顺铂单独或联合单克隆抗程序性死亡配体-1抗体对肺腺癌细胞系SPCA-1和T淋巴细胞的作用

[Effect of cisplatin alone or combined with monoclonal anti-programmed death ligand-1 antibody on lung adenocarcinoma cell line SPCA-1 and T lymphocytes].

作者信息

Pan Xue, Xing Yufei, Shi Minhua, Zhou Tong, Qian Bin, Chen Yongjing

机构信息

Department of Respiratory Medicine, the Second Affiliated Hospital of Soochow University, Suzhou 215004, China.

Email:

出版信息

Zhonghua Jie He He Hu Xi Za Zhi. 2014 Jun;37(6):416-20.

PMID:25200040

Abstract

OBJECTIVE

To observe the effect of cisplatin alone or combined with anti-programmed death ligand 1 monoclonal antibody (anti-PD-L1 mAb) on the co-culture system of lung adenocarcinoma SPCA-1 cells and T lymphocytes, and therefore to study the immunotherapeutic effect of anti-PD-L1 mAb on lung cancer.

METHODS

Human adenocarcinoma SPCA-1 cell line was selected by flow cytometry (FCM) due to its high expression of membranous programmed death ligand-1 (PD-L1). The concentration of cisplatin was determined by CCK-8 method depending on the inhibition rate of SPCA-1 cell, which was set to less-than-or-equal-to 20% (IC20). After treatment with different concentrations of cisplatin, cell proliferation (A value) of SPCA-1 cells and T lymphocytes were detected by CCK-8 method and cell cycle of SPCA-1 cells and cell apoptosis of T lymphocytes were analyzed using PI staining. Treated with different concentrations of cisplatin alone or in combination with anti-PD-L1, T lymphocyte proliferation in co-culture system was determined by CCK-8 method, and cytokines such as IFN (interferon)-γ, IL-2, IL-10 and TNF-α were detected with enzyme linked immunosorbent assay (ELISA) method.

RESULTS

The IC20 of cisplatin on SPCA-1 cells was ≤ 0.78 mg/L. The proliferation of SPCA-1 cells were inhibited with different concentrations of cisplatin in a concentration-dependent manner (0.78∼12.5 mg/L) (P < 0.001). Compared with the group treated with high-dose of cisplatin (12.5 mg/L), the proliferation of T lymphocytes treated with low-dose of cisplatin (0.78 mg/L) was higher (t = 3.508, P < 0.05) and the number of late apoptotic and dead T lymphocytes in the co-culture system was reduced (t = 17.55, P < 0.001). Compared with the group of co-culture system, cisplatin (0.78 mg/L) combined with anti-PD-L1 (1.5 mg/L) significantly enhanced the proliferation of T lymphocytes in the co-culture system (t = 4.419, P < 0.01). Also, the levels of T helper cell type-1 (Th1) cytokines such as IFN-γ, IL-2 and TNF-α were up-regulated (t = 25.79-55.15, P < 0.01) and the T helper cell type-2 (Th2) cytokine IL-10 was down-regulated (t = 18.38, P < 0.01).

CONCLUSION

Low-dose of cisplatin combined with anti-PD-L1 could effectively promote the proliferation of T lymphocytes in the microenvironment and increase the secretion of Th1 type cytokines. This may reduce the toxic effect of high-dose antineoplastic agents on immune cells and help eradication of tumor cells.

摘要

目的

观察顺铂单独或联合抗程序性死亡配体1单克隆抗体（抗PD-L1 mAb）对肺腺癌SPCA-1细胞与T淋巴细胞共培养体系的影响，进而研究抗PD-L1 mAb对肺癌的免疫治疗效果。

方法

通过流式细胞术（FCM）筛选出膜程序性死亡配体-1（PD-L1）高表达的人腺癌SPCA-1细胞系。根据SPCA-1细胞抑制率，采用CCK-8法确定顺铂浓度，设定为小于或等于20%（IC20）。用不同浓度顺铂处理后，采用CCK-8法检测SPCA-1细胞和T淋巴细胞的增殖（A值），并用PI染色分析SPCA-1细胞的细胞周期和T淋巴细胞的细胞凋亡。单独或联合抗PD-L1用不同浓度顺铂处理后，采用CCK-8法测定共培养体系中T淋巴细胞增殖情况，并用酶联免疫吸附测定（ELISA）法检测干扰素（IFN）-γ、白细胞介素（IL）-2、IL-10和肿瘤坏死因子（TNF）-α等细胞因子。

结果

顺铂对SPCA-1细胞的IC20≤0.78 mg/L。不同浓度顺铂（0.78～12.5 mg/L）以浓度依赖方式抑制SPCA-1细胞增殖（P<0.001）。与高剂量顺铂（12.5 mg/L）处理组相比，低剂量顺铂（0.78 mg/L）处理的T淋巴细胞增殖更高（t=3.508，P<0.05），共培养体系中晚期凋亡和死亡T淋巴细胞数量减少（t=17.55，P<0.001）。与共培养体系组相比，顺铂（0.78 mg/L）联合抗PD-L1（1.5 mg/L）显著增强了共培养体系中T淋巴细胞的增殖（t=4.419，P<0.01）。此外，辅助性T细胞1型（Th1）细胞因子如IFN-γ、IL-2和TNF-α水平上调（t=25.79～55.15，P<0.01），辅助性T细胞2型（Th2）细胞因子IL-10水平下调（t=18.38，P<0.01）。