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Human xenogeneic immune responses to HD antigen in xenotransplantation

Sunghoon Hurh¹, Inho Choi², Bumrae Cho^1,3, Eun Mi Lee¹, Hwa Jung Kim¹, Hye Jung Yeom¹, Juho Hong¹, Kyu Hyun Han¹, Eun Won Lee¹, Jong-Ik Hwang⁴, Jae Young Kim⁵, Jaeseok Yang^1,6, Curie Ahn^1,6,7

¹Transplantation Research Institute, Seoul National University Hospital, Seoul; ³Department of Theriogenology and Biotechnology, Seoul National University College of Veterinary Medicine, Seoul; ⁴Graduate School of Medicine, Korea University, Seoul; ⁵Department of Biological Science, Gachon University of Medicine and Science, Incheon; ⁶Transplantation Center, Seoul National University Hospital, Seoul; ⁷Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea; ²Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, United States

Background: The development of α-1,3-galactosyl transferase (α-1,3-GalT) gene knock-out pigs prolonged the survival of the porcine heart and kidney xenograft. However, acute vascular rejection still occurred due to induced antibodies to non-Gal antigens in α-1,3-GalT^-/- pig-to-baboon kidney xenotransplantation. N-glycolylneuraminic acid (Neu5Gc) epitope, so-called Hanganutziu-Deicher (HD) antigen, is one of non-Gal antigen, which expressed in all mammals except human. Recently, Neu5Gc-expressing islets transplanted from CMAH^+/+ mice into CMAH^-/- mice were reported to be rejected. However, it is still controversial whether HD antigen will be important in pig-to-human clinical xenotransplantation.

Methods & Results: We had cloned pig CMAH gene into a mammalian expression vector, pCMV-Tag 3, and introduced into HEK293 cells. After the establishment of Neu5Gc-expressing stable cell line (HEK293-pCMAH), we measured the binding of human peripheral blood IgM and IgG, and checked antibody-mediated complement-dependent cytotoxicity against Neu5Gc with human serum. Natural antibodies (both IgM and IgG) were bound to Neu5Gc and lead to complement-dependent cytotoxicity. However, the levels of antibody binding and the dominant antibody types were variable among individuals. We also verified human innate immune response to Neu5Gc using THP-1 human monocytoid cells or CD14 ⁺ peripheral blood monocytes. The activation of THP-1 cells in the presence of Neu5Gc peptides were checked by RT-PCR. The multiplex cytokine analysis showed that pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6 were significantly increased in the culture supernatant of CD14⁺ peripheral blood monocytes co-cultured with HEK293-pCMAH. The knockdown study of pig CMAH using shCMAH (pig CMAH-targeted shRNA) expression vector shows that diminished expression of pig CMAH significantly decreased the binding of human natural antibodies and complement-dependent cytotoxicity in pig aortic endothelial cell line or α-1,3-GalT^-/- pig aortic endothelial cell line.

Conclusion: In summary, Neu5Gc in human cells (HEK293-pCMAH) induced human natural antibody binding, complement-dependent cytotoxictity, and activation of human monocytes.On the other hand, knockdown of the pig CMAH attenuated the Neu5Gc-induced human immune responses. Therefore, the pig Neu5Gc is one of potential targets to be overcome for xenotransplantation.