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 of γ-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. The target cell death was measured. Results: The percentages of CD3(+) CD4(+) IL-9(+) Th9 cells in PBMCs of control group and PBMCs of gastric cancer group were (1.21±0.25)% and (1.14±0.19)%, respectively. The difference was not statistically significant (P=0.280). The percentage of CD3(+) CD4(+) IL-9(+) Th9 cells in TILs of gastric cancer group was (2.30±0.55)%, which was higher than those in PBMCs of control group and PBMCs of gastric cancer group (P<0.001). The plasma IL-9 level in control group and gastric cancer group were (5.04±1.51) and (4.93±1.25) ng/ml. The difference was not statistically significant (P=0.787). The relative levels of IL-9 mRNA in PBMCs of control group and PBMCs of gastric cancer group were 1.33±0.39 and 1.36±0.27. The difference was not statistically significant (P=0.691). The relative level of IL-9 mRNA in TILs of gastric cancer group was 2.90±0.75, which was higher than those in PBMCs of control group (P<0.001) and PBMCs of gastric cancer group (P<0.001). The relative levels of PU.1 mRNA in PBMCs of control group and PBMCs of gastric cancer group were 1.21±0.12 and 1.20±0.11. The difference was not statistically significant (t=0.21, P=0.833). PU.1 mRNA relative level in TILs of gastric cancer group was 2.81±0.65, which was higher than those in PBMCs of control group (P<0.001) and PBMCs of gastric cancer group (P<0.001). Recombinant human IL-9 stimulation did not affect the proliferation of PBMCs and TILs of gastric cancer patients (P>0.05), but elevated the phosphorylation level of STAT6 and induced the secretions of γ-IFN, IL-17, and IL-22 by TILs (P<0.05). In direct contact culture system, IL-9 stimulation promoted tumor-infiltrating CD8(+) T cells-induced autologous gastric cancer cell death [(20.62±2.27)% vs. (16.08±2.61)%, P<0.01)]. In indirect contact culture system, IL-9 stimulation did not increase CD8(+) T cell-induced autologous gastric cancer cell death [(5.21±0.70)% vs. (5.31±1.22)%, P=0.998)]. However, the secretion levels of γ-IFN were elevated in response to IL-9 stimulation in both culture systems [direct contact culture system: (100.40±12.05) pg/ml vs. (76.45±8.56) pg/ml; indirect contact culture system: (78.00±9.98) pg/ml vs. (42.09±10.71) pg/ml; P<0.01]. The TNF-α secretion level did not significantly changed (P>0.05). In direct contact culture system, the percentage of target cells was (22.01±3.05) % and γ-IFN secretion level was (104.5±12.84) pg/ml in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells group, which was higher than (16.08±2.61)% and (76.45±8.56) pg/ml in CD8(+) T cells+ gastric cancer cells group (P<0.01). However, the percentage of target cells was (14.47±3.14)% and γ-IFN secretion level was (70.45±19.43) pg/ml in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells+ anti-IL-9 neutralizing antibody group, which were lower than those in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells group (P<0.01). Conclusion: Tumor-infiltrating Th9 cells and the secreting IL-9 promote the activity of CD8(+) T cells in gastric cancer patients, and enhance anti-tumor immune response.
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<0.001)和胃癌组PBMCs(P<0.001)。对照组和胃癌组PBMCs中PU.1 mRNA相对表达量分别为1.21±0.12和1.20±0.11，差异无统计学意义(t＝0.21，P＝0.833)。胃癌组TILs中PU.1 mRNA相对表达量为2.81±0.65，高于对照组PBMCs(P<0.001)和胃癌组PBMCs(P<0.001)。重组人IL-9对胃癌患者PBMCs和TILs的增殖无明显影响(均P>0.05)，但可提高STAT6的磷酸化水平，促进TILs分泌γ-IFN、IL-17和IL-22(均P<0.05)。在直接接触共培养系统中，IL-9刺激使肿瘤浸润性CD8(+) T细胞诱导的自体胃癌细胞死亡比例增加[(20.62±2.27)%比(16.08±2.61)%，P<0.01)]。在间接接触共培养系统中，IL-9刺激并未增加CD8(+) T细胞诱导自体胃癌细胞死亡比例[(5.21±0.70)%比(5.31±1.22)%，P＝0.998]。但在两种共培养系统中，IL-9刺激后γ-IFN分泌水平均增加[直接接触共培养系统：(100.40±12.05)pg/ml比(76.45±8.56)pg/ml；间接接触共培养系统：(78.00±9.98)pg/ml比(42.09±10.71)pg/ml；P<0.01]，TNF-α分泌水平均无明显变化(均P>0.05)。在直接接触共培养系统中，CD4(+) CCR4(-) CCR6(-) CXCR3(-)细胞+CD8(+) T细胞+胃癌细胞组的靶细胞死亡比例为(22.01±3.05)%，γ-IFN水平为(104.5±12.84)pg/ml，均高于CD8(+) T细胞+胃癌细胞组[分别为(16.08±2.61)%和(76.45±8.56)pg/ml，均P<0.01]，但CD4(+) CCR4(-) CCR6(-) CXCR3(-)细胞+CD8(+) T细胞+胃癌细胞+抗IL-9中和抗体组的靶细胞死亡比例为(14.47±3.14)%，γ-IFN水平为(70.45±19.43)pg/ml，均低于CD4(+)CCR4(-)CCR6(-)CXCR3(-)细胞+CD8(+) T细胞+胃癌细胞组(均P<0.01)。 结论： 肿瘤浸润性Th9细胞及其分泌的IL-9可促进胃癌患者CD8(+) T细胞活性，增强抗肿瘤免疫应答。.
Keywords: Anti-tumo; CD8(+) T cells; Gastric neoplasms; Th9 cells.