阻断乳腺癌细胞PD-L1减弱对共培养树突状细胞成熟的抑制作用

Blocking programmed death-ligand 1 attenuates maturation inhibition of dendritic cells by co-cultured breast cancer cells

目的探讨表达PD-L1的乳腺癌细胞是否通过激活树突状细胞的PD-L1/PD-1信号通路抑制树突状细胞成熟。方法人单核细胞用GM-CSF和IL-4诱导为不成熟树状突细胞,再用TNF-α诱导为成熟树状突细胞;表达PD-L1的乳腺癌细胞系MDAMB-231与树状突细胞接触共培养;PD-L1阻断抗体处理共培的的乳腺癌细胞和树状突细胞;重组人PD-L1蛋白处理TNF-α诱导的树突状细胞;流式细胞仪检测乳腺癌细胞膜PD-L1的表达和树突状细胞的成熟分化标志HLA-DR和CD83。结果乳腺癌细胞MDA-MB-231细胞系中,细胞膜表面PD-L1阳性细胞高达(99.7±0.15)%;HLA-DR和CD83阳性细胞在成熟树状突细胞对照组分别为(88.8±6.96)%和(18.36±3.07)%,在MDA-MB-231共培养实验组树状突细胞群分别降至(42.76±10.52)%和(9.93±2.74)%,两组比较差异有统计学意义(P<0.01,P<0.05);HLA-DR和CD83阳性细胞在PD-L1抗体同型对照组分别为(45.17±10.19)%和(10.15±2.54)%,在PD-L1抗体处理组分别升至(63.46±1.72)%和(16.46±2.58)%,两组相比差异均有统计学意义(P<0.05);和成熟树状突细胞对照组相比,人重组PD-L1蛋白处理组HLA-DR和CD83阳性细胞率较低,组间统计学差异有统计学意义(P<0.05)。结论 PD-L1抗体对三阴性乳腺癌患者的治疗的效果,可能部分基于抗体阻断乳腺癌细胞表面的PD-L1,从而减弱其对肿瘤微环境中的树突状细胞成熟的抑制作用。

Objective To study if programmed death-ligand 1（PL-L1） expression in breast cancer cell activates PD-L1/PD-1 pathway in dendritic cells to inhibit dendritic cell maturation. Methods Human monocytes were induced to differentiate into immature dendritic cells using GM-CSF and IL-4, and further to mature dendritic cells using TNF-α. PD-L1-expressing breast cancer cell line MDA-MB-231 was co-cultured in contact with the dendritic cells to observe the effects of the breast cancer cells on the maturation of the dendritic cells. A PD-L1 blocking antibody was applied to the co-culture, and the changes in the inhibitory effect of the MDA-MB-231 cells on dendritic cell maturation was observed. TNF-α-induced dendritic cells were treated with a recombinant human PD-L1 protein to study the effect of PD-L1/PD-1 pathway activation on the maturation of dendritic cells. The expression of PD-L1 in MDA-MB-231 cells and the dendritic cell maturation marker HLA-DR and CD83 were analyzed using flow cytometry. Results MDA-MB-231 cell line showed PD-L1 positivity on the cell membrane cells at a rate as high as（99.7±0.15）%. In mature dendritic cells, the positivity rates for HLA-DR and CD83 were（88.8±6.96）% and（18.36±3.07）%, respectively, but in the co-culture system, the positivity rates of the dendritic cells were significantly decreased to（42.76±10.52）%（P〈0.01） and（9.93±2.74）%（P〈0.05）, respectively, indicating that MDA-MB-231 cells inhibited the maturation of dendritic cells. Following treatment with a PD-L1 antibody isotype control, the percentages of HLA-DR-and CD83-positive cells in the co-culture were（45.17±10.19）% and（10.15±2.54）%, which were significantly increased to（63.46±1.72）% and（16.46±2.58）% after treatment with PD-L1 antibody, respectively（both P〈0.05）. Compared with the mature dendritic cell controls, the cells treated with the recombinant human PD-L1 protein exhibited significantly lowered percentages of HLA-DR-positive[from（84.23±4.18）% to（2.56±2.39）%, P〈0.05] and CD83-positive cells [（87.26±1.54）% to（60.67±1.63）%, P〈0.05]. Conclusion The effect of PD-L1 antibody therapy on triple negative breast cancer can be partially mediated by blocking PD-L1 expression on breast cancer cell membrane, which attenuates the inhibition of dendritic cell maturation in the cancer microenvironment.