The Effect And Mechanism Of Soluble PD-L1 In The Immune Escape Of Lung Cancer Cells

With the rapid advancement of tumor molecular biology and genetics, it is generally recognized that the occurrence, progression, local invasion and distant metastasis of malignant tumor is associated with a series of complicated biological processes. Multiple regulatory genes were involved in this process with accumulative effect in a multi-step manner. In addition, the status of host immune function is closely. It has been observed there exist in patients with malignant tumor complicated heterogeneous microenvironment which promotes the predominance growth of some subpopulation of tumor cells phenotype of metastasis. It is still unclear how these tumor cells with high potential for metastasis manage to escape from the surveillance of human immune system and resist the cytotoxic effect of tumor antigen specific T lymphocyte. Therefore, it is important to develop novel molecular biological techniques for early diagnosis of lung cancer as well as to further our understanding on signal transduction pathways which play important roles in the growth, development and immune escape of lung cancer.Recent studies has found that abnormal expression of costimulatory molecules take part in immune imbalance of the host and closely involved in immune escape of tumor cells. Among the new members of CD28/B7 costimulatory superfamily, PD-1/PD-L1 can mediate negative costimulatory signals which inhibit the activation the proliferation of T and B lymphocyte as well as secretion of IL-2, IL-10 and IFN-γ. These negative immune regulatory pathways assume important position in tumor immunity, autoimmunity, virus infection and transplantation immunity.More other studies have demonstrated that many costimulatory molecules, e.g. . CD40, 4-1BBL, OX40L, CD86, CD80 and CTLA-4, have two isoforms, membrane-bound form and soluble form. Expression levels of soluble costimulatory molecules possess diagnostic and predictive potential in various diseases. PD-L1 expressed by tumor tissue may also assist in evaluating tumor response to therapeutics in clinical field. Up till now, there are few studies concerning serum sPD-L1 due to the lack of specific analytic method. It is still unknown and remains to be clarified if sPD-L1 exists in healthy individuals and increased in patients with lung cancer.In this study, tissue sample was collected from patient with lung squamous cell carcinoma(SCC) and SCC cell line was established by repeated subcloning and culture in vitro. Meanwhile, we found lung squamous cancer cell line highly expressed PD-L1 and could inhibit T lymphocyte function via PD-1/PD-L1 inhibitory pathway. Using the self-developed specific sPD-L1 ELISA kit, we observed the high expression level of sPD-L1 in peripheral blood of patients with lung cancer and analyzed its relationship with tumor’s clinical phenotype so as to unravel the role of sPD-L1 in occurrence and progression of lung cancer. In addition, we also undertook the study if sPD-L1 could be served as a parameter in clinical diagnosis, response predicting and prognosis evaluation. Furthermore, the mechanism of sPD-L1 in regulating immune cells remains to be investigated so as to find out if PD-L1 could be served as a target for immune intervention in clinical treatment of lung cancer in the future.Part I Establishment of human lung squamous cell carcinoma cell line and its interaction with T lymphocyte via PD-1/PD-L1 inhibitory pathwayObjective: To investigate the expression of PD-L1 on lung cancer cells and to clarify the biological function of PD-1/PD-L1 pathway in regulating the functions of T lymphocyte using human squamous lung carcinoma cells established by separating tumor cells from surgical resection specimen, subcloning and culture in vitro.Methods: Freshly resected tumor specimen obtained from pathologically confirmed patients with squamous lung carcinoma were ground and re-suspended into single cell suspension. After repeated subcloning and cultured in vitro, tumor cell line were established which could proliferate steadily. PD-L1 expression on these tumor cells were analyzed with labeled monoclonal antibody and flow cytometry. T lymphocytes from peripheral blood of human beings were collected using Ficoll density gradient centrifugation and cell purification kit. The inhibition of proliferation and activation of T lymphocyte with PD-L1 were studied using CCK-8 incorporation methods. Cytokines released by Th1 and Th2 were determined with ELISA method. The results were analyzed to probe the mechanism underlying immune escape of lung cancer cells.Results: After specicmen separation, subcloing, culture and screening in vitro, specific cell line which could proliferate steadily in vitro were established with tissue sample from lung squamous cell carcinoma patient. This cell line manifested all the characteristics of tumor cell in growth and morphology, which could survive and proliferate in vitro. PD-L1 were found to be expressed constitutively on human lung squamous cell carcinoma cell line. These tumor cells could inhibit the proliferation and activation of T lymphocytes via PD-l/PD-L1 pathway. It was also found SC cells expressing PD-L1 could inhibit the production of IL-2, IL-4, IL-10 and IFN-γ. With the use of anti-human PD-L1 blocking antibody to contradict the negative costimulatory signals, it was found the capacity of T lymphocytes for cytokine production could be partly restored.Conclusion: Establishment and verification of human lung squamous cell carcinoma cell line from resected human lung cancer tissue specimen is an initial step to prepare the object for this study. SC Cell line constitutively expressing PD-L1 will push forward the study to unravel in vitro how PD-1/PD-L1 pathway underlie the immune escape of human lung squamous cell carcinoma cells.Part II Increased serum level of sPD-L1 in serum in patients with lung cancer and its clinical significanceObjective: Using self-developed ELISA kit which is able to detect human sPD-L1 specifically, we investigated the existence and characteristics of sPD-L1 in serum patients with lung cancer. Expression level of sPD-L1 and its correlation with clinical phenotype of patients were studied and its clinical significance was discussed.Methods: sPD-L1 in serum from healthy volunteers as well as treatment na?ve lung cancer patients was analyzed using ELISA method with our previously developed sPD-L1 detection kit. sPD-L1 concentration from malignant pleural effusion was also determined in some of the above-mentioned cases using the same method. Western-blot analysis was used to quantify sPD-L1 protein in peripheral blood. Statistical method was employed to analyze the diagnostic and prognostic value of serum sPD-L1 and its correlation with response to treatment over time and 1-year survival rate. Labeled monoclonal antibody and cytometry were used to analyze changes in lymphocyte subsets in the peripheral blood of healthy controls and lung cancer patients. The role of sPD-L1 in the regulation of PD-1/PD-L1 signal pathway was explored.Results: ELISA study showed sPD-L1 expression existed in serum of healthy controls. A higher level of sPD-L1 level was found in patients with lung cancer group compared with control group. Statistical studies found the abnormally increased level of sPD-L1 was closely correlated with clinical staging, lymph node and distal metastasis, which indicating its relationship with progression of lung cancer. sPD-L1 in malignant pleural effusion was found to be increased compared with corresponding serum concentration. In the follow up of these patients, the dynamic changes of serum sPD-L1 seemed to be correlated with serum tumor markers and could reflect object response to chemotherapy according to RECIST criteria. A prolonged PFS and increased 1-year survival rate were observed in lung cancer patients without elevated serum sPD-L1 concentration. sPD-L1 possessed the efficiency in evaluating the potential of immune escape similar to that in evaluating tumor burden for other tumor markers. In addition, abnormal changes of T and B lymphocytes and their subsets were noticed, in which, the percentage of CD4+PD-1+ T lymphocyte and CD8+PD-1+ T lymphocyte was increased. The percentage of CD8+PD-1+ T lymphocyte was significantly higher than that in the control group, which signified impairment in the function of tumor antigen specific killer cells.Conclusion: The abnormally increased serum sPD-L1 in patients with lung cancer participates in the negative costimulatory signals transduction mediated via PD-1/PD-L1. sPD-L1 may play an important role in local and systemic tumor immune toleration and escape. Serum sPD-L1 is closely correlated in the occurrence, progression and prognosis of lung cancer and has predictive value for treatment and prognosis.Part III The effect of sPD-L1 in regulating CD28/B7.1 pathway and its biological significance in the immune escape of lung cancer cellObjective: To investigate the effect of sPD-L1 on the interaction between PD-1/PD-L1 and CD28/B7.1 pathway as well as its regulation on cellular immune function of T lymphocyte.Methods: Immunofluorescence labeling techniques was employed to study the CD28 and PD-1 expression on resting and activated T lymphocyte. Transfected L929/PD-1 and L929/B7.1 together with PD-L1Ig and CD28 Ig were used with indirect immunofluorescence and flow cytometry to determine the binding affinity of PD-L1Ig with membrane molecule PD-1 and B7.1. Also, soluble form and stationary phase PD-L1 were studied on their synergistic stimulating effect of T lymphocyte. ELISA method were used to find the level of soluble PD-L1 in supernatant of the culture with lung cancer cell. Effect on T cell proliferation was analyzed by WST (CCK-8) incorporation method.Results: Constitutive expression of CD28 and low or no expression of PD-1 was found on resting T lymphocyte from human peripheral blood with flow cytometry. Up-regulate PD-1 and slightly down-regulated CD28 were found on the surface of activated T lymphocyte. The direct ELISA study showed PD-L1Ig could bind specifically with tranfected L929/B7.1 cells with weakened binding force compared with that between CD28/B7.1. Soluble PD-L1Ig protein could promote proliferation of T lymphocytes. PD-L1Ig pre-coated on culture plate inhibited the growth of the cell and this effect could be attenuated with blocking anti-human PD-L1 monoclonal antibody. It was also found lung cancer cell lines expressing membrane-bound PD-L1 could generate soluble PD-L1 as well. No soluble sPD-L1 could be found in supernatant of the culture with lung cancer cell line without expressing PD-L1 on cell surface. Supernatant of culture from PD-L1+ lung cancer could inhibit T cell proliferation whereas pre-removal of sPD-L1 from supernatant showed a weaken effect on the growth of T cells.Conclusion: The large amount of sPD-L1 generated during the proliferation of cancer cells may take part in the regulation of CD28/B7.1 stimulatory signal transduction. The binding of sPD-L1 with B7.1 result in inaccessible of B7.1 stimulatory signal for resting T cells which constitutively expressing CD28 molecule. This will further lead to inactivation of tumor antigen specific T lymphocyte and immune escape of lung cancer cells.To summarize, this study has demonstrated the following results: (1) Tumor cell line derived from surgical resection specimen of lung squamous cell carcinoma could be established with isolation, subcloning screening and culture. This lung cancer cell line, as well as commercial lung squamous cell carcinoma cell line, high expression of PD-L1 present which could inhibit the activation, proliferation and cytokine secretion of T lymphocytes in mixed culture system. The PD-L1 blocking antibody could partly reverse the capacity of cytokine production by T cell. These phenomena signify membrane-bound PD-L1 on the surface of tumor cells may convey negative costimulatory signal to T cells by binding PD-1. (2) Using self-developed ELISA kit for sPD-L1, we found there exist high expression of sPD-L1 in the serum of lung cancer patients which is closely correlated with burden and extent of the tumor. In addition, level of sPD-L1 in tumor environment such as malignant pleural was observed to increase compared with serum concentration correspondingly. Follow up studies found that the expression of sPD-L1 was negatively correlated with clinical response. Lung cancer group with high sPD-L1 expression exhibited longer progression free survival (PFS) and higher 1-year survival rate, indicating better prognosis. (3) In studying the mechanism underlying sPD-L1 mediated negative signal transduction, we found human sPD-L1 protein could bind B7.1 protein and exert different biological effect as membrane-bound PD-L1. sPD-L1 may inhibit the proliferation of T lymphocytes by blocking CD28/B7.1 stimulatory signal pathway. Abundant sPD-L1 generated during culture in vitro inhibit the activation and proliferation of T lymphocyte.This study indicates that soluble PD-L1 exist in cancer patients and exert negative regulation on cellular immunity by inhibiting T cell proliferation and activation similar to membrane-bound PD-L1. Soluble PD-L1 reflects the scale of tumor immune escape both locally and systemically. Our investigation demonstrated sPD-L1 is correlated with clinical staging, response to treatment and length of survival, indicating its potential as a potent molecular target or marker in developing future treatment against cancer. Therefore, tumor target therapy via specifically blockage of the inhibitory PD-1/PD-L1 pathway is expected.