The Effect Of Soluble PD-L1Expressed By Lung Cancer Cells In Regulating The Functions Of T Lymphocyte Via PD-1/PD-L1Pathway And Its Clinical Implication In Patients With Lung Cancer

The incidence and mortality of lung cancer rates rose rapidly, and has become one ofthe most common malignancies, ranking the leading cause of death of various types ofcancer. Tumor cells may escape immune destruction of the host defense system, and progresscontinuously. Costimulatory molecule PD-L1(the Programmed death-1of ligand1, alsonamed B7-H1) was highly expressed in a variety of human solid tumor cells and tissues,including lung cancer, and was related with the tumor immune escape. Some studiesdemonstrated that many costimulatory molecules, e.g. OX40、OX40L、CD40L andCTLA-4, have two isoforms, membrane-bound form and soluble form. Expression levelsof soluble costimulatory molecules possess diagnostic and predictive potential in variousdiseases. PD-L1expressed by tumor tissue may also assist in evaluating tumor response totherapeutics in clinical field.Up till now, there are few studies concerning serum sPD-L1due to the lack of specificanalytic method. It is still unknown and remains to be clarified if sPD-L1exists in healthyindividuals and increased in patients with lung cancer. In this study, we found solublePD-L1could be found in supernatant of the culture with lung cancer cell line expressingPD-L1on cell surface and could inhibit T lymphocyte function via PD-1/PD-L1pathway,playing a negative regulatory role in cellular immunity. Using the self-developed specificsPD-L1ELISA kit, we observed the high expression level of sPD-L1in peripheral blood ofpatients with lung cancer and analyzed its relationship with tumor’s clinical phenotype soas to unravel the role of sPD-L1in development and progression of lung cancer. Inaddition, we also undertook the study if sPD-L1could serve as a parameter in clinicaldiagnosis, response prediction and prognosis evaluation. Furthermore, the mechanism of sPD-L1in regulating immune cells remains to be investigated so as to find out if PD-L1could be served as a target for immune intervention in clinical treatment of lung cancer inthe future.Part I The effect of soluble PD-L1expressed by lung cancer cellsthrough the biological function of PD-1/PD-L1pathway in regulating thefunctions of T lymphocyteObjective To explore the expression of soluble programmed death ligand-1on lungcancer cells and to clarify the biological function of PD-1/PD-L1pathway in regulating thefunctions of T lymphocyte.Medthods Both PD-L1expression on lung cancer cells and PD-1expression onhuman T cell were analyzed with labeled monoclonal antibody and flow cytometry.Thelevel of sPD-L1in the supernatant of lung cancer cells was determined by ELISA kit. Theinhibition of proliferation of T lymphocyte with mPD-L1and sPD-L1were studied usingCCK-8incorporation methods.Results Low or no expression of PD-1was found on resting T lymphocyte fromhuman peripheral blood with flow cytometry, but up-regulate PD-1was found on thesurface of activated T lymphocyte. Soluble PD-L1could be found in supernatant of theculture with lung cancer cell line expressing PD-L1on its cell surface. It was found T cellproliferation could be inhibited by co-culture with lung cancer cells expressing PD-L1.Using anti-human PD-L1blocking antibody to contradict the negative costimulatorysignals, the inhibition of T lymphocytes for proliferation could be partly restored.Supernatant of culture from PD-L1~+lung cancer also could inhibit T cell proliferationwhereas pre-removal of sPD-L1from the supernatant showed a weaken effect on thegrowth of T cells.Conclusion Lung cancer cells surface expressing membrane PD-L1can secretesoluble PD-L1, and both of them can inhibit the proliferation of T lymphocytes in mixedculture system and play a negative role on the cell-mediated immunity. This may impair the activation of tumor antigen-specific T cell to eradicate tumor cells timely, leading toimmune escape of lung cancer cells.Part II The level of soluble PD-L1in serum and its clinical implicationsin patients with lung cancerObjective To detect the expression serum level of soluble PD-L1in patients with lungcancer and explore its clinical implications.Methods Fifty-five male and twenty-six female lung cancer patients (mean age65±6, range34-87years) were selected from hospitalized patients at the Department ofRespiratory Diseases in The Second Affiliated Hospital of Soochow University from June2009to March2011. All lung cancer patients were newly diagnosed, treatment-na ve,confirmed by histopathology or cytopathology. Eight-eight healthy volunteers whichmatched in sex, age from Healthcare Center of the hospital were also enrolled as control.Each patient’s peripheral serum was collected and clinical data before and after treatmentrecorded and followed up. The sPD-L1protein expression in serum was determined bywestern-blot and self-developed ELISA kit. Statistical software was used to analyze thedata obtained. Labeled monoclonal antibody and cytometry were used to analyze changesin CD4~+PD-1~+T cell and CD8~+PD-1~+T cell and PD-L1on CD14~+monocytes in theperipheral blood of healthy controls and lung cancer patients.Results A higher level of sPD-L1level in patients with lung cancergroup(2.04±1.42)ng/ml，was found compared with control group(1.04±0.67) ng/ml，(P<0.001). High expression of sPD-L1in serum of lung cancer patients was closelycorrelated to lymph node metastasis and the presence of distant metastasis. Compared withhealthy individuals, PD-1expression levels on both CD4~+T and CD8~+T cells inperipheral blood of lung cancer patients were increased, and the level of PD-L1expressionon monocytes was also significantly increased. In the follow up of these patients, thedynamic changes of PD-1/PD-L1seemed to be correlated with the response to treatment.The expression level of PD-1on CD4~+T and CD8~+T cells remained high in lung cancer patients with objective response compared with healthy control, but was significantlylower than the average level of lung cancer patients. PD-1expression level was found toincrease further in the patients who had no response or even progressed during treatmentThe expression of PD-L1on monocyte surface we observed was similar.Conclusion The elevated expression level of sPD-L1in lung cancer patients and isclosely correlated with lung cancer staging, metastasis and clinical response. sPD-L1may become an important anti-tumor target in individualized treatment of lung cancer.Abnormal expression of PD-1and PD-L1existed in T cells and peripheral bloodmononuclear cells of lung cancer patients, prompting PD-1/PD-L1signal may inhibit Tcell proliferation during the interaction of T cells and monocytes, resulting in lung cancercells to escape T cell destruction and participating the development of lung cancer.To summarize, from the study, we can come to the following results:（1）SolublePD-L1could be found in supernatant of the culture with lung cancer cell line withexpressing PD-L1on cell surface. T cell proliferation can be inhibited by lung cancer cellsexpressing PD-L1. Using anti-human PD-L1blocking antibody to contradict the negativecostimulatory signals, the inhibition of T lymphocytes for proliferation could be partlyrestored. These phenomena signify membrane-bound PD-L1on the surface of tumor cellsmay convey negative costimulatory signal to T cells by binding PD-1.(2) Usingself-developed ELISA kit for sPD-L1, we found there exist high expression of sPD-L1inthe serum of lung cancer patients which is closely correlated with the TNM stage and thelymph node metastasis. Abnormal expression of PD-1and PD-L1on Lung cancerpatients’ T cells and peripheral blood mononuclear cells prompt PD-1/PD-L1signal mayinhibit T cell proliferation during the interaction of T cells and monocytes, resulting inlung cancer cells to escape T cell destruction, and participate in the development of lungcancer.