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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.

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 gamma IFN 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.

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.

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 (Ed) 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.

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.

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.

Indirect allorecognition is an important component of allotransplant rejection. Although the initial indirect alloresponse is limited to a few dominant determinants on donor major histocompatibility complex (MHC) molecules, subsequent spreading to additional determinants on recipient and donor antigens is common. Gilles Benichou and colleagues discuss the mechanisms by which immunodominance is acquired or disrupted in indirect alloresponses, and examine the implications for the design of peptide-based selective immunotherapy in transplantation.

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 Dd 61-80 is dominant in syngeneic BALB/c mice (H2d). 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. Here, we show that transplantation of BALB/c mice with allogeneic B10.A (H2a) splenocytes led to an autoimmune T cell response toward the dominant self-peptide Dd 61-80. No T cell responses to Dd 61-80 peptide were observed after transplantation of C57BL/6 (H2b) splenocytes into BALB/c recipients. In addition, we provide evidence indicating that the breakdown of tolerance to Dd 61-80 self-peptide resulted from the presentation of the donor crossreactive peptide Kk 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.

Autologous proteins are continuously processed and presented in the form of peptides associated with self major histocompatibility (MHC) molecules at the surface of antigen-presenting cells for interaction with autoreactive T cells. During thymic selection, the presentation of self peptides is an essential element in the establishment of the T cell repertoire. Developing T cells which recognize self peptide/self MHC complexes with sufficient affinity are clonally deleted. However, we and others have recently demonstrated that a variety of self peptides, despite their high binding affinity to MHC molecules, never reach the threshold of presentation to ensure negative selection (cryptic self peptides). This mechanism may have been selected to avoid excessive purging of T cell repertoire during ontogeny. However, T cells directed to cryptic self determinants represent a continuous threat for the initiation of autoimmunity in adults. Supporting this view, recent studies have documented the involvement of cryptic self peptide presentation in different autoimmune diseases. In this article, we examine the factors that govern the selection of self peptides for presentation to autoreactive T cells in vivo and discuss their contribution to both the induction and the maintenance of self tolerance. In addition, we analyze the mechanisms by which the hierarchy of determinants on a self protein can be disrupted, thereby leading to the presentation of previously cryptic self peptides and the induction of an autoimmune T-cell-mediated process.

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-2d) (Kd, A(d), E(d), Ld, Dd) were injected into Ld-deficient BALB/c-dm2 (dm2) mutant mice (Kd, A(d), E(d), -, Dd). 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 Ld molecule, Ld 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 Ld 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.

Recent progress in gene therapy may provide a new strategy for prevention of allograft rejection. Oligonucleotides have been shown to inhibit specific gene transcription in both cell-free and living-cell systems. In our previous studies, a 26-mer oligonucleotide (T2) designed to form a triple helix with the X/X2 box promoter region of human MHC class II (DRA) gene was shown to prevent the induction by IFN-gamma of HLA-DR molecules. Here, we show that this oligonucleotide downregulates two other IFN gamma-inducible molecules, the adhesion molecule ICAM-1 and the Fc receptor for IgG on the surface of human cells. T2 has no effect on TNF alpha- and IL-1-mediated ICAM-1 upregulation, showing its specificity for IFN gamma. T2 oligonucleotide is shown to inhibit IFN gamma-mediated induction of Fc receptor on human blood monocytes as assessed by flow cytometry. Furthermore, pretreatment of monocytes with T2 resulted in suppression of anti-CD3-mediated peripheral blood T cell proliferation. The presented data suggest that oligonucleotide T2 blockade of IFN gamma-induction of different immune receptors on accessory cells is associated with inhibition of T cell proliferative responses.