|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1995 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1994 : ¥1,000,000 (Direct Cost : ¥1,000,000)
In our preveious study, the predominant ganglioside in rat thymocytes and spleen T cells was identified as GD1c (NeuGc, NeuGc). Our study also clarified that the amount of GD1c (NeuGc, NeuGc) in the thymocytes greatly increases upon cell activation and proliferation. Furthermore, another ganglioside GD1b (NeuGc, NeuGc), which was barely recognizable in resting thymocytes, was found to increase in activated thymocytes to the same extent as GD1c (NeuGc, NeuGc). In the present study, monoclonal antibodies specific for each of those two gangliosides have been established and used in flow cytometry analyzes of activated rat thymocytes and T cells.
The monoclonal antibody designated AB1 was produced by immunizing C3H/HeN mice with purified GD1b (NeuGc, NeuGc). In enzyme-linked immunosorbent assay, AB1 reacted strongly with GD1b (NeuGc, NeuGc) and moderately with GD1b (NeuAc, NeuAc). It did not show any activity with the other gangliosides investigated including GD1c (NeuGc, NeuGc). The monoclonal antibody AC1, obtained by immunization with purified GD1c (NeuGc, NeuGc), recognized GD1c (NeuGc, NeuGc), cross-reacting weakly with GD1b (NeuGc, NeuGc) and very slightly with GD3 (NeuAc, NeuAc). The other gangliosides did not react with AC1. The isotype of both the antibodies was IgG3 (kappa).
Flow cytometry analyzes using those antibodies showed that AC1^+ cells were mainly in a part of CD4^+ cells in resting thymocytes and T cells, while AB1^+ cells were not detected. After activation with TPA and calcium ionophore A23187, the ratio of AC1^+ cells in the both cell types largely increased. In contrast, AB1^+ cells appeared only in teh activated thymocytes. These results suggested that GD1b (NeuGc, NeuGc) associates specifically with the thymocyte activation, whereas GD1c (NeuGc, NeuGc) is involved in cell activation of rat thymocytes and mature T cells.