Xenotransfusion of Triple-Knockout Porcine Red Blood Cells: Effects of hCD55/hCD39 Expression and Anti-CD154 Therapy in Non-Human Primates 2234209

Abstract Introduction Global aging and emerging infectious diseases threaten the stability of blood supplies, underscoring xenotransfusion as a potential alternative. Triple-gene knockout (TKO) pig red blood cells (pRBCs), which lack α-Gal, Neu5Gc, and SDa, can avoid acute hemolysis but exhibit shorter survival than allogeneic RBCs. We investigated the roles of complement activation and antibody induction in the clearance of pRBCs in non-human primates (NHPs). Methods Blood from O-type TKO pigs and TKO pigs expressing human CD55 and CD39 (TKO/hCD55.hCD39) was used for pRBC preparation. After 25% blood volume withdrawal, NHPs received one of the following: TKO pRBCs alone (TKO_XTf, N = 8), TKO pRBCs with PG-405 [anti-CD154(Fab)-NTIG-IL-10; 30 mg/kg IV] followed by repeat administration of PG-405 3 days later (TKO_XTf_aCD154, N = 4), or TKO/hCD55.hCD39 pRBCs (TKO/hCD55.hCD39_XTf, N = 4). Hematologic, biochemical, cytokine, complement, and antibody responses were evaluated at multiple time points. Results No acute hemolytic reactions or other severe adverse events were observed in any group, and transfusion promptly restored vital signs compromised by blood loss. In the TKO/hCD55.hCD39_XTf group, the post-transfusion rise in plasma factor Bb was delayed compared with other groups. In the TKO_XTf_aCD154 group, the rapid increase in anti-pRBC antibodies post-transfusion was markedly suppressed. Although both complement activation and antibody induction were effectively inhibited, the hematologic benefits of transfusion persisted for more than 24 hours but were lost within 72 hours across all groups, providing no significant survival advantage over TKO_XTf controls. Conclusion Complement and antibody inhibition alone are insufficient to prevent clearance of pRBCs in xenotransfusion, indicating the need for additional strategies. Further studies are required to elucidate the molecular pathways mediating interactions between splenic macrophages and pRBCs, which likely play a critical role in clearance. (Grant No. 22-CM-EC-18) Funding Source the Defense Acquisition Program Administration and the Ministry of Trade, Industry, and Energy of the Korean Government (grant no. 22-CM-EC-18) Topic Categories Translational and Interventional Immunology (TI)