| Bretscher and Cohn proposed "co-stimulatory signal" based on two-signal in T cell activation model. The absence of costimulatory molecules providing the second signal will result in T cell anergy or apoptosis of specific immune tolerance even. Positive and negative regulation of costimulation and the balance between the immune response in the whole process play an important regulatory role. PD-1/PD-L1 is one of important negative costimulatory molecules. Human PD-L1 gene located at chromosome 9p24.2, encoding 290 amino acid residues, belonging to I-transmembrane protein. PD-1/PD-L signal to play on the proliferation of lymphocytes in negative regulation, which prevents excessive immune injury and autoimmune disease, help maintain the immune homeostasis. PD-L1 constitutively expressed in monocytes / macrophages and dendritic cells (DC), which was upregulated after activation. Meanwhile, a large number of studies confirm that a variety of tumor cell membrane and cytoplasm both expressed PD-L1. PD-Ll on the surface of tumor cells play an important role in the process of apoptosis of tumor cells to induce specific CTL. PD-1/PD-L1 pathway is closely related to immune escape of tumor cells. Blocking this pathway can enhance the body's anti-tumor immune response. Therefore, PD-L1 molecules become hot in immunology in recent years, which is expected to become an effective immune intervention target molecule to cancer, autoimmune diseases and viral infectious diseases.In our study, a stable human PD-L1 transfected cell line L929/PD-L1 was used as an antigen to immunize BALB/c mice. By means of the cell fusion technique, multiple cell subcloning and repeated screening with L929/PD-L1 as target cells while L929/mock was used as the negative control, the hybridomas specifically secreting mouse anti-human PD-L1 monoclonal antibody were generated, named as 11G8,2G5 and 5C3. Western-blotting analysis showed that three antibodies can specifically recognize the proteins of L929/PD-L1 cells, but can not recognize L929/mock. Antigenic sites competitive inhibition experiment suggested that three monoclonal antibodies and commercialization of anti-PD-L1-PE mAb recognize different antigen binding sites. Biological functions of the three antibody findings suggest that three antibodies were able to identify activated T cells expressing PD-L1 molecules, and in vitro can significantly promote the T cell proliferation.Use of independently developed sPD-L1 ELISA detection system detected the soluble PD-L1 expression in human peripheral blood, peripheral blood and pleural effusion of lung cancer serum. The results showed that the sPD-L1 expression of lung cancer patients was significantly higher than that of healthy volunteers. Expression level of sPD-L1 in pleural effusion in patients with lung cancer is higher than that in peripheral blood of patients with lung cancer lung cancer patients. Age, sex and lung cancer I-III,IV in peripheral blood of patients with lung cancer,sPD-L1 had no effect on expression. The sPD-L1 expression of adenocarcinoma and squamous cell carcinoma patients was significantly higher than that of small cell carcinoma patients. The abnormal high expression of sPD-L1 in the peripheral blood of lung cancer patients showed that the factor further depressed the immune function, so that antigen-specific PD-1+ T cells disabled. Therefore, the abnormal high expression of sPD-L1 participated the regulation of PD-1/PD-L1inhibitory pathway, increasing the complexity and multi-faceted of tumor cell immune escape.In summary, our study successfully developed three specific mouse anti-human PD-L1 functional monoclonal antibody. And the three monoclonal antibodies can be used for FACS, Western blot and ELISA, etc., and can promote T cell proliferation. On analysis of human soluble PD-L1 expression in peripheral blood, peripheral blood and pleural effusion of lung cancer serum showed that sPD-L1 in the peripheral blood of untreated patients with lung cancer was highly expressed. |
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