Deficiency of N-glycolylneuraminic acid and Galα1-3Galβ1-4GlcNAc epitopes in xenogeneic cells attenuates cytotoxicity of human natural antibodies

Background: A number of carbohydrate structures apart from Gal alpha 1-3Gal beta 1-4GlcNAc (Gal) have been implicated as potential xenoantigens. Epitopes of another carbohydrate structure, namely N-glycolylneuraminic acid (NeuGc), are widely expressed on the surfaces of endothelial cells of all mammals, except humans, and are likely targets of anti-non-Gal antibodies (Abs). The purpose of this study is to assess whether deficiency of NeuGc and Gal epitopes in xenogeneic cells attenuates the cytotoxicity of naturally occurring antibodies in human sera. Methods: We generated mice deficient in both alpha 1,3-galactosyltransferase (GalT) and cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). Flow cytometric and immunohistochemical studies confirmed the complete absence of Gal and NeuGc expression in hematopoietic cells and tissue sections of the heart, lungs, liver, kidney, and pancreas in GalT-/- CMAH-/- double knockout (DKO) mice. The thymocytes obtained from wild-type (WT), GalT-/-, CMAH-/-, and DKO mice were used to capture xenoreactive Abs present in human sera from healthy volunteers of each blood group. The titers and complement-dependent cytotoxicity (CDC) of these Abs were determined by flow cytometry and the 51Cr release assay, respectively. Results: In all sera, IgM and IgG Abs bound to the thymocytes of WT, GalT-/-, CMAH-/-, and DKO mice. The average values of IgM Abs against thymocytes of WT and CMAH-/- mice were similar, but were statistically higher than those against thymocytes of GalT-/- and DKO mice. The average value of IgG Abs against WT mouse thymocytes was similar to that against GalT-/- mouse thymocytes, but was significantly higher than that against thymocytes of CMAH-/- and DKO mice. Remarkable CDC of human sera was observed against the thymocytes of WT and CMAH-/- mice, whereas thymocytes of GalT-/- and DKO mice were more resistant to lysis. CDC of human sera against CMAH-/- mouse thymocytes was significantly lower than that against WT mouse thymocytes. In addition, CDC against DKO mouse thymocytes, which appeared undetectable even at lesser serum dilutions, was significantly lower than that against GalT-/- mouse thymocytes. Conclusions: A DKO mice strain lacking both Gal and NeuGc antigens in several tissues has been generated. Comparing CDC of human sera against mouse thymocytes, the DKO strain shows a statistically significant but a small additional reduction in CDC compared to that already achieved in GalT-/- mice.