Flow cytometry complement-mediated cytotoxicity assay detects baboon xenoantibodies directed to porcine epitopes undetected by hemolytic assay

Abstract

The pig-to-primate model is increasingly being utilized as the final preclinical means of assessing therapeutic strategies aimed at allowing discordant xenotransplantation. To obtain information about the nature of cytotoxic response in pig-to-baboon xenotransplants, we sought to determine if serum cytotoxicity in this model was assay dependent. Sera from nine kidney or heart xenotransplanted baboons were obtained before transplantation and at the time of acute humoral xenograft rejection (AHXR). Cytotoxicity was measured by an anti-pig haemolytic assay (APHA) and by a flow cytometry complement-dependent assay (FCCA), using pig blood lymphocytes (PBLs). Serum samples showing inter-assay differences were absorbed with pig erythrocytes and assayed by APHA and FCCA, as well as by measuring anti-alphaGal and total anti-pig xenoantibodies. The results showed that in four AHXR samples, FCCA cytotoxicity was higher than APHA cytotoxicity. Absorption with pig erythrocytes diminished FCCA and removed APHA cytotoxicity. Residual FCCA activity was due to total anti-pig and IgM anti-alphaGal and non-Gal antibodies. Our results indicate that some cytotoxic antibodies present in the sera of xenotransplanted baboons at time of AHXR are IgM antibodies directed against pig PBL antigens not detected by APHA.