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Background: Galectin-9 serves opposing roles in the innate and adaptive immune systems. Galectin-9 triggers T-cell immunoglobulin mucin-3 (Tim-3) on T helper type 1 (Th1) cells, thereby terminating Th1 immunity and protecting allografts from host immune attacks. Meanwhile, galectin-9 promotes the maturation of dendritic cells (DCs) that deliver proinflammatory signals. We previously showed that galectin-9 significantly prolongs cardiac allograft survival in mice but failed to induce tolerance. This study aimed at improving the administration protocol to induce allograft tolerance. We examined whether rapamycin can reverse the proinflammatory effects of galectin-9 on DCs and whether rapamycin synergizes with galectin-9 to induce cardiac allograft tolerance., Methods: Monocytes/DCs from cardiac allografts were assessed for Tim-3 expression by flow cytometry. Costimulatory molecules CD80/CD86 were measured on galectin-9/rapamycin-treated bone marrow-derived DCs by flow cytometry. We performed heterotopic cervical cardiac transplantation using BALB/c donors and C57BL/6 recipients and assessed graft survival time. T cells of long-term surviving recipients were immunoassayed for interferon-γ and interleukin-4 secretion., Results: Allograft-infiltrating monocytes/DCs expressed high Tim-3 levels (47.3%±5.6%). Expression of CD80/CD86 was up-regulated on galectin-9-treated bone marrow-derived DCs, which was reversed by rapamycin. Combined treatment with galectin-9 and rapamycin promoted the permanent acceptance of fully mismatched grafts (survival time >180 days; n=6). However, treatment with galectin-9 or rapamycin alone was not sufficient to induce tolerance. Galectin-9/rapamycin-induced tolerance was associated with low donor-specific interferon-γ and interleukin-4 secretion., Conclusions: Rapamycin inhibits proinflammatory effects of galectin-9 on DCs. Combined treatment of galectin-9 and rapamycin promotes allografts tolerance, which is associated with reduced Th1 and Th2 responses.

Background: The involvement of T-cell immunoglobulin mucin-3 (Tim-3) in the regulation of solid organ transplantation rejection is largely unknown. We used galectin-9 (Tim-3 ligand) to evaluate the effect and mechanisms of Tim-3/galectin-9 pathway in an allogeneic heart transplant model., Methods: The murine cardiac transplant model from BALB/c (H-2d) to C57BL/6 (H-2b) was built. The recipients were administered with galectin-9 for 7 days from day 1 or day 3 posttransplant. The complete cessation of cardiac contractility was defined as the observation endpoint. The effect of galectin-9 on cell proliferation was assessed by mixed lymphocyte reaction. Histology and immunohistochemistry were performed to estimate the severity of rejection. The phenotype and cytokine profile of lymphocytes were analyzed by flow cytometry., Results: In vitro, galectin-9 suppressed the proliferation of lymphocytes mixed lymphocyte reactions in a dose- and beta-galactoside-dependent manner. In vivo, galectin-9 treatment from day 1 to day 3 posttransplant achieved similar survival time of grafts (median survival time, 22.7+/-1.2 vs. 23.0+/-1.0 days). The prolonged survival time was associated with reduced infiltration of CD4 and CD8 lymphocytes in allografts. Furthermore, the intragraft transcriptional profiling in galectin-9-treated group showed reduction of IFN-gamma and IL-17 mRNA but elevation of Ebi-3 and galectin-9 mRNA. Flow cytometry analysis indicated that galectin-9 treatment reduced the ratio of T helper (Th) 1 and Th17 cells in the draining lymph nodes and peripheral blood., Conclusions: A short-course administration of galectin-9 significantly prolonged the survival of fully allogeneic cardiac allografts, which was associated with the suppression of Th1 and Th17 immune responses.

Background: Rejection mediated by antibody recognition of the alpha-Gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) is a major barrier in porcine-to-human xenotransplantation. Because the synthesis of alpha-Gal is dependent on alpha1,3 galactosyltransferase (alpha1,3GT), methods of blocking this enzyme are needed. RNA interference induced by small interfering RNA (siRNA) is a powerful technique for allowing the silencing of mammalian genes with great specificity and potency. In this study, we use siRNA for silencing of alpha1,3GT with the purpose of reducing expression of the alpha-Gal epitope and subsequently decreasing immunogenicity of porcine endothelial cells., Methods: alpha1,3GT-specific and control siRNAs were transfected into the porcine aortic endothelial cell line, PED. alpha-Gal expression was assessed by Western blotting, flow cytometry, and immunofluorescence. Protection from human-complement and natural killer (NK)-cell-mediated cytotoxicity was evaluated by Cr-release assays after incubation of PED with normal human serum (NHS) and NK92 cell, respectively., Results: RNA interference was successfully achieved in PED as witnessed by the specific knock-down of alpha1,3GT mRNA levels. Flow cytometric analysis using the Griffonia simplicifolia isolectin B4 lectin confirmed the suppression of alpha1,3GT activity as evidenced by decreased alpha-Gal. Functional relevance of the knock-down phenotype was illustrated by the finding that silenced PED were protected from cytotoxicity of NHS. Protection from NK-mediated cytotoxicity was not observed., Conclusions: Our data are the first to demonstrate that RNA interference is a potent tool to down modulate alpha-Gal expression and to protect endothelial cells from complement-mediated cytotoxicity. Gene silencing by siRNA may represent a new approach for overcoming hyperacute and acute vascular rejection.

Background: Antibodies targeting recipient major histocompatibility complex (MHC) class II molecules have been demonstrated to be effective at prolonging allograft survival. However, antigen-presenting cell depletion would explain this effect and has not been definitively excluded as the mechanism of action of such antibodies. We have studied an anti-MHC class II antibody (OX6) proven to be noncytotoxic in the recipient strain used., Methods: Antibody was administered the day before, 2 hr before, and the day after grafting., Results: Antibody administration on the day before, 2 hr before, and the day after grafting significantly prolonged vascularized cardiac allograft survival. Importantly, treatment recognizing recipient MHC was effective, whereas a similar regimen recognizing donor MHC was not., Conclusions: Noncytotoxic recipient MHC class II-specific antibodies modify allograft rejection. Possible mechanisms for this therapeutic effect are discussed.

There is a need to derive donor-specific tolerance in clinical organ transplantation, where potential benefits remain overshadowed by chronic rejection and side effects of continual immunosuppressive therapy. It is known that the mature immune system in mice can be reprogrammed to accept a foreign graft as if it were "self." Here we show that, once generated, this state of operational tolerance becomes self-sustaining, imposing itself on new cohorts of lymphocytes as they arise. These new cohorts retain specificity for the tolerizing antigen and can be selectively amplified to tolerate new antigens that have linked expression with the original tolerogen. Regulation is critically dependent upon the continuous presence of tolerizing antigen and is mediated by the CD4+ lymphocyte population. We propose that such natural mechanisms of immune regulation may eventually be exploited for transplantation tolerance, even in fully immune-competent recipients.