The Role Of Soluble PD-1 And PD-L1 In The Regulation Of PD-1/PD-L1 Inhibitory Pathway And Their Biological Significance

According to the two-signal model, naive T cells require two necessary signals to become fully activated. Costimulatory signals play important roles in the regulation of immune response at different steps of initiation, effection and termination. It is well established that engagement of PD-1/PD-L1 delivers a potent coinhibitory signal to T cells, resulting in T cell exhaustion and dysfunction timely to prevent excessive immune injury and maintain self-balance of the immune system. It has been reported that PD-1/PD-L1 pathway plays a critical role in tumor immunity, autoimmune, transplantation and chronic viral infectious diseases.Programmed death-1 (PD-1, CD279), a negative costimulatory molecule, is a type I glycoprotein and is inducibly expressed on T cells, B cells, natural killer T cells and activated monocytes. The intracellular domain of PD-1 contains two tyrosine-based signaling motifs (ITIM and ITSM), which can recruit the phosphatases SHP-1 and SHP-2. The two PD-1 ligands, PD-L1 (CD274) and PD-L2 (CD273), are structurally related to the B7 superfamily. The PD-Ls have different expression patterns and distinct binding properties to PD-1. PD-L1 is constitutively expressed on a wide variety of immunocytes and nonhematopoietic cell types. However, the expression of PD-L2 is mainly restricted on dendritic cells (DCs), macrophages and bone marrow-derived mast cells. Engagement of PD-1 by its two ligands down-regulates T cell activation and cytokines secretion.It has been reported that PD-1/PD-L pathway is closely associated with many kinds of clinical diseases. Deletion or down-regulation of PD-1 negative signal can result in many autoimmune diseases such as experimental autoimmune encephalitis (EAE), multiple sclerosis and rheumatoid arthritis. Conversely, enhanced PD-1 inhibitory signal contributes to the development of many tumor disease and chronic virus infectious disease. Thus, in view of its critical function in regulating immune homostasis, PD-1 signaling has aroused great attention in immunodiagnosis and therapy.Mounting data demonstrated that many costimulatory molecules assume two forms of expression. Except membrane-bound forms, soluble forms have been found for several members of B7 family including CD28, CTLA-4, CD80, CD86 and B7-H3. The soluble protein is either shed from the membrane or produced by the special mRNA. Many soluble proteins are valuable for clinical diagnosis. However, the functions and biological significance of sPD-1, sPD-L1 and sPD-L2 remain unknown.In this study, we intend to generate and characterize monoclonal antibodies against human PD-1, PD-L1 or PD-L2. Then the mAbs were used to develope three sandwich enzyme-linked immunosorbent assay (ELISA) systems for the detection and quantification of sPD-1, sPD-L1 and sPD-L2. The levels of sPD-1 and sPD-L1 were determined by the established ELISA. The production of sPD-1 and sPD-L1 from different immunocytes were evaluated and characterized. The expression mechanisms of the soluble factors were also studied in this work. Moreover, the study focus on the roles of sPD-1 and sPD-L1 in the regulation of PD-1/PD-L1 inhibitory pathway and their biological significance in autoimmune disease and lung cancer.Part ? Generation and characterization of monoclonal antibodies against human PD-1 or PD-L1Objective: To prepare mouse anti-human PD-1 and PD-L1 monoclonal antibodies for the study of human PD-1/PD-L1 inhibitory pathway.Methods: BALB/c mice were immunized with L929/PD-1 or L929/PD-L1 transfectants respectively. The splenocytes of immunized mice and the SP2/0 cells were fused with PEG1500. The monoclonal antibodies produced by the hybridomas were screened by flow cytometry or ELISA. After multiple screening and subcloning, several monoclonal antibodies against human PD-1 or PD-L1 were generated. Fast-strip analysis was performed to identify Ig subclass of the antibodies. The specificities of the antibodies were confirmed by immunostaining and Western blot. The antigen epitopes recognized by the mAbs were analyzed by competition assays. The expression patterns of PD-1 and PD-L1 on different human cell lines were determined with the obtained mAbs by immunostaining and flow cytometry. The antibody-mediated functions on human T cells or DCs were studied in vitro.Results: Four novel antibodies against human PD-1, named as 9H1,4B9,1F8 and 8F5, were generated and characterized successfully. The results of flow cytometry showed that the mAbs could bind specially to human PD-1 protein. The mAbs could be applied to FACS and Western blot study. In addition, 4B9 and 9H1 mAbs could be used for ELISA detection. The results of competitive inhibition assays indicated that the four anti-PD-1 mAbs recognized at least three new epitopes of PD-1. It was found that 9H1 could completely block the interaction of PD-1/PD-Ls. The functional 9H1 mAb could block the inhibitory signal of PD-1/PD-Ls and promote T cell proliferation with a dose-dependent manner. A specific mouse anti-human PD-L1 monoclonal antibody, named as 10D7, was obtained after being screened by ELISA. The 10D7 mAb recognizes a novel epitope of PD-L1 and can be applied to FACS, Western blot, ELISA and IP.Conclusion: The generation of monoclonal antibodies with distinct characteristics against human PD-1,PD-L1 and PD-L2 provide essential reagents to the study of PD-1/PD-L inhibitory pathway.PartПDevelopment of two sandwich ELISA systems for evaluating human soluble PD-1 and PD-L1Objective: To establish specific ELISA systems with high sensitivity to detect human soluble PD-1 and PD-L1 proteins respectively.Methods: Mouse anti-human PD-1 mAb (4B9), used as coating antibody, was precoated in the ELISA plate with the CBS. Another biotin-anti-PD-1 mAb (9H1) was used as detecting antibody to recognize the 4B9-bound soluble PD-1 protein. Then the Streptavidin-HRP was added to the reaction system. Finally, TMB substrate was added for colorable reaction. The absorbance was measured by the microplate reader. With the same methods, the sandwich sPD-L1 ELISA was developed with the anti-human PD-L1 mAb (2H11) as coating antibody and biotin-anti-human PD-L1 mAb (10D7) as detecting antibody.Results: The results showed that the ELISA system for detecting human sPD-1 was established successfully. It could be used to detect human sPD-1 protein specially without any cross-reaction with other proteins. Standard curve of the system displayed favorable linear correlation when the concentration of sPD-1 was 1.56-100ng/ml. The ELISA system had favorable stability, precision and specificity, and the variations (CV%) were <6.24% and <8% respectively. Another ELISA system was also developed successfully to detect human sPD-L1 protein. The detection threshold was 0.39-25ng/ml, and the sPD-L1 ELISA system had favorable stability and precision.Conclusion: Two ELISA systems with high specificity and sensitivity for detecting human sPD-1 and sPD-L1 provide useful methods to study these soluble factors in the regulation of PD-1/PD-L1 inhibitory pathway.PartШThe characterization of sPD-1 and the role of this factor in the regulation of PD-1/PD-L1 pathway as well as the biological significance in autoimmune diseasesObjective: To study sPD-1 expression levels and characteristics in human serum, reveal the generation mechanism of sPD-1, and evaluate the biological significance of sPD-1 in the patients with autoimmune disease.Methods: The expression level of sPD-1 in human serum was analyzed by the established sPD-1 ELISA. Human T cells were stimulated with anti-CD3 mAb or PHA in vitro, and then the mPD-1 expression and the sPD-L1 production were determined by flow cytometry and the ELISA. Human flPD-1, PD-1Δex3 and sPD-1 genes were cloned with RT-PCR and TP-PCR, then the recombinant eukaryotic expression vectors pIRES2-EGFP/flPD-1, pIRES2-EGFP/PD-1Δex3 and pIRES2-EGFP/sPD-1 were constructed respectively, the target genes were expressed transiently in the 293T cells by Lipfect2000 transfection. The cells were collected at indicated times and the mPD-1 expression were analyzed by flow cytometry. Meanwhile, the sPD-1 production in the supernatants were determined by the ELISA. Furthermore, PD-1 intracellular peptide was used to immune New Zealand rabbit to generate special anti-human PD-1 polyclonal antibody. sPD-1 protein in human serum was immunoprecipitated and identified by Western blot, using the rabbit anti-human PD-1 polyclonal antibody. The binding ability of natural sPD-1 to PD-Ls was analyzed by the competitive inhibition assays. The level of sPD-1 in the serum of patients with autoimmune disease (Graves', RA and HSP) was determined quantitatively and qualitatively by the sPD-1 ELISA and Western blot.Results: The results showed that sPD-1 exists in human serum. Both mPD-1 expression and sPD-1 production of human T cells stimulated with anti-CD3 mAb or PHA were markedly upregulated. Human PD-1Δex3 gene and recombinant expression vectors were constructed successfully, the results of immunostaining showed that the 293T cells transfeced with flPD-1 could highly express mPD-1, while the cells transfected with PD-1Δex3 or sPD-1 genes had no PD-1 protein on the cell surface. As expected, the supernatants of PD-1Δex3 and sPD-1 transfected cells contained high levels of sPD-1 protein. PD-1Δex3 protein can effectively block PD-1/PD-L interaction. The results of Western blot indicated that the rabbit anti-human PD-1 polyclonal antibody could recognize the sPD-1 protein from human serum. It suggested that sPD-1 in human serum was encoded by the PD-1Δex3 gene. Furthermore, the data showed that the level of sPD-1 in the serum of patients with autoimmune disease increased markedly than the healthy donors.Conclusion: This work lay a foundation for further explore of sPD-1 in blocking the PD-1/PD-L inhibitory signal and the biological significance of this soluble factor in autoimmune disease. Part IV Characterization of sPD-L1 and the role of this factor in the regulation of PD-1/PD-L1 pathway as well as the biologicalsignificance in tumor immune escapeObjective: To further explore the existence of sPD-L1 and evaluate the physiological and pathological significance of this circulating factor in human serum.Methods: Human serum from different years old volunteers were collected and detected by the established sPD-L1 ELISA. The monocyte-drived dendritic cells were incuced by IL-4 plus GM-CSF and stimulated by anti-CD40 mAb, LPS or TNF-α, the mPD-L1 and sPD-L1 were analysised by flow cytometry and sPD-L1 ELISA respectively. Different human cell lines were cultured and the mPD-L1/sPD-L1 were matched to analysis by the flow cytometry and ELISA. The sPD-L1 protein bands were determined by immunoprecipitation and Western blot. The matrix metalloproteinase inhibitor (MMPI) was added to the culture medium of L929/PD-L1 and Mo-DCs to evaluate the production of sPD-L1. The coinhibition of sPD-L1 on T cell proliferation and activation in intro were analysised with CCK-8 and PE-anti-CD25 mAb. The level of sPD-L1 in lung cancer patients was also detected by the ELISA system, and the results together with the clinical data were statistically analysised by the GraphPad Prism 5.0.Results: The sPD-L1 protein is detectable in human serum and the concentration increases in an age-dependent manner. The monocyte-drived dendritic cells release abundant sPD-L1 during the induction of maturation. sPD-L1 is mainly produced by the mPD-L1+ cell lines and the sPD-L1 level in the supernatants of mPD-L1- cells below the detection limit. Human sPD-L1 has a unique protein form in the serum and matrix metalloproteinase inhibitor (MMPI) could suppress sPD-L1 production. The sPD-L1 could specially bind to its receptor PD-1 and coinhibit the proliferation and activation of T cells stimulated with anti-CD3 mAb. Furthermore, sPD-L1 is highly expressed in the serum of lung cancer patients, and the level of sPD-L1 is closely associated with node metastasis. Conclusion: These results demonstrate that the existence of circulating sPD-L1 in human serum might play a potentially important role in immune tolerance.In summary, this paper has successfully accomplished the investigations as follows: Four novel mAbs against human PD-1 and a specific anti-human PD-L1 mAb were generated after being screened by flow cytometry or ELISA. Two ELISA systems were also developed successfully to detect human sPD-1 and sPD-L1 proteins respectively. The results of ELISA showed that sPD-1 exists in human serum, and the soluble protein was produced by the PD-1Δex3 mRNA. Interestingly, it was found that the level of sPD-1 in the serum of patients with autoimmune disease increased markedly than the healthy donors. Functional sPD-L1 protein was detectable in human serum and the concentration increased in an age-dependent manner. sPD-L1 is released from the mPD-L1+ cell membrane with the contribution of matrix metalloproteinases. Importantly, sPD-L1 is highly expressed in the serum of lung cancer patients, and the level of sPD-L1 is closely associated with node metastasis.