过表达IL-12的恶性黑色素瘤细胞在肿瘤免疫微环境重建过程中抑制T细胞表面PD-1的表达  

Interleukin-12 over-expression in malignant melanoma B16 cells reduces programmed death-1 expression on T cells in mice with immune reconstitution

作　　者：刘严友葓 徐虹铃 赖楠 杨子科 康世均[1] LIU Yanyouhong;XU Hongling;LAI Nan;YANG Zike;KANG Shijun(Department of Oncology,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China;Department of Ultrasound,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China;Department of Oncology,Zhongshan Hospital of Xiamen University,Xiamen 361004,China)

机构地区：[1]南方医科大学南方医院肿瘤科,广东广州510515 [2]南方医科大学南方医院超声科,广东广州510515 [3]厦门大学附属中山医院肿瘤内科,福建厦门361004

出　　处：《南方医科大学学报》2020年第6期856-863,共8页Journal of Southern Medical University

基　　金：广东省科技计划项目(2016A020215103);南方医院院长基金(2018Z006);厦门市科技计划项目(3502Z20194018)。

摘　　要：目的探讨过表达IL-12的恶性黑色素瘤细胞B16对肿瘤免疫微环境重建过程中T细胞表面PD-1表达水平的影响。方法利用慢病毒感染小鼠黑色素瘤细胞B16以构建稳定过表达IL-12的B16细胞株;利用qRT-PCR、ELISA检测IL-12的表达情况;通过平板细胞克隆形成实验验证转染病毒对B16细胞增殖能力的影响;利用ELISA检测B16/IL-12细胞在体内表达IL-12的能力;将健康的C57BL/6小鼠随机分为2组,分别接种B16细胞及B16/IL-12细胞,14 d后用流式细胞术检测肿瘤引流区淋巴结中高表达PD-1的T细胞比例;利用650cGy剂量的放射线照射C57BL/6小鼠以构建免疫重建模型,并将造模后的小鼠随机分为2组,分别接种B16细胞及B16/IL-12细胞,将未接受放疗处理的小鼠作为对照组接种B16细胞,记录各组小鼠肿瘤生长情况,接种细胞14 d后用流式细胞术分别检测肿瘤引流区淋巴结以及肿瘤组织中PD-1^+T细胞比例。结果B16/IL-12细胞相比于B16细胞显著增加IL-12的表达(P<0.001);转染IL-12过表达慢病毒并未对B16细胞增殖能力造成显著影响(P>0.05);接种B16/IL-12细胞的何瘤小鼠在其肿瘤组织中含有更高水平的IL-12(P<0.01);常规荷瘤小鼠模型中,B16/IL-12组小鼠较B16组小鼠具有更低比例的CD4^+PD-1^+T细胞(P<0.01),而CD8^+T细胞群中,PD-1表达水平差异不显著(P>0.05);免疫重建小鼠模型中,接种B16细胞的小鼠肿瘤引流区淋巴结和肿瘤组织中的CD4^+PD-1^+T细胞(P<0.05)及CD8^+PD-1^+T细胞比例(P<0.01)均高于正常B16组小鼠,接种B16/IL-12细胞的免疫重建小鼠肿瘤引流区淋巴结及肿瘤组织中的CD4^+PD-1^+T细胞比例(P<0.01)和CD8^+PD-1^+T细胞比例(P<0.001)相比于接种B16细胞的免疫重建小鼠均有显著降低;在小鼠肿瘤生长过程中,接种B16/IL-12细胞的免疫重建小鼠的肿瘤生长受到明显抑制(P<0.001)。结论肿瘤区域过表达IL-12后可减少免疫重建过程中肿瘤区域T细胞表面PD-1的表达,达到良好的肿�Objective To investigate whether interleukin-12(IL-12)over-expression in malignant melanoma B16 cells affects the expression level of programmed death-1(PD-1)on T cells in mice during immune microenvironment reconstruction.Methods B16 cells were transfected with an IL-12 expression lentiviral vector,and IL-12 over-expression in the cells was verified qPCR and ELISA.Plate cloning assay was used to compare the cell proliferation activity between B16 cells and B16/IL-12 cells.The expression of IL-12 protein in B16/IL-12 cells-derived tumor tissue were detected by ELISA.C57BL/6 mice were inoculated with B16 cells or B16/IL-12 cells,and 14 days later the proportion of T cells with high expression of PD-1 in the tumor-draining lymph nodes was detected by flow cytometry.Mouse models of immune reconstitution established by 650 cGy X-ray radiation were inoculated with B16(B16+RT group)or B16/IL-12(B16/IL-12+RT group)cells,with the mice without X-ray radiation prior to B16 cell inoculation as controls.Tumor growth in the mice was recorded at different time points,and on day 14,flow cytometry was performed to detect the proportion of T cells with high PD-1 expression in the tumor-draining lymph nodes and in the tumor tissue.Results B16 cells infected with the IL-12-overexpressing lentiviral vector showed significantly increased mRNA and protein levels of IL-12(P<0.001)without obvious changes in cell viability(P>0.05).B16/IL-12 cells expressed higher levels of IL-12 than B16 cells in vivo(P<0.01).In the tumor-bearing mouse models,the proportion of CD4^+PD-1^+T cells was significantly lower in B16/IL-12 group than in B16 group(P<0.01).In the mice with X-ray radiation-induced immune reconstitution,PD-1 expressions on CD4^+T cells(P<0.05)and CD8+T cells(P<0.01)were significantly higher in B16+RT group than in the control mice and in B16/IL-12+RT group(P<0.01 or 0.001);the tumors grew more slowly in B16/IL-12+RT group than in B16+RT group(P<0.001).Conclusion During immune microenvironment reconstruction,overexpression IL-12 in t

关 键 词：白细胞介素12 PD-1 肿瘤免疫微环境 恶性黑色素瘤 免疫治疗 

分 类 号：R739.5[医药卫生—肿瘤]