| Objective: Lung cancer is one of the malignant tumors worldwidely,and small cell lung cancer is the most aggressive type of lung cancer.Although it is more sensitive to chemotherapy and radiotherapy,only a minor percentage of SCLC patients reach complete response and they are more likely to experience recurrence with a poor prognosis.Due to lacking an effective treatment,specific driving gene or pathogenesis,although there has been made too much efforts in the studies of small cell lung cancer,there were no important therapeutic clinical advances for 30 years,leading SCLC to be designated a intractable cancer.At present,anti-tumor immunotherapy,especially immune checkpoint inhibitors,have played an vital role in melanoma,non-small cell lung cancer and so on.However,the studies about immune checkpoints in SCLC were less.Our study based on the specific immune chekpoints which influencing the survival of SCLC patients,and studied the relationship between the expression of the specific immune checkpoints and IFN-γ,we also detected the functions of cytotoxic CD8+T lymphocytes with anti-CD155 or/and anti-PD-L1.In this way,this study could broden the knowledge of the immune checkpoints expressed on SCLC and provided new viewpoint in appling immune checkpoint inhibitors in anti-tumor therapy.Methods: 1.The survival analysis of PD-1,PD-L1,CD155,TIGIT and Galectin-9 in SCLC patients.We collected 48 SCLC patients with their OS and clinicopathological datas.We deteced the expression of PD-1,PD-L1,CD155,TIGIT and Galectin-9 by IHC,and studied the survival curves with high/low expression of the immune checkpoints by Kaplan-Meier(Log Rank).We also studied the expression of PD-1 and TIGIT of tumor infiltrated CD8+T lymphocytes by double fluorescent staining.2.Whether IFN-γ or LPS could induce the immune checkpoints up-regulate.We studied the expression of PD-L1,CD155 and Galectin-9 with IFN-γ or LPS stimulating on SCLC cell lines,NCI-H446 and NCI-H1668 by qRT-PCR,Western Blot,IHC and IF.Then,we studied the expression of MAPK pathway,NF-κB pathway,PI3K-AKT-mTOR and TLR-4/MyD88/TRAF-6 with IFN-γ or LPS stimulating by qRT-PCR and Western Blot.We next transfection cDNA and ShRNA of huamn MyD88 to increase or decrease the expression of MyD88,and anlysized the expression of the immnue checkpoints in different groups by Western Blot.3.The correlationship among IFN-γ,the number of CD8+T lymphocytes,and the expression of the immune checkpoints.We firstly studied the expression of IFN-γ and CD8 in the tumor tissue of 48 SCLC patients by IHC.And we respectively analysed the relationship between the expression of IFN-γ and the immune checkpoints,and the relationship between the expression of IFN-γ and the number of CD8+T lymphocytes by Pearson correlationship analysis.We next studied the survival analysis of IFN-γ in SCLC patients by Kaplan-Meier analysis.4.The influence on fuctions of CD8+T lymphocytes with anti-PD-L1 and/or anti-CD155.We co-cultured SCLC cell and CD8+T lymphocytes with or without anti-PD-L1 and/or anti-CD155.By flow cytometry,we studied the proliferation ability of CD8+T lymphocytes with CFSE fluorescence strength and the cytotoxic ability with apoptotoc ratio of tumor cells.Results: 1.The expression of PD-L1,CD155 and Galectin-9 were risk factors of SCLC patients.High-expressed PD-L1,CD155 and Galectin-9 patients had shorted OS than low-expressed patients,respectively.SCLC patients with high-expressed PD-L1 and CD155 had shorter OS than SCLC patients with PD-L1 or CD155 expression,and SCLC patients with low-expressed PD-L1 and CD155 had longest OS(P<0.001).However,there were no obvious relationship between the expression of PD-1 or TIGIT with the OS of SCLC patients.2.Both PD-1 and TIGIT were expressed on CD8+T lymphocytes in SCLC tissue.3.IFN-γ could induce the up-regulation of PD-L1,CD155 and Galectin-9 on SCLC cell lines.We found the expressions of the immune checkpoints,PD-L1,CD155 and Galectin-9,upregulated as elevated concentration of IFN-γ.However,the expression of the immune checkpoints decreased when the concentration of IFN-γ exceeded.PD-L1 and CD155 could up-regulated as the concentration of IFN-γ elevated at the same time.4.IFN-γ could active TLR-4 and downstream pathways as LPS,in the process of PD-L1,CD155 and Galectin-9 up-regulation.Based on the fact that IFN-γ could induce the up-regulation of PD-L1,CD155 and Galectin-9 of SCLC cell lines,we further analysed the pathways activated in this process.We found that LPS,the classical TLR-4 activator,also could up-regulate the expression of the immune checkpoints.MAPK pathway,NF-κB pathway,PI3K-AKT-mTOR and TLR-4/MyD88/TRAF-6 were up-regulated as IFN-γ or LPS elevated.In this way,we thought that IFN-γ could actived TLR-4 and downstream pathways as LPS,in the process of PD-L1,CD155 and Galectin-9 up-regulation.5.MyD88 was the key signal transductor in the process of PD-L1,CD155 and Galectin-9 up-regulation,regardless of IFN-γ or LPS stimulating.We found MyD88,one of classical signalling transduction molecules activated by LPS,also participated in the process with stimulating by IFN-γ.By comparing the expression of the immune checkpoints in the groups of IFN-γ stimulating,LPS stimulating,up-regulating MyD88,down-regulating MyD88,and down-regulating MyD88 with IFN-γ or LPS stimulating.We found the immune checkpoints also upregulaed when up-regulating MyD88,though the expression less than IFN-γ or LPS stimulating,and down-regulating MyD88 with IFN-γ or LPS stimulating could decreased the up-regulation of the immune checkpoints stimulated by IFN-γ or LPS.Therefore,we thought MyD88 was the key signal transductor in the process of PD-L1,CD155 and Galectin-9 up-regulation,regardless of IFN-γ or LPS stimulating.6.The correlationship among IFN-γ,the number of CD8+T lymphocytes,and the expression of the immune checkpoints.We found that high expression IFN-γ related to low expressed immune checkpoints and more accumulated CD8+T lymphocytes,we also found high concentrated IFN-γ could induce more tumor cell apoptosis.In this way,we believed that low concentration IFN-γ induced high-expression of the immune checkpoints to evade immune surveillance,with less apoptotic tumor cells and less cytotoxic T lymphocytes.High concentration IFN-γ suppresses expression of the immune checkpoints to assist immunological recognition,with more apoptotic tumor cells and more cytotoxic T lymphocytes.We believe these may be some of the reasons that the OS of high IFN-γ patients were longer than low IFN-γ patients.7.The influence on fuctions of CD8+T lymphocytes with anti-PD-L1 and/or anti-CD155.We found that the proliferation ability and cytotoxic function of CD8+T lymphocytes obviously increased when blocking both CD155 and PD-L1 rather anti-CD155 itself.Conclusion: 1.High-expressed PD-L1,CD155 and Galectin-9 SCLC patients had shorter OS than low-expressed patients,respectively.2.Both PD-1 and TIGIT were expressed on CD8+T lymphocytes in SCLC tissue.3.IFN-γ could induce the up-regulation of PD-L1,CD155 and Galectin-9 on SCLC cell lines.4.IFN-γ could active TLR-4 and downstream pathways(MAPK pathway,NF-κB pathway and PI3K-AKT-mTOR)as LPS,in the process of PD-L1,CD155 and Galectin-9 up-regulation.And MyD88 was the key signal transductor in these processes.5.High concentration IFN-γ suppressed the expression of the immune checkpoints to assist immunological recognition,with more apoptotic tumor cells and more cytotoxic T lymphocytes.6.Combination blocking CD155 and PD-L1,could increase the prolifertion and cytotoxic ability of CD8+T lymphocytes. |
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