Introduction: Wild type (WT) porcine organs are promptly rejected in primate recipients by hyperacute rejection (HAR) that results from uncontrolled activation of the complement and coagulation cascades. Vigorous complement activation is triggered by binding of xenoreactive preformed antibodies in the recipients' blood to xenoantigens on WT pig cells. Three major carbohydrate antigens, αGal, Sda, and Neu5Gc, have been described so far in pigs. Deletion of them in triple-knockout (3KO) porcine donors greatly reduces preformed antibody binding and complement activation, prevents HAR and prolongs graft survival. Therefore, the 3KO background is considered the ideal genetic background for engineering porcine donor organs for clinical use. Despite the reduction in antibody binding and complement fixation, a low but substantial amount of residual antibody and complement deposition remains detectable on 3KO cells. The initial steps that lead to complement fixation by 3KO cells, however, are incompletely understood. In our working hypothesis we rationalize the contribution of both the classical and the lectin pathways driven by preformed antibody binding or by altered, xenogenic glycan structures present on 3KO porcine cells, respectively, that both can fuel alternative pathway activation.

Methods: To dissect activation mechanisms, we measured C4b and C3b deposition on 3KO porcine cells by flow cytometry after incubating them in either human serum preparations depleted for individual complement components or primate (human, baboon, cynomolgus and rhesus macaque) serum treated with pathway specific complement inhibitors.

Results: We found that complement activation was almost completely abolished when 3KO cells were incubated in sera depleted for components of the classical pathway (C1q, C1r, C1s, C2, C4) or in normal primate sera treated with a specific classical pathway inhibitor (anti-C1s), as compared with untreated controls. Interestingly, inhibition of the lectin pathway in primate sera had only a negligible effect on complement activation. Further experiments showed that, unexpectedly, 3KO porcine cells do not permit amplification or direct activation of the alternative pathway in primate serum.

Conclusion: Together, our data suggest that complement activation is predominantly driven by the classical pathway on 3KO porcine cells in the absence of substantial amplification by the alternative pathway. These observations can help refine strategies of complement regulation in preclinical models of xenotransplantation or in clinic trials in the future.