The Level Of Soluble Programmed Death1Ligand1in Tuberculous Pleural Effusion And Its Biological Implications

Tuberculosis is a chronic epidemic disease which has been doing long-term seriousharms to the human health and it is among one of the high-mortality infectious diseases.Tuberculous pleurisy is a clinical phenotype of extrapulmonary tuberculosis, accountingfor about10%-20%of patients with pulmonary tuberculosis, of which about10%-30%ofthe patients have the manifestation of pleural effusion. Tuberculous pleural effusion is oneof the common types of exudative pleural effusion in which Th1cell-mediated immuneresponses play an important role. However, some studies showed that Th2, Th9, Th17,Th22immune cell subsets and the costimulatory molecules also paly an important role inthe occurrence and development of tuberculous pleurisy. Costimulatory molecule PD-L1(Programmed death ligand1, also named B7-H1) was highly expressed on activated Tlymphocytes, B lymphocytes, monocyte-macrophages and other immune cell subsets,which was correlated with the immune suppression of the host. It has been demonstratedthat expressions of PD-1and PD-L1in peripheral blood were higher in patients withpulmonary tuberculosis and they are correlated with the immune suppression and chronicinflammation in tuberculosis. It has been demonstrated that many costimulatory molecules,such as OX40, OX40L, B7-H3and CTLA-4, have two isoforms, membrane-bound formand the soluble form. Our previous data have showed the existence of circulating solubleform of PD-L1(sPD-L1) in human serum and we got the idea that the level of sPD-L1inpatients with lung cancer was closely related to lung cancer staging, metastasis and clinicalresponse to treatment. However, whether sPD-L1exists in pleural effusion and the role ofsPD-L1in pleural effusion remain unknown.In this study, we observed the expression of sPD-L1in patients with tuberculouspleural effusion by using the self-developed specific sPD-L1enzyme linkedimmunosorbent assay (ELISA) kit and its clinical implications in the process werediscussed. In this study, we revealed the potential relation of sPD-L1and PD-1/PD-L1 pathway in tuberculous pleural effusion. In addition, we explored the biological function ofsPD-L1in tuberculous pleural effusion and provide evidence for applying newimmunological method of intervention in the treatment of tuberculous pleural effusion.PartIThelevelofsolubleprogrammeddeathligand1inpleuraleffusionandperipheralbloodofpatientswithtuberculouspleurisyanditsclinical implicationsObjective To analyze the level of soluble programmed death ligand1(sPD-L1) inpleural effusion and peripheral blood of patients with tuberculous pleural effusion andexplore its clinical implications.Methods Sixty-eight patients with newly diagnosed pleural effusion in ward ofrespiratory diseases of the Second Affiliated Hospital of Soochow University from June2012to March2013were enrolled. Of the68patients,24were diagnosed as tuberculouspleural effusion (TPE),30were determined as malignant pleural effusion (MPE) and14were classified as non-tuberculous non-malignant (non-TPE non-MPE). Samples of pleuraleffusion and peripheral blood were collected and the clinical data was recorded. The levelof sPD-L1in the supernatant of pleural effusion and peripheral blood were analyzed usingELISA kit. The immune cell subsets of CD4+, CD8+, CD4+-PD-1, CD8+-PD-1, CD14+monocytes and CD14+-PD-L1monocytes were analyzed by flow cytometry. The level ofPD-L1and MMP-3were detected by reverse transcription PCR analysis in pleuraleffusions of different etiology. Receiver operating characteristic (ROC) curve was used toanalyze the diagnostic value of sPD-L1in tuberculous pleural effusion.Results The level of sPD-L1in tuberculous pleural effusion was higher than in MPEand non-TPE non-MPE (P<0.0001). Elevated sPD-L1level in pleural effusion was foundcompared with in peripheral blood for the same patient (P=0.0033). The proportion ofCD8+subset and CD14+-PD-L1monocytes in TPE was higher than in MPE and non-TPEnon-MPE (P=0.0001, P<0.0001). The expression of PD-L1was up-regulated in TPE atmRNA level and it was positively correlated with the expression of MMP-3(r=0.887,P<0.0001). Receiver operating characteristic (ROC) curve analysis showed that sPD-L1had a sensitivity of82.6%, specificity of82.9%and AUC of0.840for differentialdiagnosis of TPE from others. Conclusion Levels of sPD-L1differ in pleural effusions with various etiologies. Theexpression of sPD-L1is higher in tuberculous pleural effusion and it is possibly related tothe occurrence and development of the disease. Detection of sPD-L1alone might benefitthe diagnosis of TPE.Part II The expressionof IFN-γ andIFN-intuberculous pleural effusionandthe immunologicalfunction of PD-1/PD-L1pathway in tuberculous pleurisyObjective To detect the level of IFN-γ and IFN-in tuberculous pleural effusion andanalyze their clinical implications. To explore the immunological function of PD-1/PD-L1pathway in tuberculous pleurisy.Methods The expression of IFN-γ and IFN-in pleural effusions which werecollected from68patients was detected by ELISA kits. Peripheral blood mononuclear cells(PBMCs) were obtained from healthy donors by Ficoll-Hypaque density centrifugation.Different concentrations of tuberculous pleural effusions were co-cultured with PBMCsand changes of PD-L1on CD14+monocytes were evaluated by flow cytometry, theproliferation of PBMCs was detected by CCK-8kit. T lymphocytes and CD14+monocyteswere separated by using immunomagnetic beads. The proliferation of T cells was detectedby CCK-8kit.Results The level of IFN-γ in tuberculous pleural effusion was higher than in MPEand non-TPE non-MPE (P<0.0001). However, there was no difference in the level ofIFN-in pleural effusions (P>0.05). The level of sPD-L1in pleural effusion waspositively correlated to IFN-γ and adenosine deaminase (ADA) based on Spearman’scorrelation result (P=0.0004, P<0.0001). A combination of sPD-L1, CD14+-PD-L1, IFN-γand ADA showed an AUC of0.981with sensitivity87%and specificity100%by using amultivariate analysis in differenating tuberculous pleural effusion. High expression ofPD-L1on monocytes in pleural effusions could bind to PD-1on T cells to inhibit theproliferation of T cells via PD-1/PD-L1pathway. But this effect could be partly reversedby blocking PD-1/PD-L1pathway with anti-PD-L1antibody.Conclusion The expression of IFN-γ is higher in tuberculous pleural effusion and it ispositively correlated to the expression of sPD-L1in pleural effusions. Detection of sPD-L1,IFN-γ and other clinical biomarkers might benefit the diagnosis of TPE. High-expression of Th1cell cytokine IFN-γ is possibly correlated to the immunological function ofPD-1/PD-L1pathway in tuberculous pleurisy.In summary, we can arrive to the following results in this study:(1) Expression ofsPD-L1, Th1cell cytokine IFN-γ, CD8+subset and CD14+-PD-L1monocytes were higherin tuberculous pleural effusion and sPD-L1alone or combined with other clinicalbiomarkers could benefit the diagnosis of tuberculous pleural effusion.(2) Tuberculouspleural effusion may increase the expression of PD-L1on CD14+monocytes and theproliferation of PBMCs in vitro. In this way, high-expression of PD-L1could bind to thereceptor PD-1on T cells to induce the anergy of T cells and it is possibly related to theoccurrence and development of tuberculous pleural effusion.(3) Blocking PD-1/PD-L1pathway by anti-PD-L1antibody could partially restore the proliferation of T cells inmicroenvironment and it provides the basis of immunological therapy in tuberculouspleural effusion.