self-peptide. Analyses of T cell responses to the self-peptide in several H-2 recombinant mice revealed that the presentation of Ld 61-80 was controlled by genes that mapped to a 170-kb portion of the MHC class II region. This study shows that (a) endogenously processed self-peptides presented by MHC class II molecules are involved in shaping the CD4+ T cell repertoire in the thymus; (b) The selection of self-peptides for presentation by MHC class II molecules to nascent autoreactive T cells is influenced by nonstructural MHC genes that map to a 170-kb portion of the MHC class II region; and (c) the MHC locus of H-2a mice encodes factors that prevent or abrogate the presentation by MHC class II molecules of the self-peptide Ld 61-80. These findings may have important implications for understanding the molecular mechanisms involved in T cell repertoire acquisition and self-tolerance induction.