GITRL对肺癌移植瘤小鼠髓源性抑制细胞免疫抑制功能的调控及其分子机制

目的: 研究糖皮质激素诱导的肿瘤坏死因子受体相关配体（glucocorticoid induced TNF receptor ligand, GITRL）对Lewis肺癌移植瘤小鼠来源的髓源性抑制细胞（myeloid-derived suppressor cells, MDSCs）免疫抑制功能的调控作用，并探究其分子机制。方法: 采用C57BL/6小鼠构建Lewis肺癌移植瘤模型，分选荷瘤小鼠脾脏中MDSCs，采用流式细胞术检测其表面GITR的表达；GITRL（5 μg/mL）处理MDSCs 48 h后，流式细胞术检测MDSCs对羧基荧光素二醋酸盐琥珀酰亚胺酯（CFSE）标记的CD4⁺ T细胞增殖的影响，比色法检测MDSCs胞内精氨酸酶1（arginase-1, Arg-1）的活性，Griess偶联法检测效应分子一氧化氮的释放量，并通过蛋白质印迹法检测MDSCs胞内信号分子的表达变化。结果： Lewis肺癌移植瘤小鼠脾脏中MDSCs表达GITR分子；与对照组相比，GITRL处理的MDSCs对CD4⁺ T细胞增殖的抑制能力下降，Arg-1活性降低（P<0.05），一氧化氮释放量减少（P<0.01）；GITRL蛋白下调了MDSCs胞内JAK2和STAT3 （P<0.05或P<0.01）信号的磷酸化蛋白水平。结论： GITRL蛋白通过抑制MDSCs胞内JAK2/STAT3信号通路，下调MDSCs的免疫抑制功能。

Regulation of GITRL on the immunosuppressive function of MDSCs from Lewis lung carcinoma and its molecular mechanism

Objective: To investigate the effect of glucocorticoid induced TNF receptor ligand (GITRL) on the immunosuppressive capacity of myeloid-derived suppressor cells (MDSCs) from mice bearing Lewis lung carcinoma (LLC), and further explore the underlying mechanism. Methods: C57BL/6 mice were implanted with LLC cells to establish the tumor-bearing model. MDSCs sorted from the spleen of tumor-bearing mice were used to detect the expression of GITR by flow cytometry (FCM). GITRL-treated MDSCs were cocultured with CFSE-labeled CD4⁺ T cells, and then the proliferation of CD4⁺ T cells were analyzed by FCM. The activity of arginase 1 (Arg-1) was measured by colorimetric assay and the expression of nitric oxide (NO) was detected by Griess assay. The intracellular signals of GITRL-treated MDSCs were analyzed by Western blotting. Results: GITR was expressed on MDSCs from tumor-bearing mice. Compared with the control group, MDSCs treated with GITRL significantly reduced the suppressive capacity of MDSCs on the proliferation of CD4⁺ T cells, and the activity of Arg-1 (P<0.05) as well as the production of NO were also significantly decreased (P<0.01). Additionally, GITRL treatment remarkedly downregulated the phosphorylation of JAK2 and STAT3 (P<0.05 or P<0.01) in MDSCs. Conclusion: GITRL inhibited the immunosuppressive function of MDSCs from tumor bearing mice via downregulating JAK2/STAT3 signaling pathway. ［Key words］glucocorticoid induced TNF receptor ligand; myeloid derived suppressor cells; anti-tumor immunity; immunosuppression