*Purpose: We have achieved >6 months survival of a life-supporting kidney with a vascularized thymus (VT) in a pig-to-baboon model. While improved survival has been achieved with GalT KO pig xenografts in primates, non-Gal natural antibody determinants, including β1,4 N-acetylgalactosaminyl transferase (B4) and N-glycolylneuraminic acid (NeuGc) are recognized by human sera on GalT KO pig cells. In this study, we compared preformed anti-pig natural antibodies (nAb) against GalT KO alone (GalTKO) and triple knockout (TKO) pigs that were GalT, B4, and NeuGc KO, in baboons to test (1) if TKO induced new antigens that are recognized by baboon preformed nAb, and then assessed (2) if these new antigens were immunogenic in vivo.

*Methods: Preformed nAb against GalTKO or TKO PBMC in 10 naïve baboon sera were assessed by FCM Ab binding assays using anti-human IgM or IgG ab. Among these baboons, five received pig kidneys and VT grafts from porcine CMV negative TKO pigs. All baboons received anti-CD40 mab, ATG and Rituximab. Graft renal function (sCre and histology), thymic emigrants from pig thymic grafts (chimerism) as well as immunologic assays (anti-pig ab and cellular assays) were performed.

*Results: Among 10 naïve baboon sera, two had less Ab binding against TKO than GalTKO cells. Four baboon sera had similar binding to TKO and GalTKO cells. Notably, 4 baboon sera had higher ab binding to TKO than GalTKO cells, suggesting that new antigens were revealed in association with the additional KO. Although all baboons (n=5) had stable renal function in the first 11 days (Cre <1.5mg/dl), two baboons with higher non-Gal preformed IgG against TKO than GalTKO (high preformed nAb against TKO) rejected their kidney plus VT grafts at POD 20. In contrast, three baboons, including one that had similarly high IgG binding against both GalTKO and TKO, maintained renal function >86, 43* and >30 days with Cre <1.0mg/dl (> ongoing. * died from anesthetic complication). Chimerism (thymic emigrants from VT grafts) after the first 7 days was markedly lower in baboons with high preformed nAb against TKO than in those without. Immunohistology showed predominant IgG binding in the excised rejected kidneys.

*Conclusions: These data indicate the TKO may induce new antigenic specificities that led to delayed vascular xenograft rejection in a pig-to-baboon kidney plus VT model. Prescreening for preformed IgM and IgG nAb using both GalTKO and TKO PBMC is essential to avoid early loss of pig xenografts.

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