Studies on the activation-associated ganglioside in rat T cell

在我们之前的研究中，大鼠胸腺细胞和脾脏T细胞中主要的神经节苷脂被鉴定为GD1c （NeuGc, NeuGc）。我们的研究还明确了胸腺细胞中GD1c （NeuGc, NeuGc）的数量在细胞激活和增殖过程中显著增加。此外，另一种在静止胸腺细胞中几乎不被识别的神经节苷脂GD1b （NeuGc, NeuGc）被发现在激活的胸腺细胞中增加的程度与GD1c （NeuGc, NeuGc）相同。在本研究中，已经建立了针对这两种神经节苷的单克隆抗体，并将其用于活化大鼠胸腺细胞和T细胞的流式细胞术分析。该单克隆抗体AB1是用纯化的GD1b （NeuGc, NeuGc）免疫C3H/HeN小鼠产生的。在酶联免疫吸附试验中，AB1与GD1b （NeuGc, NeuGc）反应强烈，与GD1b （NeuAc, NeuAc）反应中等。它没有显示出与其他神经节苷脂的活性，包括GD1c （NeuGc, NeuGc）。用纯化的GD1c （NeuGc, NeuGc）免疫获得的单克隆抗体AC1识别GD1c (NeuGc, NeuGc)，与GD1b （NeuGc, NeuGc）有弱交叉反应，与GD3 （NeuAc, NeuAc）有极轻微交叉反应。其他神经节苷脂不与AC1反应。两抗体的同型均为IgG3 （kappa）。流式细胞术分析显示，在静止胸腺细胞和T细胞中，AC1^+细胞主要存在于部分CD4^+细胞中，而AB1^+细胞未检出。经TPA和钙离子载体A23187活化后，两种细胞类型中AC1^+细胞的比例均显著增加。相比之下，AB1^+细胞仅出现在激活的胸腺细胞中。这些结果表明，GD1b （NeuGc, NeuGc）与胸腺细胞激活特异性相关，而GD1c （NeuGc, NeuGc）参与大鼠胸腺细胞和成熟T细胞的细胞激活。

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.