Establishment Of Stable Transfectant Cell Line Of Human PD-L1 Gene And Its Impact On The Function Of CD8~+T Cells In Malignant Pleural Effusion Of Lung Cancer

Programmed death-1 ligand (PD-L1) is one of the important members of B7/CD28 costimulatory super family. It's receptor is PD-1. PD-1/PD-L1 pathway functions as a negative co-inhibitor in T cells proliferation and plays a pivotal role in the evasion of tumor immunity. The purpose of our experiment is to establish human PD-L1 transgenic cells and to study the impact of PD-L1 signal on CD8+T cells in malignant pleural effusion of lung cancer, hoping to evaluate how PD-1/PD-L1 pathway mediates tumor immune escape.PartⅠ: The Expression of PD-1 on CD8+T Cells in Malignant Pleural Effusion of Lung CancerObjective: To detect the expression of PD-1 on CD8+T cells in malignant pleural effusion of lung cancer.Methods: 21 samples of malignant pleural effusion of lung cancer were collected. The pleural effusion was separated by ficoll density gradient centrifugation to get mononuclear cells. FCM was used to analyze the expression of PD-1 on CD8+T cells from mononuclear cells.Results: The expression of PD-1 was observed on CD8+T cells in malignant pleural effusion of lung cancer. Expression level of PD-1 was 12.30±7.72% (mean±SD; median, 9.2%), and high expression of PD-1(≥9.2%) were detected in 11 cases out of all 21 cases. No relationship was found between the expression of PD-1 and either sex, age, smoking status or pathological types (P>0.05).Conclusion: The expression of PD-1 was observed on CD8+T cells in malignant pleural effusion of lung cancer, which suggested that PD-1 on CD8+T cells may combine with PD-L1 on tumor cells, inhibit the function of CD8+T cells, and mediate tumor immune escape.PartⅡ: Construction of Eukaryotic Expressing Vector of Human PD-L1 and Establishment of Stable Transfectant HEK293 Cell LineObjective: To construct eukaryotic expressing vector of human PD-L1 gene and transfect HEK293 cells so as to establish stable HEK293 cell line, so to provide a useful tool for studying the impact of PD-L1 signal on CD8+T cells in malignant pleural effusion of lung cancer.Methods: PD-L1 gene was amplified by polymerase chain reaction from the human heart cDNA library by PCR and confirmed by DNA-sequence analysis. The PD-L1 gene was then digested with the restriction endonucleases Xho1 and EcoR1 and inserted into eukaryotic expressing vector pIRES2-EGFP. pIRES2-EGFP/PD-L1 was transfected into HEK293 cell by lipofectamine AMINE. After screening culture by G418, stable transfected HEK293 cell line was established, and the expression of PD-L1 was identified by FCM, RT-PCR and Western blotting.Results: The eukaryotic expressing vector pIRES2-EGFP/PD-L1 was constructed, stable transfected HEK293 cell line was established, and PD-L1 gene was expressed successfully. FCM analysis identified that the expression level of green fluorescence protein(EGFP) report gene in the transfected cells was up to 99%. The expression of PD-L1 protein(≥90%) on the surface of HEK293/PD-L1 transfected cells was detected by commercialized mouse anti-human PD-L1 mAb. Transcription of PD-L1 gene identified by RT-PCR indicated that recombinant pIRES2-EGFP/PD-L1 had been successfully inserted into eukaryotic genome. Western blot results revealed that PD-L1 mAb could specifically bind the PD-L1 molecule extracted from HEK293/PD-L1 transfected cells.Conclusion: The construction of eukaryotic expressing vector pIRES2-EGFP/PD-L1 and the establishment of stable transfected HEK293 cell line have provided a useful tool for further studies on the function of PD-L1. PartⅢ:The Impact of PD-L1 Signal on the Function of CD8+T Cells in Malignant Pleural Effusion of Lung CancerObjective: To investigate the regulatory effect of PD-1/PD-L1 pathway on the function of purified CD8+T cells in malignant pleural effusion of lung cancer.Methods: Collected malignant pleural effusion of lung cancer was firstly separated by ficoll density gradient centrifugation to get mononuclear cells. The purified CD8+T cells were separated from mononuclear cells by magnetic bead isolation technique. Transgenic cells HEK293/PD-L1 and CD8+T cells stimulated by PHA were co-cultured as experimental group, and the same treatment was done on HEK293/mock cells as control group. Immunophenotypes and apoptosis of CD8+T cells were analyzed by FCM, proliferation by MTT and cytokine production of IFN-γby ELISA.Results: Compared with the control group accoding to 1:10 as stimulator-to-effector ratio, CD8+T cells stimulated by HEK293/PD-L1 transgenic cells got a decrease of prolife- ration([0.337±0.022) vs (0.229±0.018),P<0.05], and the levels of IFN-γsecrection was significantly decreased too[(355.3±28.6)pg/ml vs (106.2±17.5)pg/ml,P<0.01]. Meanwhile, the expression of immunophenotype CD25 of CD8+T cells of the experimental group was down-regulated about 20% compared with the control group(P<0.01), but there was no obvious difference of the expression of immunophenotype CD69 between two groups(P>0.05). At the same phase, the apoptosis rate of CD8+T cells stimulated by HEK 293/PD-L1 transgenic cells was enhanced compared with the control group[(10.1±3.1)% vs (21.5±2.4)%,P<0.05].Conclusion: Transgenic cells HEK293/PD-L1 significantly down-regulated the stimulatory phenotypes and proliferation of CD8+T cells in malignant pleural effusion of lung cancer, decreased levels of IFN-γsecrection, and induced CD8+T cells apoptosis. PD-1/PD-L1 pathway has a negatively regulatory effect on CD8+T cells in malignant pleural effusion of lung cancer.