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Prophylaxis of antibody-mediated rejection (AMR) caused by donor-specific antibodies remains challenging. Given the critical roles of complement activity in antibody-mediated graft injury, we developed a lipid nanoparticle (LNP) formulation of small-interfering RNA against complement C5 (C5 siRNA-LNP) and investigated whether C5 siRNA-LNP could downregulate the complement activity and act as an effective treatment for AMR.Lewis recipient rats were sensitized by skin grafting from Brown Norway donor rats. Kidney transplantation was performed at 4 wk post-skin grafting.C5 siRNA- or control siRNA-LNP was administered intravenously, and the weekly injections were continued until the study's conclusion. Cyclosporin (CsA) and/or deoxyspergualin (DSG) were used as adjunctive immunosuppressants. Complement activity was evaluated using hemolysis assays. The deposition of C5b9 in the grafts was evaluated using immunohistochemical analysis on day 7 posttransplantation.C5 siRNA-LNP completely suppressed C5 expression and complement activity (hemolytic activity ≤ 20%) 7 d postadministration. C5 siRNA-LNP in combination with CsA and DSG (median survival time: 56.0 d) prolonged graft survival compared with control siRNA-LNP in combination with CsA and DSG (median survival time: 21.0 d; P = 0.0012; log-rank test). Immunohistochemical analysis of the grafts revealed that downregulation of C5 expression was associated with a reduction in C5b9-positive area ( P = 0.0141, Steel-Dwass test).C5 siRNA-LNP combined with immunosuppressants CsA and DSG downregulated C5 activity and significantly prolonged graft survival compared with control siRNA-LNP with CsA and DSG. Downregulation of C5 expression using C5 siRNA-LNP may be an effective therapeutic approach for AMR.

Background Both dendritic cells (DC) and T-regulatory cells (Treg) have been implicated in regulation of alloimmune responses and transplant tolerance. Methods We analyzed B7 coregulatory molecule expression on circulating DC subset precursors, together with CD4+CD25(hi) Foxp3+ Treg by rare event, flow cytometric analysis in operationally tolerant pediatric liver transplant recipients (TOL), those undergoing prospective immunosuppressive drug weaning (PW) or maintenance immunosuppression (MI), and normal healthy individuals (controls). Results Use of DC subset-specific monoclonal antibodies confirmed elevated precursor plasmacytoid DC/myeloid DC ratios in TOL and PW compared with MI. In addition, Treg frequencies were higher in TOL than in PW and MI, but not controls. While there was no difference in levels of costimulatory and coinhibitory molecules on precursor myeloid DC between the groups, the programmed death ligand-1 (PD-L1=B7-H1):CD86 (B7-2) ratio on precursor plasmacytoid DC was significantly higher in TOL than MI and correlated with the Treg frequency. There was no relation between prednisone or tacrolimus dose or tacrolimus trough level and either the PD-L1/CD86 ratio on plasmacytoid DC or the Treg frequency. Moreover, clinically relevant concentrations of dexamethasone or tacrolimus did not affect these values in short-term culture. Conclusion These novel findings suggest a possible functional relationship between the enhanced incidence of precursor plasmacytoid DC, their comparatively high relative expression of the coinhibitory molecule PD-L1, and the elevated frequency of Treg in operational liver transplant tolerance.

Background. Immature dendritic cells (DC) can promote long-term transplant survival in rodents. We assessed the impact of stably immature, donor-derived DC on alloimmune reactivity in rhesus macaques. Methods. CD14 + monocytes isolated from leukapheresis products of Macacca mulatta were cultured in granulocyte-macrophage colony stimulating factor plus interleukin (IL)-4 ± vitamin (vit) D3, and IL-10. Major histocompatibility complex class II and cosignaling molecule expression was determined on CD11c + cells by flow cytometry. T-cell allostimulatory capacity of the DC, including DC exposed to proinflammatory cytokines, was determined in mixed leukocyte reaction. To test their influence in vivo, purified DC were infused intravenously into allogeneic recipients, either alone or followed by CTLA4Ig, 24 hr later. Proliferative responses of recipient CFSE-labeled T cells to donor or third party DC, cytokine production by stimulated T cells, and circulating alloantibody levels were determined by flow cytometry, up to 100 days postinfusion. Results. VitD3/IL-10-conditioned, monocyte-derived DC were resistant to maturation and failed to induce allogeneic T cell proliferation in vitro. After their infusion, an increase in anti-donor and anti-third party T-cell reactivity was observed, that subsequently subsided to fall significantly below pretreatment levels (by day 56) only in animals also given CTLA4Ig. No increase in circulating immunoglobulin (Ig) M or IgG anti-donor alloantibody titers compared with pretreatment values was detected. With DC+CTLA4Ig infusion, alloreactive IL-10-producing T cells were prevalent in the circulation after day 28. Conclusions. Maturation-resistant rhesus DC infusion is well-tolerated. DC + CTLA4Ig infusion modulates allogeneic T-cell responses and results in hyporesponsiveness to donor and third party alloantigens.

Background. This study was performed to determine the usefulness of intraoperative near-infrared spectroscopy (NIRS) for evaluating the extent of congestion in the anterior segment of the graft after living-donor liver transplantation using right lobe grafts that do not have the middle hepatic vein.

An understanding of the mechanisms that suppress the human anti-pig cellular response is key for xenotransplantation. We have compared the ability of human regulatory T cells (Tregs) to suppress xenogeneic and allogeneic responses in vitro.Human peripheral blood mononuclear cells (PBMC), CD4+ T cells, or CD4+ CD25- T cells were stimulated with irradiated human or wild type (WT) or alpha1,3-galactosyltransferase gene-knockout (GT-KO) pig PBMC in the presence or absence of human CD4+ CD25 high Tregs. In separate experiments, 5- (and 6)-carboxyfluorescein diacetate succinimidyl ester-labeled human CD4+ T cells were stimulated with human or pig PBMC. The expansion and precursor frequencies of allo- and xenoreactive Tregs were assessed by labeling with FoxP3 mAb and flow cytometric analysis.The responses of human PBMC, CD4+ T cells, and CD4+ CD25- T cells to pig PBMC were stronger than to human PBMC (P0.05). Human anti-GT-KO responses were weaker than anti-WT responses (P0.05). Human CD4+ CD25 high Tregs suppressed proliferation of CD4+ CD25- T cells to both human and pig PBMC stimulator cells with the same efficiency. Alloreactive CD4+ CD25+ FoxP3 high responder T cells proliferated more than their xenoreactive counterparts (P0.05), although xenoreactive CD4+ CD25+ T cells proliferated more than alloreactive cells (P0.05). There was no difference in precursor frequency between allo- and xeno-reactive CD4+ CD25+ FoxP3 high cells.Human T-cell responses to pig cells are stronger than to allogeneic cells. The human response to GT-KO PBMC is weaker than to WT PBMC. Although human Tregs can suppress both responses, expansion of CD4+ CD25+ FoxP3 high cells against pig PBMC is weaker than against human PBMC. More human Tregs may be required to suppress the stronger xenogeneic response.

This study was performed to determine the usefulness of intraoperative near-infrared spectroscopy (NIRS) for evaluating the extent of congestion in the anterior segment of the graft after living-donor liver transplantation using right lobe grafts that do not have the middle hepatic vein.Fifteen patients undergoing living-donor liver transplantation using a right lobe graft without the middle hepatic vein were enrolled in this study. During the course of harvesting and implantation, in vivo NIRS was performed on the liver grafts to determine hemoglobin (Hb) and cytochrome oxidase content in the hepatic tissues.The 15 cases were divided into three groups according to the caliber of the middle hepatic vein tributaries in the right lobe grafts: the small group (4 mm), the intermediate group (4-7 mm), and the large group (7 mm). After implantation, congestion (increase in tissue Hb) in the anterior segment was more severe than that in the posterior segment in the intermediate and large groups. However, well-preserved mitochondrial cytochrome oxidase redox state was observed in both segments except for two cases in the large group with severe congestion in the anterior segment. The extent of postoperative congestion in the anterior segment was significantly correlated with the tissue content of remaining Hb in that segment after ex vivo flushing.Intraoperative NIRS enables quantification of the extent of congestion in the anterior segment after implantation of a right lobe liver graft and even enables prediction of such congestion at the phase of ex vivo perfusion.