Claudin-2 enhances human antibody-mediated complement-dependent cytotoxicity of porcine endothelial cells by modulating antibody binding and complement activation

Weilong Li ¹ Fang Yang ¹ Dexing Yang ¹ Zhuoheng Song ¹ Zigan Xu ¹ Jinmei Wu ¹ Yanmei Li ¹ Zixi Chen ¹ Peishan Chen ¹ Yeye Yu ² Ting Xie ² Cuishan Yang ¹ Liying Zhou ¹ Shaodong Luan ¹ Hanchao Gao ^1*

• ¹ Shenzhen Longhua District Central Hospital, Shenzhen, China
• ² First Affiliated Hospital of Jinan University, Guangzhou, Guangdong Province, China

Immune rejection represents a significant barrier to transplantation, especially in the context of xenotransplantation. Endothelial cells (ECs) derived from pigs serve as the initial barrier against the human immune system in xenotransplantation. Tight junction proteins are essential components of endothelial cell (EC) tight junctions; however, their role in xenotransplantation has been less thoroughly investigated. Here, we evaluated the role of Claudin-2 through an in vitro model of human antibody-mediated complement-dependent cytotoxicity (CDC). The loss of Claudin-2 protected porcine aortic endothelial cells (PAECs) and porcine iliac endothelial cells (PIECs) from human antibody-mediated complement-dependent cytotoxicity (CDC), while the overexpression of Claudin-2 enhanced the cytotoxicity in PAECs and PIECs within the same model. Unexpectedly, the loss or overexpression of Claudin-2 did not influence the mRNA expression levels of complement regulators CD46, CD55, CD59, Factor H, and Factor I. Importantly, the loss of Claudin-2 significantly decreased the deposition of the C5b-9 complex, commonly referred to as the membrane attack complex (MAC), whereas the overexpression of Claudin-2 enhanced the deposition of the C5b-9 complex, indicating that Claudin-2 facilitates complement activation. Furthermore, the loss of Claudin-2 resulted in a decrease in the deposition of C3c and C9 on PIECs. Moreover, Claudin-2 enhanced human antibody binding to porcine ECs, as evidenced by increased IgG and IgM binding. In conclusion, our findings indicate that Claudin-2 enhances the cytotoxicity of porcine ECs through modulating antibody binding and complement activation. The deficient of Claudin-2 in genetically modified pigs is likely to protect porcine ECs and enhance xenograft survival in pig-to-human organ or tissue xenotransplantation.