Effect Of Anti-PD-L1 Monoclonal Antibody And EGFR-TKI On PD-L1 Expressed By EGFR Mutated Lung Cancer Cells And Function Of T Lymphocytes

Objective:To investigate the serum level of soluble programmed death-1 ligand 1(s PD-L1)in lung cancer patients and to explore its clinical significance;To study the expression of membrane programmed death-ligand 1(PD-L1) and s PD-L1 on EGFR mutated lung cancer cells and to elucidate the effect on tumor cells and T cells in co-culture system using anti-PD-L1 monoclonal antibody and EGFR-TKI.Methods:1. Using ELISA kit, we tested the s PD-L1 protein expression in lung adenocarcinoma patients including 22 EGFR mutation cases, 18 WT EGFR cases, 28 lung squamous cell carcinomas cases and 40 healthy volunteers. The relationship of s PD-L1 with clinicopathologic features were also analyzed.2. Flow cytometry was used to analyze the expression of membrane PD-LI and ELISA method was used to determine supernatant level of s PD-L1 in EGFR mutated and EGFR wild type lung cancer cells before and after erlotinib treatment.3. After treatment with anti-PD-L1 monoclonal antibody alone and in combination with erlotinib, the proliferation and apoptosis of T cells in co-culture system was measured using Cell Counting Kit-8(CCK-8) assay and flow cytometry. Using ELISA method, IFN-γwas determined in cell supernatant to observe the changes of T lymphocyte function. The expression of PD-L1 on tumor cells and T cells treated with erlotinib in co-culture system were analyzed by flow cytometry.Results:1. A higher level of s PD-L1 in patients with lung adenocarcinoma group and squamous cell carcinomas group was found compared with control group(P<0.01, P<0.01).A similar expression level of s PD-L1 was found in EGFR mutation lung adenocarcinoma group and EGFR wild lung adenocarcinoma group(P>0.05). High level of s PD-L1 inadvanced lung adenocarcinoma was closely correlated to smoking history and distant metastasis(P<0.05), in contrast to sex, age and TNM staging(P>0.05).2. A higher expression of PD-L1 was observed on EGFR mutated lung cancer cells(PC9, HCC827) cells in contrast to EGFR wild type lung cancer cells(H1299, A549).Treatment with EGFR inhibitor erlotinib down-regulated the expression of membrane PD-L1(P<0.05) and level of s PD-L1(P<0.01) in EGFR mutated cell lines but not in EGFR wild type cell lines.3. In the co-culture system composed of T cell and EGFR mutated lung cancer cells,treatment of erlotinib alone promoted the proliferation of T cells and decrease the apoptosis rate of T cell(P<0.05), in cantrast to the co-culture system composed of T cell and EGFR wild type cell lines(P>0.05). Combined treatment of anti-PD-L1 monoclonal antibody with erlotinib promoted the proliferation of T cells and decrease the apoptosis rate of T cell in the co-culture system still further than using erlotinib alone(P<0.05).This phenomenon was not seen in the co-culture system composed of T cell and EGFR wild type cell lines(P>0.05). After treatment of erlotinib, IFN-γ level in the supernatant of the co-culture system was significantly increased(P<0.01) and PD-L1 expressed by EGFR mutated HCC827 cell was down-regulated(P<0.001) in the co-culture system composed of T cell and HCC827 cells, compared with the co-culture system composed of T cell and A549cells(P>0.05).Conclusions:1. Elevated expression of serum level of s PD-L1 was found in advanced lung adenocarcinoma, which is closely correlated with smoking history and distant metastasis.Expression level of s PD-L1 has no significant correlation with the EGFR sensitive mutations.2. After treated with erlotinib, the expression of m PD-L1 and s PD-L1 in EGFR mutated cell lines is decreased, which signifies PD-L1 expression may be regulated by EGFR signaling pathway.3. After treatment of erlotinib, IFN-γ was significantly increased in the co-culture system composed of T cell and EGFR mutated lung cancer cells, which signifies EGFR TKIs can promote the immune function of T cells.4. Combined with anti-PD-L1 monoclonal antibody and EGFR-TKI could effectively enhance proliferation and reduce apoptosis of T lymphocyte in the EGFR mutated lungcancer microenvironment.