NKG2D配体RAE1ε对乳腺癌细胞4T1衍生MDSC功能的影响

Effects of NKG2D ligand RAE1ε on the function of 4T1 mammary cancer cells-derived MDSC

目的:探讨表达小鼠NKG2D配体之一视黄酸早期转录因子1ε(retinoic acid early transcript 1ε,RAE1ε)的原B淋巴细胞Ba F3对乳腺癌细胞株4T1成瘤小鼠来源的髓系抑制性细胞(myeloid-derived suppressor cell,MDSC)功能的影响。方法:以小鼠原B淋巴细胞株Ba F3为基础,构建表达RAE1ε的Ba F3-RAE1ε细胞以及表达空质粒的Ba F3-mock对照细胞。通过4T1原位肿瘤模型诱导产生CD11b+Gr-1+MDSC,将Ba F3-mock细胞和Ba F3-RAE1ε细胞作为刺激细胞,分别与脾MDSC共培养,流式细胞术检测MDSC表面CD40、CD80、F4/80、CD11c的表达和MDSC内活性氧(ROS)的水平;ELISA法检测共培养上清液IL-10、IL-4和IFN-γ的含量;Griess法检测共培养上清液一氮化氮(NO)的浓度。磁珠分选共培养体系中的MDSC,检测裂解后精氨酸酶的活性;另外,将分选后的MDSC与抗CD3/抗CD28抗体活化的脾细胞共培养,CFSE法检测活化的CD3+CD8+T细胞增殖情况。结果:成功获得4T1原位肿瘤模型来源的小鼠脾MDSC。与Ba F3-mock细胞相比,Ba F3-RAE1ε细胞刺激对MDSC分泌IL-4、IFN-γ、IL-10和NO的水平没有明显影响(P>0.05);对MDSC表达CD40、CD80、F4/80、CD11c和ROS也没有显著影响(P>0.05)。与Ba F3-mock细胞相比,Ba F3-RAE1ε细胞刺激显著提高MDSC的精氨酸酶活性(156.166±1.209 vs 110.135±7.356,P<0.01),并明显增强MDSC对CD8+T细胞增殖的抑制作用。结论:RAE-1ε在体外增强4T1成瘤小鼠来源的MDSC的抑制功能。

Objective： To explore effect of BaF3 primary B lymphocyte that expresses retinoic acid early transcript 1ε （RAE1ε）, ligand of mouse NKG2D, on function of myeloid-derived suppressor cell （MDSC） originated from mouse. Methods： Based on mouse primary B lymphocyte BaF3 line, a BaF3-RAE1ε cell that expressed RAE1ε and a BaF3-mock control cell with empty plasmid were structured. CD11b ^＋ Gr^-1 ＋ MDSC was produced through introduction of 4T1 tumor model in situ. Spleen MDSC was respectively cocultured with BaF3-RAE1？ cell and BaF3-mock cell that acted as stimulating cells. Flow cytometry assay was used to detect expressions of CD40, CD80, F4/80 , CD11c and content of reactive oxygen species （ROS） in the MDSC. ELISA assay was used to test contents of IL-10, IL-4 ans IFN-γ in supernatant of the co-culture. Concentration of nitric oxide （NO） in the supernatant was examed by Griess assay. Magnetic beads were used to separate the MDSC in the co-culture system, activity of arginase was detected after splitting. In addition, the separated MDSC co-culture with splenocyte activated by anti-CD3/CD28 an-tibody. Proliferation of the activated CD3 ＋ CD8 ＋ T cell was examed by CFSC assay. Results： The MDSC of mouse spleen derived from 4T1 in situ tumor model was successfully obtained. Comparing with the BaF3-mock cell, effecting of the BaF3-RAE1ε cell stimulation on amounts of IL-4, IFN-γ, IL-10 and NO secreted by the MDSC was not significant （P〉0.05）, and on amounts of CD40, CD80, F4/80 ,CD11c and ROS expressed by the MDSC also not sig- nificant （P〉0.05）. Stimulation of the BaF3-RAE1ε cell did more increase activity of arginase in the MDSC than that of the BaF3-mock cell obviously （156.166±1.209 vs 110.135±7.356, P〈0.01）, and evidently enhanced inhibitory effect of the MDSC on proliferation of CD8 ^＋ T cell. Conclusion： RAE1ε could enhance inhibitory function in vitro of the MDSC derived from 4T1 mouse with tumor.