Expression Of PD-L1 On The Surface Of Dendritic Cells Derived From Human Malignant Pleural Effusion And It's Impact On The Function Of T Cells In Malignant Pleural Effusion

Dendritic cell (dendritic cell.DC) is the most potent antigen-presenting cell (antigen presenting cell, APC) in vivo. They can stimulate the naive T cell activation and induce CD8+CTL to kill the tumor cells directly or indirectly and achieve anti-tumor effect. Programmed death ligand -1 (PD-L1) is an important member of the B7/CD28 co-stimulatory factor super family, PD-L1 with its receptor PD-1 interaction could regulate immune response negatively in order to inhibit T cell activating, proliferating and cytokine secreting, play an important role in the evasion of tumor immunity. The purpose of this study is to detect the expression of PD-L1 on the surface of dendritic cells derived from human malignant pleural effusion and to study the impact of DC and PD-L1 in lung cancer, to evaluate how PD-1/PD-L1 pathway mediates tumor immune escape.PartⅠ: The differentiation of tuberculosis and malignant pleural effusion in the proportion of lymphocyte subsetsObjective: To compare tuberculous and malignant pleural effusion in the proportion of lymphocyte subsets and IFN-γlevelsMethods: 16 samples of tuberculous pleural effusion and 22 samples of malignant pleural effusion of lung cancer were collected before treatment. The pleural effusion was separated by ficoll density gradient centrifugation to get mononuclear cells. Mononuclear cells were counted and classified, FCM was used to analyze the expression of CD3, CD4 and CD8 on T cells from mononuclear cells. Calculated the ratios of CD4+T and CD8+T. Cytokine levels of IFN-γwere analyed by ELISA.Results: The dominant T helper cells in the tuberculous pleural effusion were the CD4+T cells.The percentages of CD3+T, CD4+T-cell(P < 0.01) and the ratios of CD4+T / CD8+T(P < 0.05) were higher than those in malignant pleural effusion.The levels of INF-γin two group were: 1140.16±266.82pg/ml in the tuberculous pleural effusion group and 29.14±22.36 pg / ml in the malignant pleural effusion group. The level of IFN-γin the tuberculous pleural effusion was significant higher than that in the malignant pleural effusion (P < 0.01)Conclusion: Tuberculous pleural effusion manifested mainly Th1 immune response; while patients with malignant pleural effusion appeard an obvious cellular immune dysfunction. IFN-γcan be used as a maker for differential diagnosis between tuberculous pleural effusion and malignant pleural effusion.PartⅡ: Expression of PD-L1 on the surface of dendritic cells derived from human malignant pleural effusionObjective: To study the expression of PD-L1 on the DCs derived from malignant pleural effusion.Methods: Collected malignant pleural effusion of lung cancer was firstly separated by double ficoll density gradient centrifugation to get mononuclear cells. The purified CD14+cells were separated from mononuclear cells by magnetic bead isolation technique. The monocyte-macrophage cells were cultured by adding granulocyte / macrophage colony-stimulating factor (GM-CSF), interleukin 4 (IL-4) and tumor necrosis factor-α(TNF-α)in vitro. The cell morphology was observed by light microscopy and imunophenotypes of DCs were analyzed by FCM.Results: Mononuclear cells from malignant pleural effusion were isolated by CD14-positived magnetic bead selection, the purity of CD14+ mononuclear cells was more than 90% which were detected by FCM. DC precursor cells can be cultured into mature DC by GM-CSF, IL-4 and TNF-αin vitro. The mature DC from malignant pleural effusion demonstrated its unique morphological characteristic of burr-like protuberance, and high expression of CD80(90.3±5.7%),CD83(73.8±6.9%),HLA-DR(94.4±2.8%) and PD-L1(92.9±4.3%),moderate expression of CD1α(31.5±6.0%).Compared with DC derived from normal peripheral blood mononuclear cells, there is no significant difference(P >0.05).Conclusion: DC precursor cells from malignant pleural effusion can be induced to mature DC with normal morphology and immunophenotypes. Mature DC high expression of PD-L1.PartⅢ: The impact of PD-L1 expressed on the surface of DC on the function of T cells in malignant pleural effusionObjective: To investigate the regulatory effection of DC which express PD-L1 on the function of T cells in malignant pleural effusion of lung cancer.Methods: Collected the mature DC and recovered T lymphocytes in frozen.Mixtured DCs and T lymphocytes according to the ratio of 1:10.Experiment were divided into experimental groups: T+PHA group,T+PHA+DC group,T+PHA+DC+MAb group and control group: T cell group. Immunophenotypes of T cells were analyzed by FCM, proliferation by MTT and cytokine production of IFN-γby ELISA 3 days later.Results: Compared with T+PHA group, The proliferation Rate of T cells in T+PHA+DC group got an increase [(0.191±0.078)vs (0.372±0.065),P<0.01], and the levels of IFN-γsecrection were increased too[(135.45±9.47) pg/ml vs (342.16±42.74) pg/ml,P<0.01]. Compared with T+PHA+DC group, The proliferation Rate of T cells in T+PHA+DC+MAb group got an increase [(0.372±0.065) vs (0.536±0.144), P<0.05] and the levels of IFN-γsecrection were significantly increased too[(342.16±42.74) pg/ml vs (507.56±57.89) pg/ml, P<0.05]. Meanwhile, the expression of immunophenotype CD69 of T cells of the T+PHA+DC+MAb group was up-regulated about 16% compared with the T+PHA group (P<0.05).There was no obvious difference in the expression of immunophenotype CD25 between experimental groups.Conclusion: DC with high expression of PD-L1 could stimulate T cell proliferation and activation, and this function improved by PD-L1 monclonal antibody. It might suggested that PD-1/PD-L1 pathway has a negatively regulatory effect on T cells in malignant pleural effusion of lung cancer.