Background: alpha1,3-galactosyltranferase knockout (GalT-KO) pigs have been established to avoid hyperacute rejection in GalT-KO pig-to-human xenotransplantation. GalT-KO pig heart and kidney glycolipids were studied focusing on elimination of Gal-antigens and whether novel antigens would appear. Non-human primates are used as pre-clinical transplantation experimental models. Therefore, sera from baboons transplanted with GalT-KO hearts were compared with human serum regarding reactivity with pig glycolipids.
Methods: Neutral and acidic glycolipids were isolated from GalT-KO and WT pig hearts and kidneys. Glycolipid immune reactivity was tested on TLC plates using human affinity-purified anti-Gal Ig, anti-blood group monoclonal antibodies, lectins, and human serum as well as baboon serum collected before and after GalT-KO pig heart transplantations. Selected glycolipid fractions, isolated by HPLC, were structurally characterized by mass spectrometry and proton NMR spectroscopy.
Results: GalT-KO heart and kidney lacked alpha3Gal-terminated glycolipids completely. Levels of uncapped N-acetyllactosamine precursor compounds, blood group H type 2 core chain compounds, the P1 antigen and the x(2) antigen were increased. Human serum antibodies reacted with Gal-antigens and N-glycolylneuraminic acid (NeuGc) in WT organs of which only the NeuGc reactivity remained in the GalT-KO tissues. A clear difference in reactivity between baboon and human antibodies with pig glycolipids was found. This was most pronounced for acidic, not yet identified, compounds in GalT-KO organs which were less abundant or lacking in the corresponding WT tissues.
Conclusions: GalT-KO pig heart and kidney completely lacked Gal glycolipid antigens whilst glycolipids synthesized by competing pathways were increased. Baboon and human serum antibodies showed a different reactivity pattern to pig glycolipid antigens indicating that non-human primates have limitations as a human pre-clinical model for immune rejection studies.