The alternative complement pathway contributes to the development of IBMIR and the early loss of grafts in NHPs with porcine ITx. Complement inhibition during the peritransplant period may be beneficial for the survival of islet grafts.

BackgroundAnti-Gal is a major antibody induced in non-human primates (NHPs) after xenotransplantation. To understand the mechanism of graft rejection, we investigated the association between anti-Gal responses and graft failure in NHP recipients of porcine islet transplantation (PITx).MethodsIntraportal PITx was performed in 35 diabetic NHPs, and graft function was monitored. Early graft failure (EGF) was defined as loss of graft function within a month after PITx. Seven, 19, nine NHPs received immunosuppression (IS) without CD40 pathway blockade (Group I), with anti-CD154 (Group II), and with anti-CD40 (Group III), respectively. The anti-Gal levels on day 0 and day 7 of PITx were measured by ELISA.ResultsThe frequency of EGF was significantly lower in Group II (26.3%) than in Group I (100%, P=0.0012) and Group III (77.8%, P=0.0166). While levels of anti-Gal IgG in Group I and anti-Gal IgM in Group III increased on day 7 compared with day 0 (P=0.0156 and 0.0273), there was no increase in either on day 7 in Group II. The ratio of anti-Gal IgM or IgG level on day 7 to that on day 0 (Ratio7/0) was significantly higher in recipients with EGF than without EGF (P=0.0009 and 0.0027). ROC curve analysis of anti-Gal IgM Ratio7/0 revealed an area under the curve of 0.789 (P=0.0003).ConclusionsIS with anti-CD154 suppressed anti-Gal responses and prevented EGF in PITx. Anti-Gal IgM Ratio7/0, being associated with EGF, is a predictive marker for EGF.

In NHP recipients of PITX, anti-PA and anti-BA IgG antibodies are elicited by porcine serum included as a supplement in porcine islet preparation. IS including CD40-CD154 blockade and sirolimus suppresses these antibody responses in naïve recipients, but not in sensitized recipients. The elicitation of anti-xenogenic albumin antibodies, a humoral response to a model protein antigen, is distinct from that of anti-Gal antibodies, a response to carbohydrate antigen.

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Human TNFα level increases in human blood after contact with PECs, which is attributed to xenoreactive antibody binding and subsequent complement activation. Human TNFα induces procoagulant changes in PECs with increased TF expression. This study suggests that human TNFα may be one of the mediators linking complement activation with procoagulant changes in the xenoendothelium.

Intravascular coagulation, in association with immune responses against xenografts, may partly contribute to loss of islet grafts in NHPs after PITx.

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BackgroundTriple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared between alpha-1,3-galactosyltransferase gene-knockout (GTKO) and TKO porcine cells.MethodsPrimary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) from GTKO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB++) or Mg++-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containing GTKO or TKO pECs was analyzed.ResultsThe C3 deposition level in GTKO pPBMCs or pECs in GVB++ was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg++-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg++ -EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar between GTKO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB++ increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than in GTKO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence of GTKO pECs.ConclusionTKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation.