Lack of iGb3 and Isoglobo-Series Glycosphingolipids in Pig Organs Used for Xenotransplantation: Implications for Natural Killer T-Cell Biology

Abstract

α-1,3-Terminated galactose residues on glycoproteins and glycosphingolipids are recognized by natural anti-α-1,3-galactose antibodies in human serum and cause hyperacute rejection in pig-to-human xenotransplantation. Genetic depletion of α-1,3-galactosyl- transferase-1 in pigs abolishes the hyperacute rejection reaction. However, the isoglo- botriosylceramide (iGb3) synthase in pigs may produce additional α-1,3-terminated galactose residues on glycosphingolipids. In both α-1,3-galactosyltranserase-1 knockout mice and pigs, cytotoxic anti-α-1,3-galactose antibodies could be induced; thus, a paradox exists that anti-α-1,3-galactose antibodies are present in animals with functional iGb3 synthases. Furthermore, iGb3 has been found to be an endogenous antigen for natural killer T (NKT) cells, an innate type of lymphocyte that may initiate the adaptive immune responses. It has been reasoned that iGb3 may trigger the activation of NKT cells and cause the rejection of α-1,3-galactosyltransferase-1-deficient organs through the potent stimulatory effects of NKT cells on adaptive immune cells (see ref.^[20]). In this study, we examined the expression of iGb3 and the isoglobo-series glycosphingolipids in pig organs, including the heart, liver, pancreas, and kidney, by ion-trap mass spectrometry, which has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 5 μg/mL of the total iGb3/Gb3 mixture is present (see ref.^[35]). We did not detect iGb3 or other isoglobo-series glycosphingolipids in any of these organs, although they were readily detected in mouse and human thymus and dendritic cells. The lack of iGb3 and isoglobo-series glycosphingolipids in pig organs indicates that iGb3 is unlikely to be a relevant immune epitope in xenotransplantation.

Keywords:

• Natural killer T cells
• Isoglobotriaosylceramide
• CD1d
• α1,3-Galactosyltransferase-1
• iGb3 synthase

ACKNOWLEDGMENTS

DZ is supported by MD Anderson Cancer Center and NIH grant AI079232. MD Anderson Cancer Center is supported in part by NIH grant CA16672. ICA is partly supported by NIH grants 2G12RR008124-16A1, 8G12MD007592, from the National Institutes on Minority Health and Health Disparities (NIMHD), and Biomolecule Core Facility at BBRC/UTEP. We thank the Consortium of Functional Glycomics (CFG), National Institute of General Medical Sciences, for providing glycan standards and other CFG investigators for sharing unpublished data, Paul Savage, Albert Bendelac, Luc Teyton, Sarah J Bronson and Kathryn Carnes for helpful discussion.

Notes

We previously developed a method to detect iGb3 among iGb3/Gb3 mixtures, which has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 5 μg/mL of total iGb3/Gb3 mixture is present.^{[ 35 ]} We typically analyze the permethylated GSL samples at a concentration of at least 250 μg/mL; thus, the concentration of Hex-Hex-Hex trisaccharide-ceramide GSLs per analysis is above 5 μg/mL.

By both MS and anti-Galα3Gal antibody staining, Diswall et al. found that lacto-series GSLs, but not isoglobo-series GSLs, carry the Gal α3Gal epitope in pig organs. Galα3Gal epitope-bearing GSLs such as Galα3nLc4 and Galα3nLc6 are present in wild-type pigs but completely absent in α3GalT1 knockout pigs.^{[ 36 – 39 ]}