BACKGROUNDImmune-mediated injury to the graft has been implicated in the pathogenesis of chronic rejection. However, little is known regarding the nature of the antigen(s) involved in this immune process. We demonstrated that cardiac transplantation in mice induces an autoimmune T-cell response to a heart tissue-specific protein, cardiac myosin (CM). This response contributes to transplant rejection in that its modulation affects cardiac graft survival. This study investigates whether anti-CM T cells undergo activation and expansion in mice with chronic cardiac allograft rejection.METHODSThe frequency of CM- and donor major histocompatibility complex (MHC)-specific interferon (IFN)-gamma-producing T cells were assessed by ELISPOT in BALB/c mice, which were injected with anti-CD40L (MR1) mAb (chronic rejection group) or CTLA4Ig fusion protein (tolerant group) and transplanted with C57BL/6 cardiac allografts.RESULTS AND CONCLUSIONSMR1-treated BALB/c recipients of C57BL/6 hearts with chronic rejection displayed a high frequency of activated CM-specific T cells, whereas the frequency of activated alloreactive T cells were similar to naïve, nontransplanted mice. In contrast, no activation of CM-reactive T cells was detected in tolerant recipients after CTLA4Ig treatment. Therefore, in the absence of alloimmunity, chronic rejection is associated with persistence of a T-cell response against CM. Our data indicate that anti-CM autoimmunity may be involved in the immune mechanisms of chronic rejection and suggest that tolerance strategies should target both allo- and autoimmune responses to prevent this process.

We have previously shown that cardiac allotransplantation induces autoimmunity to a heart tissue-specific antigen, cardiac myosin (CM). In a single MHC class I-mismatched donor-recipient combination, pre-transplant injection of A.TL mice with CM in IFA resulted in a prolongation of A/J heart allograft survival (10 ± 2 d vs 100 ± 25 d). However, this treatment had no effect in a fully mismatched, C57BL/6 into BALB/c, mouse model. Here, we studied whether increased graft survival observed after modulation of CM response is dependent upon the magnitude of alloresponse. ELISPOT analysis showed that in untreated A.TL mice grafted with A/J hearts, the frequency of alloreactive Th1 cells was significantly lower than in BALB/c mice that received C57BL/6 hearts. These data confirm that in the absence of MHC class II disparity, alloreactive CD4+ T cells are activated via indirect pathway and are oligoclonal. Conversely, in a full mismatch model, there is a potent polyclonal direct alloresponse. We next observed that in a single MHC class I-mismatched model, after CM/IFA treatment, the frequency of Th1 alloreactive cells was comparable to naı̈ve animals. In contrast, in a full mismatch model, a high frequency of alloreactive Th1 cells was detected. These data suggested that modulation of CM response can affect oligoclonal indirect alloresponse but not polyclonal direct alloresponse. Next, CM/IFA-treated BALB/c mice were transplanted with B6.129 (H-2b) MHC class II KO hearts. In this model, in the absense of MHC class II on donor APCs, host CD4+ T cells recognize donor-derived antigens exclusively in indirect fashion and are oligoclonal. We found that CM/IFA-injected animals grafted with MHC class II KO hearts experienced significant prolongation of heart survival as compared to untreated mice (11±1 d vs 24±2 d). We conclude that modulation of CM response can on its own achieve graft prolongation when CD4+ alloresponse is oligoclonal, i.e. mediated via indirect allorecognition. Our findings have important implications for the design of future therapies in heart transplantation.

The role of immune response to tissue-specific Ags in transplant rejection is poorly defined. We have previously reported that transplantation of cardiac allografts triggers a CD4+ Th1 cell response to cardiac myosin (CM), a major contractile protein of the heart, and that pretransplant activation of proinflammatory CM-specific T cells accelerates rejection. In this study, we show that administration of CM together with IFA (CM/IFA) can prevent acute rejection of an allogeneic heart transplant. Prolongation of cardiac graft survival is associated with activation of CM- and allo-specific T cells secreting type 2 cytokines (IL-4, IL-5) and reduction of the frequency of proinflammatory IFN-γ-secreting (type 1) alloreactive T cells. Blocking of IL-4 cytokine with Abs abrogates the prolongation. CM/IFA treatment prevents acute rejection of MHC class I-mismatched, but not fully mismatched grafts. However, if donor heart is devoid of MHC class II expression, CM-IFA administration delays rejection of fully allogeneic cardiac transplants. This finding suggests that the effect of CM modulation depends on the type (direct vs indirect) and strength of recipient’s CD4+ T cell alloresponse. Our results underscore the important role of host immunity to tissue-specific Ags in the rejection of an allograft. This study demonstrates that modulation of the immune response to a tissue-specific Ag can significantly prolong cardiac allograft survival, an observation that may have important implications for the development of novel selective immune therapies in transplantation.

In this study, we measured direct and indirect T-cell alloresponses mediated by CD4+ and CD8+ T cells in three mouse transplantation models: skin, cornea, and retina. We show that the contribution of direct and indirect antigen recognition pathways to the alloresponse to fully allogeneic grafts varies depending upon the nature of the tissue/organ transplanted. The implications of this finding for understanding the cellular mechanisms by which rejection is mediated in different transplant models are discussed.

Chronic allograft dysfunction, which is the most common cause of late allograft failure, is in part caused by an ongoing immune response orchestrated by T lymphocytes primed by the indirect pathway of allorecognition. The low frequencies of such T cells have made it difficult to study indirect alloreactivity by using currently available assays. The development of a sensitive, clinically useful method of measuring indirect alloreactivity among human renal transplant recipients was thus attempted. Furthermore, in a pilot immunologic study, the contribution of the indirect pathway was studied in two groups of renal transplant recipients, i.e., patients with no prior acute rejection episodes and stable renal function ("stable" patients) and patients with at least one previous episode of biopsy-proven acute rejection, who were thus at risk for the development of chronic rejection ("high-risk" patients). The frequencies of type 1 T helper (interferon-gamma-producing) and type 2 T helper (interleukin-5- and -10-producing) peripheral blood lymphocytes reactive with a panel of synthetic peptides (corresponding to sequences from donor HLA-DR molecules) were determined for renal transplant recipients and normal control subjects by using an enzyme-linked immunosorbent spot assay (ELISPOT). Among recipients of DR-mismatched allografts, a cut-off value of 60 interferon-gamma spots/10(6) cells significantly (P = 0.02) separated stable patients (creatinine concentration, 1.1 +/- 0.3 mg/dl) from high-risk patients (creatinine concentration, 2.3 +/- 1.7 mg/dl). This is the first demonstration that the enzyme-linked immunosorbent spot assay can be used to monitor indirect alloreactivity to donor HLA-DR peptides among renal transplant recipients. These data provide the rationale for the prospective study of indirect alloreactivity among transplant recipients, to allow predictions of which patients would be at risk for the development of chronic rejection and thus allow appropriate planning of future interventions.

The transplantation of neuronal cells and tissues represents a promising approach for the treatment of incurable neurodegenerative diseases. Indeed, it has been reported recently that retinal transplantation can rescue photoreceptor cells and delay age-related changes in various retinal layers in rodents. However, retinal grafts deteriorate progressively after placement in recipients' eyes. Here we investigated whether a host's immune response elicited toward the graft contributes to its deterioration. Using an ELISA spot assay, we measured T cell responses to retinal tissues placed in the vitreous cavity of syngeneic and allogeneic mice. We found that allogeneic retinas induced potent alloimmune responses mediated by T cells secreting type 1 cytokines (IFN-gamma and IL-2). No response was found in mice engrafted with syngeneic retinas. In addition, all syngeneic retinal grafts displayed no signs of tissue damage (at 55 days), while the majority of allogeneic retinas deteriorated as early as 12 days after placement. Next, we showed that anti-donor responses occurred within two phenotypically and functionally distinct T cell subsets: CD4+ T cells secreting IL-2 and CD8+ T cells producing IFN-gamma. Importantly, CD4+ T cells were necessary and sufficient to cause graft deterioration, while CD8+ T cells did not contribute to this process.

T lymphocytes interact with protein antigens (Ags) in the form of peptides bound to self-major histocompatibility complex (MHC) molecules displayed at the surface of antigen-presenting cells (APC) (1, 2). Dissection of the different T-cell receptor (TCR)-mediated functions elicited by peptide Ag stimulation is essential for understanding the mechanisms that govern T-cell immunity. Historically, it was believed that each individual T-cell clone recognized a single peptide, and T-cell activation was thought to be an all-or-nothing phenomenon. The current interpretation is that the exquisite specificity of T-cell recognition of a given peptide is not absolute, because of crossreactive T-cell responses with other sequentially or structurally related peptides (3-7). Additionally, recent studies (8, 10) using altered peptide ligands (APLs) or analog peptides displaying amino acid subsitutions at key TCR contact positions of the Ag peptide have revealed that TCR can interpret subtle modifications in its ligand, resulting in differential activation of T-cell functions.KeywordsAnalog PeptideSterile SyringeAltered Peptide LigandExquisite SpecificityFetal Thymic Organ CultureThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The purpose this study was to investigate the relationship of anti-myosin and anti–heat shock protein immunoglobulin G (IgG) serum antibodies to the original heart disease of cardiac transplant recipients, and also to rejection and patient survival after cardiac transplantation.Anti-myosin and anti–heat shock protein (anti-hsp) IgG antibodies were evaluated in pre-transplant sera from 41 adult cardiac allograft recipients and in sequential post-transplant serum samples from 11 recipients, collected at the time of routine endomyocardial biopsies during the first 6 months after transplantation. In addition, the levels of these antibodies were determined from the sera of 28 healthy blood donors.Higher anti-myosin antibody levels were observed in pre-transplant sera than in sera from normal controls. Moreover, patients with chronic Chagas heart disease showed higher anti-myosin levels than patients with ischemic heart disease, and also higher levels, although not statistically significant, than patients with dilated cardiomyopathy. Higher anti-hsp levels were also observed in patients compared with healthy controls, but no significant differences were detected among the different types of heart diseases. Higher pre-transplant anti-myosin, but not anti-hsp, levels were associated with lower 2-year post-transplant survival. In the post-transplant period, higher anti-myosin IgG levels were detected in sera collected during acute rejection than in sera collected during the rejection-free period, whereas anti-hsp IgG levels showed no difference between these periods.The present findings are of interest for post-transplant management and, in addition, suggest a pathogenic role for anti-myosin antibodies in cardiac transplant rejection, as has been proposed in experimental models of cardiac transplantation.

The presentation of MHC peptides by recipient and donor antigen presenting cells is an essential element in allorecognition and allograft rejection. MHC proteins contains two sets of determinants: the dominant determinants that are efficiently processed and presented to T cells, and the cryptic determinants that are not presented sufficiently enough to induce T-cell responses in vivo. In transplanted mice, initial T-cell response to MHC peptides is consistently limited to a single or a few immunodominant determinants on donor MHC molecule. However, in this article we show that under appropriate circumstances the hierarchy of determinants on MHC molecules can be disrupted. First, we observed that γIFN can trigger de novo presentation of cryptic self-MHC peptides by spleen cells. Moreover, we showed that allotransplantation is associated with induction of T-cell responses to formerly cryptic determinants on both syngeneic and allogeneic MHC molecules. Our results suggest that cross-reactivity and inflammation are responsible for the initiation of these auto- and alloimmune responses after transplantation.

Fedoseyeva, Eugenia V.1; Kishimoto, Koji2; Rolls, Hillary1; Sayegh, Mohamed2; Benichou, Gilles1 Author Information

Anosova, Natalie G.1; Boisgerault, Florence1; Fedoseyeva, Eugenia V.1; Whiteley, Simon1; Illigens, Ben1; Young, Michael1; Benichou, Gilles1 Author Information

Fedoseyeva, Eugenia V.1; Kishimoto, Koji2; Rolls, Hillary1; Sayegh, Mohamed H.2; Benichou, Gilles1 Author Information

We analyzed CD4(+) T helper responses to wild-type (wt) and mutated (mut) p53 protein in normal and tumor-bearing mice. In normal mice, we observed that although some self-p53 determinants induced negative selection of p53-reactive CD4(+) T cells, other p53 determinants (cryptic) were immunogenic, Next, BALB/c mice were inoculated with J774 syngeneic tumor cell line expressing mut p53, BALB/c tumor-bearing mice mounted potent CD4(+) T cell responses to two formerly cryptic peptides on self-p53. This response was characterized by massive production of IL-5, a Th2-type lymphokine. Interestingly, we found that T cell response was induced by different p53 peptides depending upon the stage of cancer. Mut p53 gene was shown to contain a single mutation resulting in the substitution of a tyrosine by a histidine at position 231 of the protein. Two peptides corresponding to wt and mutated sequences of this region were synthesized. Both peptides bound to the MHC class II-presenting molecule (E-d) With Similar affinities. However, only mut p53.225-239 induced T cell responses in normal BALB/c mice, a result strongly suggesting that high-affinity wt p53.225-239 autoreactive T cells had been eliminated in these mice. Surprisingly, CD4(+) T cell responses to both mut and wt p53.225-239 peptides were recorded in J774 tumor-bearing mice, a phenomenon attributed to the recruitment of low-avidity p53.225-239 self-reactive T cells.

We used signal transducer and activator of transcription 4 (STAT4) and STAT6 gene knockout (-/-) mice as recipients of fully mismatched cardiac allografts to study the role of T-cell costimulatory pathways in regulating allogeneic T-helper 1 (Th1) versus Th2 responses in vivo. STAT4(-/-) mice have impaired Th1 responses, whereas STAT6(-/-) mice do not generate normal Th2 responses. Cardiac allografts from C57BL/6 mice were transplanted into normal wild-type (WT), STAT4(-/-), and STAT6(-/-) BALB/c recipients. STAT4(-/-) and STAT6(-/-) mice rejected their grafts with the same tempo as untreated WT recipients. CD28-B7 blockade by a single injection of CTLA4Ig induced long-term engraftment and donor-specific tolerance in all three groups of recipients. CD154 blockade by a single injection of MR1 was effective in prolonging allograft survival and inducing tolerance in STAT4(-/-) mice but was only marginally effective in STAT6(-/-) recipients and WT controls. In addition, a similar protocol of MR1 was ineffective in prolonging graft survival in CD28(-/-) BALB/c recipients, suggesting that the lack of efficacy seen in WT and STAT6(-/-) mice is not due to the presence of a functional CD28-B7 pathway. Furthermore, there was a similar differential effect of CD28-B7 versus CD154-CD40 blockade in inhibiting immune responses in animals immunized with ovalbumin and complete Freund's adjuvant. These novel data indicate that Th1 and Th2 cells are differentially regulated by CD28-B7 versus CD154-CD40 costimulation pathways in vivo and may have potential implications for the development of therapeutic strategies such as T-cell costimulatory blockade in humans.

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.

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.

The presentation of donor-derived MHC peptides by recipient APCs to T cells is an essential component of the rejection of allografts (indirect allorecognition). Initial alloreactive T cell response is confined to a few well processed and presented dominant determinants on donor MHC. However, during long-term graft rejection, T cell response spreads to formerly poorly presented cryptic allogeneic MHC peptides. This phenomenon is likely to play an important role in the amplification and the perpetuation of the rejection process. Additionally, we present evidence that T cell repertoire selection to allogeneic MHC peptides is acquired via recognition of self-MHC peptides presented in the thymus during ontogeny. Supporting this view, we have shown that indirect alloresponses can lead to self-T cell tolerance breakdown to cross-reactive determinants on self-MHC molecules or alternatively that sensitization of recipients to self-MHC peptides can lead to accelerated graft rejection. It is therefore essential to determine the factors which govern the processing and presentation of self and allogeneic MHC molecules and to elucidate the mechanisms regulating subsequent T cell responses in order to design antigen-specific based immune therapies in transplantation.