Glycolipid-bound sialic acid levels were elevated 2 to 4-fold in the sera of two strains of mice bearing thymic lymphoma produced either spontaneously (AKR/J) or due to chemical carcinogenesis [Swiss mice injected with 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboximide]. The serum glycolipid-bound sialic acid level reflected the tumor burden of the AKR/J mice during the early stages of leukemogenesis. Furthermore, the elevation was found to coincide with the ontogenesis of thymic lymphoma and not to be simply an age-dependent phenomenon. TLC analysis of Florisil column-purified gangliosides from the sera of AKR/J and Swiss mice suggested presence of gangliosides with mobilities very close to GM2 and GM3 standards respectively. On the premise that the elevated glycolipid levels in circulation might interfere with normal lymphocyte functions, the immunoinhibitory properties of exogenously added mixed gangliosides were examined on tests of in vitro correlates of the immune response. Gangliosides inhibited concanavalin A and lipopolysaccharide-induced [3H]-thymidine, [14C]-leucine and [3H]-lysine uptake by normal AKR/J mouse thymocytes and spleen cells. Mixed gangliosides also suppressed the two-way mixed lymphocyte reaction of AKR/J X Swiss and AKR/J X DBA/2 spleen cells. These and other results strongly suggest a general immunologically relevant role for gangliosides in the ontogeny of thymic lymphoma of mice.

Exogenous gangliosides at concentrations found in serum inhibit the concanavalin A- (Con A) induced mitogenic response of mouse thymocytes. Of four gangliosides tested, the trisialoganglioside, GT1, was the most potent inhibitor. Ceramides, cerebrosides, and sialic acid were not inhibitory at any concentration tested. The inhibition by gangliosides was not due to interference with Con A binding as shown by direct binding studies with [3H]acetyl-Con A nor was it due to a nonspecific killing effect. Thymocytes exposed to a ganglioside concentration 5 times that required to inhibit mitogenesis were still capable of excluding trypan blue up to 44 hr after ganglioside addition. Furthermore, ganglioside inhibition could be reversed by washing the cells 4 hr after addition of the glycolipid. A productive interaction with Con A occurs in the presence of ganglioside as shown by a Con A-induced increase in carbohydrate metabolism. However, uridine and thymidine incorporation are inhibited by the presence of ganglioside. Complete inhibition could be achieved if the glycolipid were added as late as 24 to 28 hr after the Con A in a 48-hr mitogenic assay. The results are discussed in light of recent findings that elevated levels of gangliosides are found in in the sera of tumor-bearing animals, and it is suggested that gangliosides shed by tumor cells could be involved in the generalized immunosuppression observed in such animals.

When incubated for 24 hr in medium capable of supporting mitogen-induced blastogenesis, thymocytes from young, adult nonleukemic, and leukemic AKR/J mice could utilize both glucose and fatty acids to satisfy their energy requirements. At least 95% of the glucose utilized by these cells was converted to extracellular lactic acid. The rate of lactic acid production was a function of age and of the onset of leukemogenesis, i.e. , highest in thymocytes from leukemic mice, moderate in young mice, and lowest in adult, nonleukemic mice. These rates paralleled basal, nonstimulated rates of incorporation of [ methyl -3H]thymidine into nucleic acid, and similar correlations could be made for changes in the specific activities of several glycolytic enzymes. The mitogen, concanavalin A, at concentrations capable of producing blast formation in the thymus cells stimulated lactic acid and CO2 production in all three groups of mice. The exogenous addition of physiological levels of lactate to the culture medium did not affect the production of lactic acid. Oleic acid, however, was oxidized preferentially in the presence of glucose and was capable of inhibiting glycolysis in normal but not in leukemic thymocytes. Furthermore, oleate utilization was increased in the presence of concanavalin A and was higher in thymus cells from leukemic mice than it was in normal thymocytes. Physiological levels of oleate were used extensively by normal and leukemic cells, indicating that fatty acids are an important energy source for thymus cells.