Introduction: Triple knockout (TKO) pigs lacking Gal, Neu5Gc, and Sda antigens have been proposed as ideal donors for clinical xenotransplantation. However, preclinical studies in Old World nonhuman primates (OWNHPs) have revealed that knockout of the CMAH gene, which eliminates Neu5Gc, may induce the expression of novel neoantigens that elicit strong natural antibody responses. This poses a challenge not only for organ transplantation but also for cellular xenografts such as red blood cells (RBCs), islets, or hepatocytes, which may be more vulnerable to rapid antibody-mediated clearance. While the immune response in OWNHPs has been well documented, it remains unclear whether CMAH knockout truly results in neoantigen expression. In this study, we investigated this question from a new angle by evaluating natural antibody responses from CMAH-positive pigs toward CMAH-knockout (CMAHKO) pigs.

Methods: We performed in vitro assays (antibody binding, hemagglutination, and complement-dependent cytotoxicity) using sera and RBCs from CMAH-positive pigs (WT, GTKO, GTKO/β4GalNT2KO) and CMAH-KO pigs (GTKO/CMAHKO, TKO). In vivo, CFSE-labeled RBCs from CMAHKO pigs were transfused into WT or GTKO pigs. RBC survival and post-transfusion immune responses were monitored. Controls included WT-to-WT and GTKO-to-WT transfusions.

Results: Sera from WT, GTKO, and GTKO/β4GalNT2KO pigs showed strong IgM and IgG binding to GTKO/CMAHKO and TKO RBCs. Complement-dependent cytotoxicity was significantly elevated. In vivo, transfused CMAHKO RBCs were rapidly cleared (within 2–10 days), unlike WT or GTKO RBCs which persisted beyond 28 days. CMAHKO RBC transfusion induced notable increases in antibody titers and cytotoxic responses.

Conclusion: This study provides the first direct evidence that CMAH gene knockout in pigs leads to the emergence of neoantigens that are recognized by natural antibodies in CMAH-positive pigs. Although it remains to be determined whether these neoantigens correspond to those recognized by OWNHPs, the results suggest a critical vulnerability of CMAHKO cellular grafts to antibody-mediated rejection. Compared to solid organ xenografts, cellular xenografts may face greater risks due to their smaller size and lack of protective architecture. These findings emphasize the need for robust immunomodulatory strategies when using TKO pigs for preclinical cellular xenotransplantation models in OWNHPs.