Background: Pig red blood cells (RBCs) are rapidly eliminated when transfused into nonhuman primates (NHPs) because of immune reactions involving antibody binding and complement activation. We assessed the relationship between post-transfusion hemolysis and complement activation. Methods: RBCs for transfusion were prepared from wild-type (WT) and genetically modified pigs and NHPs. After the withdrawal of 25% of the blood volume, NHPs received transfusions of WT (N = 4), triple knockout (TKO, N = 8), and TKO pig RBCs expressing human CD55 and CD39 (TKO/hCD55.hCD39, N = 4). Additional groups received repeated xenotransfusions (ReXTf, N = 3), NHP RBC transfusions (N = 3), or a saline infusion (N = 4).Blood samples were collected at multiple time points to measure Hb and complement fragment (C3a, C4a, and factor Bb) levels and agglutination titers. Results: Hb levels were restored by transfusions but not by saline infusion. The degree of complement activation varied with the type of transfused RBCs, with significant increases in C3a and factor Bb levels immediately after xenotransfusions but not allotransfusions.These increases were particularly notable in ReXTf and negatively correlated with Hb levels on post-transfusion day 1 (ρ = –0.547 and –0.556; P = 0.0187 and 0.0165, respectively).In TKO/hCD55.hCD39 pig RBC transfusions, C3a and factor Bb peak levels were delayed until post-transfusion day 3, unlike in TKO pig RBC transfusions. Conclusions Post-transfusion complement activation varies depending on prior sensitization and genetic modifications in pig RBCs. Monitoring complement activation can provide insight into the survival and compatibility of transfused RBCs in NHPs.

BACKGROUND: Anti-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). METHODS: Intraportal 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. RESULTS: The 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). CONCLUSIONS: IS 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.
Animals ; Antibodies/blood/immunology ; Antigens, CD40/immunology ; Area Under Curve ; CD40 Ligand/immunology ; Disaccharides/*immunology ; Epidermal Growth Factor/blood ; Graft Rejection/*immunology ; Immunoglobulin G/blood ; Immunoglobulin M/*blood ; Immunosuppressive Agents/therapeutic use ; *Islets of Langerhans Transplantation ; Macaca mulatta ; ROC Curve ; Swine ; Transplantation, Heterologous