Evaluation of Complement‐Dependent Cytotoxicity Assays for Gene‐Edited Pig‐to‐Human Xenotransplantation

ABSTRACT Background Gene‐edited pigs for xenotransplantation usually contain one or more transgenes encoding human complement regulatory proteins (CRPs). Because of species differences, human CRP(s) expressed in gene‐edited pigs may have difficulty inhibiting the activation of exogenous rabbit complement added to a complement‐dependent cytotoxicity (CDC) assay. The use of human complement instead of rabbit complement in CDC experiments may more accurately reflect the actual regulatory activity of human CRP(s). Methods Peripheral blood mononuclear cells (PBMCs) were obtained from one GTKO pig and two GTKO/hCD55 pigs with a high or low level of hCD55 expression. After incubation of heat‐inactivated normal human sera (HINHS) with porcine PBMCs, CDC levels were measured after the addition of commercial rabbit complement or human complement. In addition, a modified one‐step CDC method was established using pooled normal human sera (NHS) without the addition of exogenous complement. Results There was no significant difference in the binding of IgM/IgG to PBMCs from the three pigs. Both rabbit and human complement mediated a significant cytotoxic effect on GTKO pig PBMCs (98.97% vs. 82.73%). Even the high expression of hCD55 only had a very limited inhibitory effect on rabbit complement‐mediated cytotoxicity (81.70% vs. 98.97%). However, regardless of whether the expression level was high or low, hCD55 had a very remarkable inhibitory effect on human complement‐mediated cytotoxicity (2.94% and 23.83% vs. 82.73%; p < 0.01). Similar results were obtained using the modified one‐step CDC method. In addition, the inhibitory effect of hCD55 on C3c and C5b‐9 deposition on pig PBMCs was positively correlated with the expression level of hCD55. Conclusion The use of human complement instead of rabbit complement in CDC assays can better reflect the actual cytotoxic effect of human xenoantibodies against pig PBMCs expressing human CRP(s), and thus may have potential application to gene‐edited pig‐to‐human xenotransplantation.