Galectin-9对活化的CD4^＋T细胞免疫调节作用的机制研究

The mechanism of Galectin-9 in the immune regulation of the active CD4＋ T cells

目的研究Galectin-9对活化的CD4＋T细胞的免疫调节作用，并进一步探讨其作用机制。方法获取野生型C57BL／6小鼠淋巴细胞，利用MACS分选CIM＋naiveT细胞，给予anti-CD3（2．5μm1）抗体、anti-CD28（5μg／ml）抗体和IL-2（100ng／ml）刺激，培养3d。将活化的CD4＋T细胞分为3组：正常对照组、Galectin-9组和Galectin-9＋α-乳糖组。利用CFSE检测T细胞增殖情况，并动态观察细胞形态变化；检测CD4＋CD69＋T细胞、Thl、Th2和Thl7细胞的比例；并利用ELISA检测淋巴细胞分泌IFN-γ、IL-4、IL-10、IL-12、IL-17A和TGF-β1等细胞因子的水平；利用Westernblot检测T-bet、GATA-3和ROR-γt等T细胞分化调控蛋白的变化。结果与正常对照组和Galectin-9＋α-乳糖组相比，Galectin-9组于2h时开始出现细胞形态改变，同时CD4＋CD69＋T细胞、Thl和Thl7细胞表达减少（P〈0．05），但Th2细胞无明显变化；培养上清液中IFN-γ、IL-12、IL-17A和TGF-β1的表达水平降低（P〈0．05），而IL-4和IL-10等Th2型细胞因子水平无明显变化；同时T-bet和ROR-γt的表达水平减少（P〈0．05）。结论Galectin-9抑制活化CD4＋T细胞的Thl和Thl7型免疫应答，而对Th2型免疫应答无影响，免疫调节的机制可能与其在转录水平影响Thl和Thl7特定转录因子的表达有关。

Objective To study the immune regulation of Galectin-9 on the active CIM＋T cells and demonstrate the mechanisms. Methods Lymphocytes were harvested from Mid-type C57BL/6 mouse, from which nayve CD4＋T cells were separated via MACS and then stimulated with anti-CD3 antibody（Ab） （2.5μg/ml）, anti-CD28 Ab（5 μg/ml） and IL-2（ 100 ng/ml） for 3 days. The active CIM＋T cells were di- vided into 3 groups： Control group, Galectin-9 group and Galectin-9＋α-lactose group. We detected the cell proliferation level by CFSE fluorescence intensity and then dynamically observed the cell morphological chan- ges. The proportion of CIM＋CD69＋T cell, Thl, Th2 and Thl7 cell was valued; Meanwhile, ELISA was used to detect the cytokine levels of IFN-γ, IL-4, IL-10, IL-12, IL-17A and TGF-β1 secreted by lympho- cytes. Also Western blot was used to observe the changes of T cell differentiation regulatory protein such as T-bet, GATA-3 and ROR-γt. Results Compared with control group and Galectin-9＋c^-lactose group, in Galectin-9 group, the cell morphology began to change at 2 h. Moreover the proportions of CIM＋CD69＋ T cell, Thl and Thl7 cells decreased（P〈0.05）, but no significant differences in Th2 cells. The level of IFN-γ, IL-12, IL-17A and TGF-β1 from the supernatant decreased （P〈0.05）, while Th2-type cytokines IL-4 and IL-10 did not change. In addition, the expressions of T-bet and ROR-γt were significantly down-regula- ted（P〈0.05）. Conclusion Galectin-9 inhibited Thl and ThlT-type immune response, while had no effect on Th2-type immune response. The mechanism of the immune regulation may be related to affect the expres- sion of Thl and Thl7 specific transcription factors at transcription level.