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

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，达到良好的肿瘤控制效果。

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

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 the tumor microenvironment can reduce the percentage of PD-1 + T cells and suppress the growth of malignant melanoma in mice.