Antibodies to unknown antigens other than swine leukocyte antigens on GTKO/β4GalNT2KO pig cells are associated with AHXR after pig-to-rhesus monkey kidney transplantation

Hongtao Jiang¹Haiyun Jiang¹Songzhe he¹Yuxiang Chen¹Jiaxiang Du²Dengke Pan²Tao Li^1*Yi Wang^1*

• ¹The Second Affiliated Hospital of Hainan Medical University, Haikou, China
• ²Chengdu Clonorgan Biotechnology Co., Ltd., Chengdu, China

Background: Although progress in experimental life-supporting pig renal xenotransplantation has been encouraging, acute humoral xenograft rejection (AHXR) is still an obstacle to survival of nonhuman primates who receive genetically-modified pig kidneys. This is possibly associated with expression of xenoantigens in addition to the 2 known xenoantigens (Gal, Sda). We attempted to clarify the effect of elicited antibodies on GTKO/β4GalNT2KO-based pig-to-rhesus renal xenotransplantation. Methods: Rhesus monkeys (n=7) received kidneys from GTKO/β4GalNT2KO (n=1) or GTKO/β4GalNT2KO/hCD55/hTBM (n=3) pigs, and recipient serum was collected. We incubated serum with GTKO/β4GalNT2KO pig red blood cells (pRBCs) to measure remaining antibodies to pig peripheral blood mononuclear cells (pPBMCs). Antibody binding and cytotoxicity of serum (either adsorbed on pig RBCs or un-adsorbed) to GTKO/β4GalNT2KO or GTKO/β4GalNT2KO/hCD55/hTBM pig PBMCs or RBCs were measured by flow cytometry. At biopsy or euthanasia, the grafts were examined by histological assessment. Results: Survival of the 7 recipients was <30 days. Serum creatinine was increased and platelet count was decreased. Anti-pig antibodies (IgG or IgM) were elevated in serum of all 7 recipients at some time-point. Histopathology of the kidneys showed features of AHXR and thrombotic microangiopathy in all grafts. Immunohistochemistry showed C3c, C4d, IgM and/or IgG, and C5b-9 deposition and CD68 infiltration in most grafts. Serum anti-pig antibodies remained elevated even after absorption on pig RBCs, which indicated that another xenoantigen, e.g., swine leukocyte antigens (SLA) or 'neoantigen I' (which also express on pig PBMCs but not express on pig RBCs), may be playing a role in AHXR. Neoantigen I is an unidentified xenoantigen expressed on PBMCs, shared with kidney xenografts but not present on RBCs. There was a difference in antibody binding to PBMCs between unabsorbed and absorbed serum, suggesting the presence of anti - 'neoantigen II' (which both express on pig RBCs and PBMCs) antibodies on PBMCs that may be important in causing AHXR. Conclusions: These data suggest that elicited antibodies to 'neoantigens', e.g., non-SLA antigens, play a role in AHXR after GTKO/β4GalNT2KO-based pig kidney transplantation in nonhuman primates.