Kentaro Ide

Role for CD47-SIRPα signaling in xenograft rejection by macrophages

We have previously proven that human macrophages can phagocytose porcine cells even in the absence of Ab or complement opsonization, indicating that macrophages present a pivotal immunological obstacle to xenotransplantation. A recent report indicates that the signal regulatory protein (SIRP)α is a critical immune inhibitory receptor on macrophages, and its interaction with CD47, a ligand for SIRPα, prevents autologous phagocytosis. Considering the limited compatibility (73%) in amino acid sequences between pig and human CD47, we hypothesized that the interspecies incompatibility of CD47 may contribute to the rejection of xenogeneic cells by macrophages. In the present study, we have demonstrated that porcine CD47 does not induce SIRPα tyrosine phosphorylation in human macrophage-like cell line, and soluble human CD47-Fc fusion protein inhibits the phagocytic activity of human macrophages toward porcine cells. In addition, we have verified that manipulation of porcine cells for expression of human CD47 radically reduces the susceptibility of the cells to phagocytosis by human macrophages. These results indicate that the interspecies incompatibility of CD47 significantly contributes to the rejection of xenogeneic cells by macrophages. Genetic induction of human CD47 on porcine cells could provide inhibitory signaling to SIRPα on human macrophages, providing a novel approach to preventing macrophage-mediated xenograft rejection.

Liver Sinusoidal Endothelial Cells Tolerize T Cells across MHC Barriers in Mice

Although livers transplanted across MHC barriers in mice are normally accepted without recipient immune suppression, the underlying mechanisms remain to be clarified. To identify the cell type that contributes to induction of such a tolerance state, we established a mixed hepatic constituent cell-lymphocyte reaction (MHLR) assay. Irradiated C57BL/6 (B6) or BALB/c mouse hepatic constituent cells (HCs) and CFSE-labeled B6 splenocytes were cocultured. In allogeneic MHLR, whole HCs did not promote T cell proliferation. When liver sinusoidal endothelial cells (LSECs) were depleted from HC stimulators, allogeneic MHLR resulted in marked proliferation of reactive CD4+ and CD8+ T cells. To test the tolerizing capacity of the LSECs toward alloreactive T cells, B6 splenocytes that had transmigrated through monolayers of B6, BALB/c, or SJL/j LSECs were restimulated with irradiated BALB/c splenocytes. Nonresponsiveness of T cells that had transmigrated through allogeneic BALB/c LSECs and marked proliferation of T cells transmigrated through syngeneic B6 or third-party SJL/j LSECs were observed after the restimulation. Transmigration across the Fas ligand-deficient BALB/c LSECs failed to render CD4+ T cells tolerant. Thus, we demonstrate that Fas ligand expressed on naive LSECs can impart tolerogenic potential upon alloantigen recognition via the direct pathway. This presents a novel relevant mechanism of liver allograft tolerance. In conclusion, LSECs are capable of regulating a polyclonal population of T cells with direct allospecificity, and the Fas/Fas ligand pathway is involved in such LSEC-mediated T cell regulation.

Antibody‐ and complement‐independent phagocytotic and cytolytic activities of human macrophages toward porcine cells

Abstract:  Background:  It has been speculated that host macrophages contribute to rapid clearance of transplanted xenogeneic cells. To address such a possibility, phagocytotic and cytolytic activities of human macrophages toward xenogeneic porcine cells were evaluated in vitro in the absence of antibodies and complement factors.

Liver Sinusoidal Endothelial Cells That Endocytose Allogeneic Cells Suppress T Cells with Indirect Allospecificity

A portal venous injection of allogeneic donor cells is known to prolong the survival of subsequently transplanted allografts. In this study, we investigated the role of liver sinusoidal endothelial cells (LSECs) in immunosuppressive effects induced by a portal injection of allogeneic cells on T cells with indirect allospecificity. To eliminate the direct CD4+ T cell response, C57BL/6 (B6) MHC class II-deficient C2tatm1Ccum (C2D) mice were used as donors. After portal injection of irradiated B6 C2D splenocytes into BALB/c mice, the host LSECs that endocytosed the irradiated allogeneic splenocytes showed enhanced expression of MHC class II molecules, CD80, and Fas ligand (FasL). Due to transmigration across the LSECs from BALB/c mice treated with a portal injection of B6 C2D splenocytes, the naive BALB/c CD4+ T cells lost their responsiveness to stimulus of BALB/c splenic APCs that endocytose donor-type B6 C2D alloantigens, while maintaining a normal response to stimulus of BALB/c splenic APCs that endocytose third-party C3H alloantigens. Similar results were not observed for naive BALB/c CD4+ T cells that transmigrated across the LSECs from BALB/c FasL-deficient mice treated with a portal injection of B6 C2D splenocytes. Adaptive transfer of BALB/c LSECs that had endocytosed B6 C2D splenocytes into BALB/c mice via the portal vein prolonged the survival of subsequently transplanted B6 C2D hearts; however, a similar effect was not observed for BALB/c FasL-deficient LSECs. These findings indicate that LSECs that had endocytosed allogeneic splenocytes have immunosuppressive effects on T cells with indirect allospecificity, at least partially via the Fas/FasL pathway.

Deficiency of N-glycolylneuraminic acid and Galα1-3Galβ1-4GlcNAc epitopes in xenogeneic cells attenuates cytotoxicity of human natural antibodies

Basnet NB, Ide K, Tahara H, Tanaka Y, Ohdan H. Deficiency of N‐glycolylneuraminic acid and Galα1‐3Galβ1‐4GlcNAc epitopes in xenogeneic cells attenuates cytotoxicity of human natural antibodies. Xenotransplantation 2010; 17: 440–448.

Immunological Property of Antibodies against N-Glycolylneuraminic Acid Epitopes in Cytidine Monophospho–N-Acetylneuraminic Acid Hydroxylase-Deficient Mice

The generation of pigs devoid of Galα1,3Galβ1,4GlcNAc (Gal) residues has stimulated interest in non-Gal Ags as potentially important targets for Ab binding leading to rejection of pig organ xenografts in humans. Although N-glycolylneuraminic acid (NeuGc) epitopes, which are widely expressed on the endothelial cells of all mammals except humans, are likely targets of anti–non-Gal Abs, this aspect has not been investigated intensively owing to the absence of an appropriate animal model. In this study, we used CMAH−/− mice, which are completely deficient in NeuGc and thus produce anti-NeuGc Abs. Sera obtained from CMAH−/− mice and healthy human volunteers having anti-NeuGc Abs initiated complement-mediated lysis against CMAH+/+ cells in vitro. The cytotoxic activity of anti-NeuGc Abs was also determined in vivo (i.e., NeuGc-expressing CMAH+/+ mouse splenocytes that had been i.v. injected were completely eliminated in syngeneic CMAH−/− mice). CMAH−/− mice rejected the islets transplanted from syngeneic CMAH+/+ mice. Thus, the anti-NeuGc Ab-mediated response may be crucially involved in xenograft loss. This is the first direct demonstration of the immunogenic property of NeuGc determinants as targets of the corresponding Abs in CMAH+/+-to-CMAH−/− transplantation setting.

Successful hepatitis B vaccination in liver transplant recipients with donor-specific hyporesponsiveness.

Currently, patients are prescribed lifelong treatment with hepatitis B immunoglobulin (HBIg) after liver transplantation (LT) for hepatitis B virus (HBV)‐related diseases in order to prevent reinfection with HBV. Active immunization with an HBV vaccine would be a preferable alternative; however, the immunosuppressive environment in LT recipients is believed to elicit a poor response to vaccination. Minimizing the exposure of the HBV‐infected LT recipients to immunosuppressants would be beneficial in inducing adaptive immunity against HBV by vaccination. In this study, in addition to efforts to minimize immunosuppression, prophylaxis with HBV vaccination combined with continuous HBIg administration was performed in 17 LT recipients who had undergone transplantation attributable to HBV‐related diseases. During the observation period, the overall response rate to HBV vaccination was 64.7%. The immune status of the recipients was evaluated by a mixed lymphocyte reaction assay in response to allostimulation. Patients showing a donor‐specific hyporesponse with a well‐maintained response to the third‐party stimulus always achieved a sustained immune response to the vaccine, whereas patients showing a hyporesponse to both the donor and the third‐party stimulus were unable to do so. Thus, inducing an anti‐donor‐specific immunosuppressive status by minimizing immunosuppression should enable post‐transplant HBV vaccination to be a promising prophylactic strategy.

Safety and feasibility of diet-treated donors with steatotic livers at the initial consultation for living-donor liver transplantation.

Background The purpose of this study was to evaluate both safety of diet-treated donors and the feasibility of their use for living-donor liver transplantation (LDLT). Methods A total of 128 living donors were enrolled in this study between April 2003 and March 2010. Of them, 41 were diagnosed with hepatic steatosis at the initial consultation. Donor selection was based on the findings of liver biopsy accompanied with normalization of liver function tests after diet treatment consisting of an 800 to 1400 kcal/day diet and a 100 to 400 kcal/day exercise without drug treatment, targeting body mass index of 22 kg/m2. Results Body mass index of diet-treated donors was significantly reduced with diet from 23.3±0.6 to 21.9±0.4 kg/m2 (P<0.0001). Liver function tests associated with fatty liver, including alanine aminotransferase, gamma-glutamyl transpeptidase, and total cholesterol levels, also improved with diet (P=0.0128, 0.0016, and 0.0004, respectively). The liver biopsy results of most of these donors showed stage 0/1 fibrosis and minimal/mild steatosis after the diet therapy. Surgical outcomes, including postoperative liver function tests, perioperative complications, and liver regeneration rates, did not significantly differ between nondiet-treated and diet-treated donors. Surgical outcomes and the overall survival did not significantly differ between recipients of grafts from nondiet-treated and diet-treated donors. Conclusion The use of diet-treated donors for living-donor liver transplantation is feasible with respect to donor safety and the outcome of the recipient when strict selection criteria are used.

Significant correlation between spleen volume and thrombocytopenia in liver transplant patients: A concept for predicting persistent thrombocytopenia

Interferon (IFN) therapy with or without ribavirin treatment is well established as a standard antiviral treatment for hepatitis C virus (HCV)–infected patients. However, susceptibility to thrombocytopenia is a major obstacle for initiating or continuing this therapy, particularly in liver transplant (LTx) recipients with HCV. Studies have reported that splenectomy performed concurrently with LTx is a feasible strategy for conditioning patients for anti‐HCV IFN therapy. However, the relationship between the severity of splenomegaly and alterations in the blood cytopenia in LTx recipients remains to be clarified. Here, we analyzed the relationship between spleen volume (SV) and thrombocytopenia in 45 patients who underwent LTx at Hiroshima University Hospital. The extent of pre‐LTx splenomegaly [the SV to body surface area (BSA) ratio in an individual] was inversely correlated with both the post‐LTx white blood cell count and platelet (PLT) count (P < 0.001). Furthermore, the PLT count of patients with thrombocytopenia (PLT count ≤ 5 × 104/mm3) increased significantly in the group without splenomegaly (SV/BSA value < 400) versus that in the group with splenomegaly (P = 0.005). Thus, if both splenomegaly and thrombocytopenia coexist (PLT count ≤ 5 × 104/mm3 and SV/BSA value ≥ 400), persistent thrombocytopenia is predictable after LTx. Liver Transpl 15:208–215, 2009.

Microsurgical hepatic artery reconstruction during living‐donor liver transplantation by using head‐mounted surgical binocular system

We have described our experience with arterial reconstruction during living‐donor liver transplantation by using Varioscope® AF3 – a head‐mounted surgical binocular system with automatic focusing and continuous zoom magnification from 3.6× to 7.2×. From July 1996 to December 2006, 91 grafts were implanted in 89 living‐donor liver transplantation recipients, including two that required retransplantation. For microsurgical reconstruction of the graft hepatic artery, a conventional operating microscope was used in the first 10 transplants and Varioscope, in the subsequent 81. The time required to complete arterial reconstruction while using a conventional operating microscope and Varioscope was 78.6 ± 44.6 min and 35.5 ± 15.5 min, respectively. No arterial complications, including hepatic artery thrombosis, occurred in any of the 89 patients during the observation period. In living‐donor liver transplantation, successful hepatic artery reconstruction can be safely carried out using Varioscope.