Immunohistological and flow cytometric analysis of glycosphingolipid expression in mouse lymphoid tissues

Expression of neutral glycosphingolipids (GSLs) and gangliosides in normal lymphoid tissues and cells has been studied mostly by biochemical and immun ochemical analysis of lipid extracts separated by thin-layer chromatography . CSLs and gangliosides involved in the GM1b biosynthetic pathway were assi gned to T-lymphocytes, whereas B-cell gangliosides and GSLs have been poorl y characterized in former publications. We used specific polyclonal antibod ies in immunohistochemistry and flow cytometry to analyze the distribution of globotriaosylceramide (Gb(3)Cer), globoside (Gb(4)Cer), gangliotriaosylc eramide (Gg(3)Cer), gangliotetraosylceramide (Gg(4)Cer), and gangliosides G M3 and GalNAc-GM1b in the mouse thymus, spleen, and lymph node. immature th ymocytes expressed epitopes recognized by all antibodies, except for anti-G b(4)Cer. Mature thymocytes bound only antibodies to GalNAcGM1b Gg4Cer, and Gb4Cer. In secondary lymphoid organs, antibodies to globe-series GSLs bound to vascular spaces of secondary lymphoid organs, whereas the ganglio-serie s GSL antibodies recognized lymphocyte-containing regions. In a Western blo tting analysis, only GalNAc-GM1b antibody recognized a specific protein ban d in ail three organs. Flow cytometric analysis of spleen and lymph node ce lls revealed that B-cells carried epitopes recognized by all antibodies, wh ereas the T-cell GSL repertoire was mostly oriented to ganglio-series-neutr al CSLs and GM1b-type gangliosides. The results of immunohistochemistry and flow cytometry were not always identical, possibly because of crossreactiv ity to glycoprotein-linked oligosaccharides and/or differences between cell surface carbohydrate profiles of isolated cells and cells in a tissue envi ronment.