108.4: Deletion of additional iGb3s in GGTA1/B4GalNT2/CMAH knock-out pig reduces lymphocyte infiltration and tissue factor deposition in pig-to-cynomolgus arteriovenous fistula model

Introduction: Solid organ transplantation, such as kidney transplantation, can cause a strong rejection response, and the rejection mechanism is not fully understood. Therefore, studying the rejection mechanism in xenotransplantation using small blood vessels that do not significantly affect survival is necessary. We used three genetically engineered pig models with knocked-out non-protein antigens, including GGTA1 KO (GTKO), GGTA1/B4GalNT2/CMAH TKO, and QKO pigs (TKO with iGb3s knocked out). Our study investigated the effect of iGb3s deletion on the patency and pathological damage of transplanted blood vessels by comparing xenovascular transplantation in QKO pigs with previously studied TKO pigs. Methods: Fifteen cynomolgus macaques received xenotransplantation of pig vessels with a diameter of 2mm or less. The recipients were divided into four groups: WT (N=3), GTKO (N=3), TKO (N=5), and QKO (N=4). Immunosuppression was maintained through four administrations of anti-CD154 Abs. Blood flow was monitored using ultrasound and Doppler devices, and histological examination was performed on one of the two grafts received by each recipient after two weeks. Blood samples were collected from the cynomolgus monkey recipients at regular intervals for immune phenotyping, and ELISA analysis, donor specific antibodies (DSA) and complement-dependent cytotoxicity (CDC) analyses. IHC analysis was conducted on the transplanted vessels obtained through grafectomy at the end of the experiment. Results: The survival of xenovascular grafts in WT pigs was significantly shorter than that of all KO pigs (P<0.0001). WT pigs had an average graft survival of 22.7±4.1 days, while GTKO, TKO and QKO pigs had average survival periods of 90.0±2.0 days, 91.2±0.5 days, and 80.8±10.9 days, respectively. This suggests that the degree of immunosuppression was appropriate. QKO pigs showed similar results in DSA and CDC to TKO pigs, indicating that the iGb3S KO gene in QKO did not affect graft patency. However, QKO had less T and B lymphocyte infiltration and less tissue factor deposition than TKO, as observed in graft IHC. Conclusion: In conclusion, our study using the pig-to-cynomolgus arteriovenous fistula model showed that GGTA1 expression is critical for xenograft patency. Although the deletion of additional iGb3S in TKO pigs reduced lymphocyte infiltration and tissue factor deposition in graft IHC, it did not affect patency. These findings suggest that the iGb3s gene may play a role in immune regulation in xenotransplantation grafts. 2020M3A9D308010711. References: 1. Lu T, Yang B, Wang R and Qin C. Xenotransplantation: Current Status in Preclinical Research. Front. Immunol. 2020; 10: 3060. 2. Yamamoto, T., Iwase, H., Patel, D. et al. Old World Monkeys are less than ideal transplantation models for testing pig organs lacking three carbohydrate antigens (Triple-Knockout). Sci Rep. 2020; 10: 9771. 3. Rhu J, Lee KW, Kim KS, Lee JS, Kim SJ, Park JB. Coagulation biomarkers in healthy male Cynomolgus macaque monkeys (Macaca fascicularis). Xenotransplantation. 2019; 26: e12457. 4. Gao H, Zhang Q, Chen J, et al. Porcine IL-6, IL-1β, and TNF-α regulate the expression of pro-inflammatory-related genes and tissue factor in human umbilical vein endothelial cells. Xenotransplantation. 2018; 25: e12408.