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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。
AIM: To investigate the myeloid-derived myeloid-derived inhibitory effect of Ba F3, a primary murine NKG2D ligand, on retinoic acid early transcript 1ε (RAE1ε) (Myeloid-derived suppressor cell, MDSC) function. Methods: Ba F3-RAE1ε cells expressing RAE1ε and Ba F3-mock control cells expressing empty plasmids were constructed based on mouse primary B lymphocyte strain Ba F3. CD11b + Gr-1 + MDSCs were induced by 4T1 in situ tumor model. Ba F3-mock cells and Ba F3-RAE1ε cells were stimulated with spleen MDSCs respectively. Flow cytometry was used to detect CD40, CD80, The levels of IL-10, IL-4 and IFN-γ in the co-culture supernatants were detected by ELISA and Grignard’s assay was used to detect the expression of FN / F4 / 80, CD11c and reactive oxygen species (ROS) Nitrogen (NO) concentration. The MDSCs in the co-culture system were sorted by magnetic beads to detect the activity of arginase after cleavage. In addition, the sorted MDSCs were co-cultured with anti-CD3 / anti-CD28 antibody-activated spleen cells, and the activated CD3 + CD8 + T cell proliferation. RESULTS: Mouse spleen MDSCs from 4T1 in situ tumor models were successfully obtained. Compared with BaF3-mock cells, stimulation of BaF3-RAE1ε cells had no significant effect on the secretion of IL-4, IFN-γ, IL-10 and NO from MDSCs (P> 0.05) / 80, CD11c and ROS also had no significant effect (P> 0.05). Ba F3-RAE1ε cells stimulated significantly increased arginase activity of MDSCs (156.166 ± 1.209 vs 110.135 ± 7.356, P <0.01) as compared with Ba F3-mock cells and significantly enhanced MDSCs inhibition of CD8 + T cell proliferation effect. Conclusion: RAE-1ε enhances the suppressive function of MDSC derived from 4T1 tumor-bearing mice in vitro.