Anna Mhoyan

Induction of allograft nonresponsiveness after intrathymic inoculation with donor class I allopeptides. I. Correlation of graft survival with antidonor IgG antibody subclasses

We have recently demonstrated that cardiac allograft rejection in the PVG.R8-to-PVG.1U rat strain combination involves the recognition of a isolated class I (RT1.Aa) molecules as peptides in the context of the recipient MHC molecules. Three synthetic peptides (P1, P2, and P3) corresponding to the alpha-helices of the RT1.Aa molecule served as T-cell epitopes for graft rejection. In this study, we demonstrate that two of these peptides (P2 and P3) are sufficient to induce immune nonresponsiveness (median survival time >237 days) to cardiac allografts when presented to the recipient immune system in the thymus 7 days before transplantation. This effect was time dependent, as intrathymic inoculation 60 days before transplantation did not prolong graft survival (median survival time=12 days). Previous studies have demonstrated a critical role for alloantibody responses in mediating graft rejection in this rat strain combination. We, therefore, studied the role alloantibody responses may play in the observed immune nonresponsiveness. The titers of alloantibody in serum samples harvested from graft recipients at different times after transplantation were measured. We used recipient primary aortic endothelial cells genetically manipulated to express the donor RT1.Aa molecule as targets in an enzyme-linked immunosorbent assay. High titers of anti-RT1.Aa IgM antibody were detected in unmanipulated controls at the time of graft rejection. The IgM antibody switched to high IgG titers in intrathymically inoculated rats with accelerated or delayed rejection. Graft rejection in intrathymically manipulated recipients that had achieved a transient state of immunological nonresponsiveness correlated with higher titers of the IgG2b alloantibody. In marked contrast, the long-term graft survivors expressed undetectable or low levels of the IgG2b antibody and moderate to high levels of the IgG1 and IgG2a subclasses. These data suggest that the IgG2b alloantibody may contribute to the rejection reaction, whereas IgG1 and IgG2a may be involved in active enhancement of graft survival.

Chronic cardiac allograft rejection in a rat model disparate for one single class I MHC molecule is associated with indirect recognition by CD4+ T cells

T-cell mediated immune responses play a critical role in chronic allograft dysfunction. The complex nature of allograft rejection, particularly with respect to the vast repertoire of alloantigens and their mode of recognition by T cells, presents a major challenge for the design of well-controlled studies into the immunobiology of chronic rejection. The purpose of this study was to develop a rat model with restricted antigenic specificity that develops chronic rejection without any immunologic manipulation to study the T-cell response. PVG.1U allogeneic hearts disparate for one single class I antigen, RT.1A(u), were transplanted into PVG.R8 rat recipients. Grafts from PVG.R8 were used as syngeneic controls. Chronic rejection was studied by histological analysis of the grafted hearts at various time points posttransplantation (20-100 days). Donor specific alloreactive response was studied in a mixed lymphocyte reaction assay. All allografts survived more than 90 days and showed extensive evidence of chronic rejection, which was characterized by interstitial fibrosis, vasculitis, and occlusive myointimal thickening. Chronic rejection was evident by day 20 and most extensive by day 100 posttransplantation. In marked contrast, syngeneic grafts remained free of chronic lesions. Lymphocytes harvested from graft recipients showed a more vigorous proliferative response to allogeneic splenocytes as compared with that of lymphocytes from nai;ve animals. The proliferative response was primarily mediated by CD4(+) T cells recognizing the RT1.A(a) molecule via the indirect pathway. A single class I disparity in this model generates chronic rejection associated with potent CD4(+) T-cell responses induced by the indirect recognition pathway. The use of this antigenically restricted model may facilitate the design of well-controlled studies for the characterization of immune mechanisms responsible for chronic rejection.

Predominant expression of Th2 cytokines and interferon-gamma in xenogeneic cardiac grafts undergoing acute vascular rejection

Background. The Th1 response has been shown to play a role in acute allograft rejection, whereas the Th2 response has been implicated in the protection of allografts. Unlike allografts, the pattern of cytokines in response to solid-organ xenografts has been the subject of limited studies. We investigated intragraft cytokine expression in a concordant cardiac xenograft model (rat-to-mouse) to test if a particular cytokine profile predominates. Methods. Intra-abdominal cardiac transplantation was performed using C57BL/10 mice as recipients of PVG.R8 rat hearts. Syngeneic grafts (C57BL/10-to-C75BL/10) served as controls. Cardiac grafts harvested on various days posttransplantation were analyzed for histology and intragraft cytokine expression using reverse-transcriptase polymerase chain reaction. Results. The grafts in this model were rejected with a mean survival time of 7±1 days and showed extensive evidence of acute vascular rejection, consisting of global distortion of myocardial architecture, fewer cellular infiltrates, interstitial hemorrhage with myocyte necrosis thrombosis, and vasculitis with neutrophils and lymphocytes infiltrating vessel walls. Cardiac xenografts predominantly expressed Th2 cytokines, interleukin (IL)-4, IL-10, and transforming growth factor-&bgr; with various kinetics. IL-10 was detectable on day 1 and reached its peak level of expression on day 6 posttransplantation. IL-4 showed minimal and undetectable expression on days 1 and 3 and significant expression on day 6 posttransplantation. Transforming growth factor-&bgr; was expressed moderately on all days examined. The expression of interferon (IFN)-&ggr;, a Th1 cytokine, was specific to xenografts and showed a gradual increase from days 3 to 6 posttransplantation. In marked contrast, IL-2 showed complete lack of expression. Conclusions. Our data demonstrate predominant expression of Th2 cytokines and IFN-&ggr; in cardiac xenografts undergoing acute vascular rejection. The Th2 cytokines may promote acute vascular rejection by regulating the humoral response, and IFN-&ggr; may delay, but not prevent, this response.

Prevention of Chronic Rejection with Immunoregulatory Cells Induced by Intrathymic Immune Modulation with Class I Allopeptides

Intrathymic immune modulation with RT1.Aa allopeptides in the PVG.R8‐to‐PVG.1 U rat strain combination leads to long‐term survival of cardiac allografts. This regimen, however, does not induce transplantation tolerance, since most long‐surviving allografts undergo chronic rejection. We investigated recipients with chronic rejection for donor‐specific immune nonresponsiveness and immunoregulatory cells as possible mechanisms responsible for long‐term graft survival. There was a significant reduction in the proliferative response of T cells from long‐term allograft recipients to donor alloantigens as compared with that of naïve T cells. Adoptive transfer of splenocytes from intrathymically manipulated primary long‐term graft survivors into minimally irradiated secondary hosts resulted in indefinite survival of > 80% of allografts, providing evidence for immunoregulatory cells. Secondary recipients had total absence of donor‐reactive cellular and humoral responses. Immunoregulation was also transferable from secondary to tertiary graft recipients. More importantly, there was a significant reduction in the incidence of chronic rejection in secondary hosts (> 85%) and complete prevention of acute and chronic rejection in tertiary hosts. This study demonstrates that intrathymic immunomodulation with class I allopeptides results in the generation of immunoregulatory cells that do not block chronic rejection in primary hosts where they develop, but prevent both acute and chronic allograft rejection when adoptively transferred into secondary and tertiary recipients.