Liu Yanyouhong, Xu Hongling, Lai Nan, Yang Zike, Kang Shijun
Department of Oncology, Southern Medical University, Guangzhou 510515, China.
Department of Ultrasound, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2020 Jun 30;40(6):856-863. doi: 10.12122/j.issn.1673-4254.2020.06.13.
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.
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.
B16 cells infected with the IL-12-overexpressing lentiviral vector showed significantly increased mRNA and protein levels of IL-12 ( < 0.001) without obvious changes in cell viability (>0.05). B16/IL-12 cells expressed higher levels of IL-12 than B16 cells ( < 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 ( < 0.01). In the mice with X-ray radiation-induced immune reconstitution, PD-1 expressions on CD4 T cells ( < 0.05) and CD8+ T cells ( < 0.01) were significantly higher in B16+ RT group than in the control mice and in B16/IL-12+RT group ( < 0.01 or 0.001); the tumors grew more slowly in B16/IL-12+RT group than in B16 + RT group ( < 0.001).
During immune microenvironment reconstruction, overexpression IL-12 in the tumor microenvironment can reduce the percentage of PD-1 T cells and suppress the growth of malignant melanoma in mice.
探讨恶性黑色素瘤B16细胞中白细胞介素-12（IL-12）过表达在免疫微环境重建过程中对小鼠T细胞程序性死亡蛋白-1（PD-1）表达水平的影响。
用IL-12表达慢病毒载体转染B16细胞，通过qPCR和ELISA验证细胞中IL-12过表达。采用平板克隆实验比较B16细胞和B16/IL-12细胞的增殖活性。用ELISA检测B16/IL-12细胞来源的肿瘤组织中IL-12蛋白的表达。将C57BL/6小鼠接种B16细胞或B16/IL-12细胞，14天后通过流式细胞术检测肿瘤引流淋巴结中高表达PD-1的T细胞比例。将经650 cGy X射线辐射建立的免疫重建小鼠模型接种B16细胞（B16+RT组）或B16/IL-12细胞（B16/IL-12+RT组），以接种B16细胞前未接受X射线辐射的小鼠作为对照。记录不同时间点小鼠肿瘤生长情况，在第14天，通过流式细胞术检测肿瘤引流淋巴结和肿瘤组织中高表达PD-１ 的T细胞比例。
感染IL-12过表达慢病毒载体的B16细胞中IL-12的mRNA和蛋白水平显著升高（<0.001），细胞活力无明显变化（>0.05）。B16/IL-12细胞中IL-12的表达水平高于B16细胞（<0.01）。在荷瘤小鼠模型中，B16/IL-12组中CD4+PD-1+T细胞比例显著低于B16组（<0.01）。在X射线辐射诱导免疫重建的小鼠中，B16+RT组CD4+T细胞（<0.05）和CD8+T细胞（<0.01）上的PD-1表达显著高于对照小鼠和B16/IL-12+RT组（<0.01或<0.001）；B１6/IL-12+RT组肿瘤生长比B16+RT组更慢（<0.001）。
在免疫微环境重建过程中，肿瘤微环境中IL-12过表达可降低PD-1+T细胞百分比，抑制小鼠恶性黑色素瘤生长。
