Glycolipids with Thy-1 activity have been isolated from mouse brain and thymocytes. An immune response assay has been found to be effective in the identification of these glycolipids. The addition of brain or thymocyte glycolipids from AKR (Thy-1.1) or C3H (Thy-1.2) mice to spleen cells resulted in the generation of antibody-forming cells specific for Thy-1.1 or Thy-1.2, respectively. This response was investigated with a modified plaqueforming cell (PFC) assay by using thymocyte target cells in agarose. Incubation of C3H brain glycolipid or Thy-1.2 brain glycoprotein with AKR spleen cells resulted in an anti-Thy-1.2 PFC response when assayed for PFC in a lawn of C3H thymocytes. Controls in which equivalent amounts of AKR glycolipid were added to these cultures resulted in no PFC response. On the other hand when C3H or AKR glycolipid or Thy-1.2 glycoprotein was incubated with C3H spleen cells, the AKR glycolipid induced an anti-Thy-1.1 PFC response in a lawn of AKR thymocytes, but C3H glycolipid and Thy-1.2 glycoprotein elicited no PFC response. These experiments demonstrated the Thy-1 specificity of the glycolipids. Absorption of the antigens with either anti-Thy-1.1 or anti-Thy-1.2 sera before addition to the cultures also demonstrated Thy-1 antigen specificity.

Development of the immune response assay has permitted further purification of Thy-1 active glycolipids and has led to the following conclusions. Brain GMl ganglioside purified by column and thin layer chromatography from AKR or C3H mice contained Thy-1.1 or Thy-1.2 active glycolipids, respectively. The Thy-1.1 or Thy-1.2 active glycolipids could be separated from GMl by a third thin layer chromatography system. The Thy-1.1 and Thy-1.2 glycolipids were estimated to be only a small percentage of the total AKR or C3H brain GMl preparations. Thy-1 active glycolipids isolated from thymocytes had similar thin layer mobility and exhibited Thy-1 specificity in the system described. We suggest that Thy-1 antigenicity lies in carbohydrate structures that are conjugated to either lipid or protein carriers because we observed Thy-1 specificity associated with both glycolipids and glycoprotein.

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