Regulatory Role Of The PD-1/PD-L1 Signaling Pathway In Treg Cells In Lung Cancer

Along with understanding the mechanism of cancer molecular pathogenesis, by intervening the process of tumor immune escape and enhancing the host’s own ability to eradicate tumor cells, immunotherapy is becoming a novel therapy strategy. The programmed death molecule-1（programmed death 1, PD-1）/PD-1 ligand（programmed death ligand, PD-L1） signaling pathway and regulatory T cell（Treg） take effect through different mechanisms in down-regulating immune response to tumors and may lead to tumor immune escape. On tumor cell surface where it acts as a ligand, high level of PD-L1 molecule can be detected. On the other hand, tumor-infiltrating lymphocytes express the PD-1 molecule on their surface as a receptor. The interaction between PD-L1 and PD-1 transmits a negative costimulatory signal resulting in down-regulation of CTL antitumor response, eventually leading to tumor cell immune escape. Additionally, in regard of tumor-infiltrating T lymphocytes, it has been found that the Treg increased significantly and they can suppress the T lymphocytes which have anti-tumor activity. Studies have shown that the PD-L1/PD-1 signaling pathway plays a role in promoting the formation of Treg and maintaining the functional stability of Treg. However, it still remains unclear in the following fields: whether the process by which lung cancer cells evade the body’s antitumor immune response is related to increased expression of PD-L1 on the tumor cell surface, the level of expression of PD-1 on Treg surface in peripheral blood in patients with lung cancer, as well as the clinical significance of these phenomena.In this study, we established co-culture system composed of lung cancer cells and T lymphocyte from peripheral blood of healthy donors in order to detect whether Treg cell differentiation can be influenced by PD-L1 expression on the lung cancer cell surface. We specifically blocked PD-L1 and further analyzed the role of PD-L1 in inducing Treg differentiation. Afterwards, in patients with lung cancer, level of surface molecule PD-1 on Treg cell from peripheral blood and their clinical significance were evaluated so as to further explore the role of the PD-1/PD-L1 signaling pathway in lung cancer.Part I Expression levels of PD-L1 molecules on the surface of lung cancer cell lines and the regulatory effect of Treg cellsObjective To detect the expression level of PD-L1 in three lung cancer cell lines and to determine whether it can induce differentiation of Treg cells.Methods PD-L1 expression on the surface of three human lung cancer cell lines was detected by flow cytometry. We then selected the cell line with the highest expression of PD-L1 for co-culture experiment. Peripheral blood mononuclear cells（PBMCs） were isolated from healthy donors and then immunomagnetic bead was used to sort T lymphocytes. We assessed the purity of separated PBMCs. Human peripheral blood T lymphocytes were stimulated with phytohemagglutinin（PHA）. The co-cultured cells to be treated were divided into three groups: Group A: lung cancer cells + T lymphocytes + PHA Group B: lung cancer cells + T lymphocytes + PHA + TGF-β, Group C: lung cancer cells+T lymphocytes + PHA + TGF-β + anti-PD-L1 antibody. Flow cytometry was used to determine the proportion of Treg cell differentiation.Results We found that SPCA-1 cell was the one with the highest surface PD-L1 expression among the three cell lines. Thus, we selected SPCA-1 cells for the co-culture experiments. In group A, the activated peripheral blood T lymphocytes differentiated into Treg cells with low degree;In group B, the activated peripheral blood T lymphocytes differentiated into Treg cells with the high degree;In group C,anti-PD-L1 Ab was used, the proportion of Treg cells reduced compared to group B.Conclusions PD-L1 is expressed at different levels on the surface of lung cancer cells. In collaboration with TGF-β, PD-L1 may induce human peripheral blood T lymphocytes to differentiate into Treg cells, thus potentially promote the occurrence and development of cancer.Part II Surface PD-1 molecules on Treg in peripheral blood of patients with lung cancer and its clinical implicationObjective To detect the level of Treg and surface PD-1 molecules on Treg from peripheral blood in patients with lung cancer and to analyze its clinical significance.Methods Newly diagnosed patients with lung cancer in Department of Respiratory Medicine, the Second Affiliated Hospital University of Soochow University were enrolled in this study from December 2014 to May 2015. Totally there included 22 patients with lung cancer and 25 gender and age-matched healthy donors as a control group. Flow cytometry was used to detect the CD4⁺ CD25⁺ CD127^low Treg cell proportions in peripheral blood mononuclear cells（PBMCs） and PD-1 level on cell surface. Flowjo software was used to analyze and the expression of PD-1 molecules on the surface and the clinicopathological parameters of the patients were analyzed.Results The level of CD4⁺ CD25⁺ CD127^low Treg cells in the peripheral blood of patients with lung cancer increased compared to the healthy controls（P < 0.01）. The level of PD-1 expressed on the surface of CD4⁺ CD25⁺ CD127^low Treg cells also increased with statistical significance（P = 0.001）. Subgroup analysis showed no significant difference according to age, sex, smoking status, tumor size and lymph node metastasis status（P>0.05） but clinical stage（P < 0.05）.Conclusions Treg cells from peripheral blood and their surface molecule PD-1 significantly increased in lung cancer compared with normal controls. Therefore, Treg cells and their surface molecule PD-1 may be involved in the occurrence and development of lung cancer. Detecting CD4⁺ CD25⁺ T cells and PD-1 expression levels in peripheral blood may signify impaired immune response against lung cancer.In summary, the results of this study were as follows:（1） PD-L1 expressed at different levels on the surface of lung cancer cells and act in combination with TGF-β in inducing human peripheral blood T lymphocytes to differentiate into Treg cells.（2） By blocking PD-1/PD-L1 pathway with an anti-PD-L1 monoclonal antibody, the number of Treg cells can reduced.（3） The number of Treg cells and their surface molecule PD-1 in peripheral blood increased in lung cancer compared with normal controls and related with stage, so Treg cells and their surface molecule PD-1 may affect the occurrence and development of lung cancer.