肿瘤浸润性辅助性T细胞9细胞对胃癌患者CD8^(+)T细胞抗肿瘤活性的调控作用  

The regulatory function of tumor-infiltrating Th9 cells to anti-tumor activity of CD8^(+) T cells in patients with gastric cancer

作　　者：孙培胜[1] 高正杰 范龙鑫 刘亚飞[1] 陈炳合[1] 穆淑真 闫争强[1] 

机构地区：[1]新乡医学院第一附属医院普通外科,新乡453100

出　　处：《中华肿瘤杂志》2022年第11期1186-1193,共8页Chinese Journal of Oncology

摘　　要：目的探讨辅助性T细胞9(Th9)细胞和白细胞介素9(IL-9)在胃癌患者中的水平,评估Th9/IL-9对胃癌患者抗肿瘤免疫应答的调控作用。方法纳入2018年10月至2019年8月在新乡医学院第一附属医院行手术治疗的胃癌患者34例,同期健康体检者20例。采集外周血,分离血浆和外周血单个核细胞(PBMCs),从手术切除的胃癌组织分离肿瘤浸润性淋巴细胞(TILs)和自体胃癌细胞,分选PBMCs和TILs中的CD4^(+)T细胞、CD8^(+)T细胞和CD4^(+)CCR4^(-)CCR6^(-)CXCR3^(-)细胞。采用酶联免疫吸附试验(ELISA)检测血浆IL-9水平,流式细胞术检测PBMCs和TILs中CD3^(+)CD4^(+)IL-9^(+)Th9细胞比例,实时荧光定量聚合酶链反应检测IL-9和转录因子富嘌呤核酸结合蛋白1(PU.1)mRNA的相对表达量。以重组人IL-9刺激胃癌患者PBMCs和TILs,采用细胞计数试剂盒8法检测细胞增殖,Western blot法检测信号转导和转录激活因子3(STAT3)和STAT6磷酸化,ELISA法检测细胞因子分泌。以重组人IL-9刺激胃癌患者TILs分选的CD8^(+)T细胞,建立CD8^(+)T细胞与自体胃癌细胞的直接接触与间接接触共培养系统,通过检测乳酸脱氢酶(LDH)水平计算靶细胞死亡比例,ELISA法检测γ-干扰素(γ-IFN)和肿瘤坏死因子(TNF-α)水平。CD4^(+)CCR4^(-)CCR6^(-)CXCR3^(-)细胞、CD8^(+)T细胞与自体胃癌细胞共培养,加入抗IL-9中和抗体,检测靶细胞死亡比例。结果对照组PBMCs和胃癌组PBMCs中CD3^(+)CD4^(+)IL-9^(+)Th9细胞比例分别为(1.21±0.25)%和(1.14±0.19)%,差异无统计学意义(P=0.280)。胃癌组TILs中CD3^(+)CD4^(+)IL-9^(+)Th9细胞比例为(2.30±0.55)%,高于对照组PBMCs和胃癌组PBMCs(均P<0.001)。对照组和胃癌组血浆IL-9水平分别为(5.04±1.51)ng/ml和(4.93±1.25)ng/ml,差异无统计学意义(P=0.787)。对照组PBMCs和胃癌组PBMCs中IL-9 mRNA相对表达量分别为1.33±0.39和1.36±0.27,差异无统计学意义(P=0.691)。胃癌组TILs中IL-9 mRNA相对表达量为2.90±0.75,高于对照组PBMCs(P<0.Objective To investigate the levels of Th9 cells and interleukin-9(IL-9),and to assess the regulatory activity of Th9/IL-9 to anti-tumor immune response in patients with gastric cancer.Methods Thirty-four patients with gastric cancer who received operation in the First Affiliated Hospital of Xinxiang Medical University between October 2018 and August 2019 were included.Twenty individuals who received physical examination in the same period were also enrolled.Peripheral blood was collected,and then plasma and peripheral blood mononuclear cells(PBMCs)were isolated.Tumor-infiltrating lymphocytes(TILs)and autologous gastric cancer cells were isolated from resected gastric cancer tissues.CD4^(+)T cells,CD8^(+)T cells,and CD4^(+)CCR4^(-)CCR6^(-)CXCR3^(-)cells were purified from PBMCs and TILs.Plasma IL-9 level was measured by enzyme linked immunosorbent assay(ELISA).The percentage of CD3^(+)CD4^(+)IL-9^(+)Th9 cells in PBMCs and TILSs was assessed by flow cytometry.The mRNA levels of IL-9 and transcriptional factors purine-rich nucleic acid binding protein 1(PU.1)were semi-quantified by real-time quantitative polymerase chain reaction(RT-qPCR).PBMCs and TILs from gastric cancer patients were stimulated with recombinant human IL-9.Cellular proliferation was measured by cell counting kit-8.The phosphorylation levels of signal transducer and activator of transcription 3(STAT3)and STAT6 were investigated by western blot.Cytokine production was measured by ELISA.Purified CD8^(+)T cells from TILs of gastric cancer patients were stimulated with recombinant human IL-9.CD8^(+)T cells and autologous gastric cancer cells were cocultured in direct contact and indirect contact manner.The percentage of target cell death was calculated by measuring the lactate dehydrogenase(LDH)level.These cretion ofγ-Interferon(γ-IFN)and tumor necrosis factor-α(TNF-α)was measured by ELISA.CD4^(+)CCR4^(-)CCR6^(-)CXCR3^(-)cells,CD8^(+)T cells,and autologous gastric cancer cells were directly cocultured,and anti-IL-9 neutralizing antibody was added.

关 键 词：胃肿瘤 TH9细胞 CD8^(+)T细胞 抗肿瘤 

分 类 号：R735.2[医药卫生—肿瘤]