Structure elucidation of marker glycolipids of alloantigen-activated murine T lymphocytes.

DBA/2 mouse spleen cells were stimulated in vitro by (a) alloantigen (mitomycin-treated CBA/J splenocytes), (b) the T cell mitogen concanavalin A (Con A), and (c) the B cell mitogen lipopolysaccharide (LPS). The cultures were pulsed for 10 h with (14)C-labeled galactose and glucosamine. Radiolabeled glycosphingolipids (GSL's) were extracted from the cells and the neutral GSL's isolated and analyzed by high-performance thin-layer chromatography. Two of the radioactive neutral GSL's, 9 and 12a, were found to be prominent in the alloantigen-stimulated cells but not in T cells stimulated by Con A. GSL 9 was also present as a minor component in LPS-stimulated B lymphocytes. GSL's 9 and 12a were purified by preparative column chromatography on Iatrobeads. The sequence and anomeric linkages of the carbohydrate moiety of these glycolipids were determined by successive degradation with exoglycosidases. The structures were shown to be Gal(alpha1-x)Gal(beta1-x)GlcCer (glycolipid 9) and GalNAc(beta1-x)Gal(alpha1-x)Gal(beta1-x)GlcCer (glycolipid 12a), respectively. The latter glycolipid may serve as a marker for alloantigen-activated T cell subpopulations.