Studies on the activation-associated ganglioside in rat T cell

在我们的研究中，大鼠胸腺细胞和脾T细胞中的主要神经节苷脂被鉴定为GD 1c（NeuGc，NeuGc）。我们的研究还阐明了胸腺细胞中GD 1c（NeuGc，NeuGc）的量在细胞活化和增殖时大大增加。此外，另一种神经节苷脂GD 1b（NeuGc，NeuGc）在静息胸腺细胞中几乎不可识别，但在活化胸腺细胞中的增加程度与GD 1c（NeuGc，NeuGc）相同。本研究用纯化的GD 1b（NeuGc，NeuGc）免疫C_3 H/HeN小鼠，制备了抗这两种神经节苷脂的单克隆抗体AB_1，并将其用于流式细胞术分析。在酶联免疫吸附试验中，AB 1与GD 1b（NeuGc，NeuGc）呈强反应，与GD 1b（NeuAc，NeuAc）呈中等反应。它与研究的其他神经节苷脂（包括GD 1c（NeuGc，NeuGc））没有任何活性。用纯化的GD 1c（NeuGc，NeuGc）免疫获得的单克隆抗体AC 1识别GD 1c（NeuGc，NeuGc），与GD 1b（NeuGc，NeuGc）有弱交叉反应，与GD 3（NeuAc，NeuAc）有极轻微交叉反应。其他神经节苷脂均不与AC 1反应。结果表明，两种抗体的同种型均为IgG 3（kappa），流式细胞仪检测结果显示，在静息胸腺细胞和T细胞中，AC 1 ^+细胞主要分布在部分CD 4 ^+细胞中，而AB 1 ^+细胞则未检测到。在TPA和钙离子载体A23187激活后，两种细胞类型中AC 1 ^+细胞的比例大幅增加。相反，AB 1 ^+细胞只出现在活化的胸腺细胞中。这些结果表明，GD 1b（NeuGc，NeuGc）特异性地与胸腺细胞活化相关，而GD 1c（NeuGc，NeuGc）参与大鼠胸腺细胞和成熟T细胞的细胞活化。

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.