The Expression Of Negative Costimulatory Molecules In Peripheral Tregs From Chronic Hepatitis B Patients And Its Clinical Significance

Chronic hepatitis B (CHB) infected by Hepatitis B virus (HBV) is a worldwidedisease and correlates intimately with the occurrence and development ofhepatocirrhosis, hepatic carcinoma as well as hepatic failure. HBV infection inducesorganism to engender diversified antivirus immune reaction mainly of cytotoxic tlymphocyte (CTL) specific immune response. HBV infection becomes chronic anddevelops into CHB which is probably caused by attenuated HBV specific CTLimmune response so long as virus can’t be cleaned up and replicate persistently. Theimmune functional deficiency of CTL is related to the up-regulated expression ofinhibitory receptor molecule, formation of inhibitory cytokines, maladjustment ofantigen presentation and negative regulation of the regulatory T cells (Treg). Thesurface makers of main Treg subsets demonstrate CD4~+CD25~+and characterized byspecific functional molecules of Foxp3expressed in cytoplasm. Researches showed thatCD4~+CD25~+FoxP3~+Treg could be capable of exerting inhibitory influences on variousimmune cells by inhibiting HBV specific CD4+and CD8+T cells activity which leads tothat virus can’t be cleaned up and infects organism constantly resulting in CHB. Theproliferation and immune inhibitory function of Treg is related to the costimulatorymolecules expressed on its own surface. Costimulatory molecule receptor interactingwith corresponding ligand mediate positive or negative costimulatory signal andregulate the proliferation of T cells, the production of cytokine, cell toxicity as well ascell apoptosis and existence, which control T cell activation. BTLA and PD-1belongingto cosuppression receptor molecules from immunoglobulin superfamily interact withcorresponding ligands HVEM and PD-L1respectively, producing negativecostimulatory signal which inhibit T cell activation, proliferation and immune function.Researches indicated that negative costimulatory signal path mediated by PD-1 negatively regulated the proliferation and immune inhibitory function of Treg inHepatitis C virus (HCV) infected individuals via inhibiting Treg signal transduction andthe phosphorylation of activating transcription factor, accordingly blocking up thefunction of IL-2. However, the expression characteristics of PD-1in Treg from CHBpatients and regulation on Treg have not been reported. In addition, it’s not reported todate that the BTLA and HVEM expression characteristics and regulation on Treg andeffect of BTLA/HVEM signaling diversification on Treg as well as its significances inCHB.This study determined the percentage of CD4~+CD25~+FoxP3~+Treg in CD4+T cellsand expression characteristics of PD-1, PD-L1, BTLA and HVEM negativecostimulatory molecules of Treg in CHB patients via flow cytometry. Also, we analyzedthe correlation between Tregs and clinical indicators of CHB and between the negativecostimulatory molecules expressed by Treg and clinical indicators, and investigated theeffect of PD-1and BTLA/HVEM negative costimulatory signal on Treg regulation andPD-L1participated in the inhibitory function of Treg as well as the influence on HBVchronic infection, thereby preliminary revealed the expression characteristics ofnegative costimulatory molecules on Treg and its significant, and providing valuableindicators to clinical diagnosis.Part I Determination of CD4~+CD25~+FoxP3~+Treg and theexpression of PD-L1and analysis of its relation to clinic indicatorsObjective: Determine the percentage of peripheral blood CD4~+CD25~+FoxP3+Tregin CD4+T cell and the expression of PD-L1from CHB patients, and analyze theircorrelations to clinical indicators and investigate the influences of Treg and PD-L1onCHB.Methods: Determine serum markers of HBsAg, HBsAb, HBeAg, HBeAb, HBcAbvia ELISA. Check HBV DNA by Realtime PCR and the positive consequences shouldmore than5×102copies/ml. Check liver function indicator using Roche automaticbiochemistry analysis meter. Isolate peripheral blood lymphocyte and fluorescence label CD4~+CD25~+FoxP3~+Treg to determine by flow cytometry, and the datas were analyzedby GraphPad Prism5software.Results: The percentage of peripheral blood CD4~+CD25~+FoxP3~+Treg in CD4+Tcell of HBV sufferer is higher than that of healthy individuals (P <0.05) and showedpositive correlation to ALT and AST in CHB patients serum (P <0.05) nonetheless, noconspicuous correlation to HBV-DNA. PD-L1revealed distinct up-regulation comparedto healthy individuals,and the expression level indicated conspicuous positivecorrelation to Treg ratio and serum markers of ALT and AST however, no distinctcorrelation to HBV DNA.Conclusion: Tregs in peripheral blood of CHB patients increased and werepositively correlated with indicators of liver injury, which may be due to its effect toenhanced HBV specific CTL immune inhibitory action, accordingly HBV inferredconstantly and deterioration continued which boosted the formation. Up-regulation ofPD-L1and its positive correlation with Treg ratio and indicators of liver injury, gainedthe possibility to participate in the HBV specific CTL immune inhibitory action of Tregwhich benefit to virus constant infection and development of CHB.Part II The expression characteristics of PD-1and BTLA/HVEMin Treg cells and correlation analysis of clinical indicator.Objective: Analyze the expression of PD-1, BTLA and HVEM inCD4~+CD25~+FoxP3~+Treg from CHB patients and its correlations with the amount ofTreg and clinical indicators. Investigate the regulatory effect of PD-1/PD-L1,BTLA/HVEM inhibitory signals to Treg and the influence on CHB, and lay theoreticalfoundation to clinical diagnoses and intervenes.Methods: Isolate human peripheral blood mononuclear cells. Determine theexpression of PD-1, BTLA and HVEM on CD4~+CD25~+FoxP3~+Treg surface with4colorfluorescence labeling and flow cytometry. Analyze the results of flow cytometrydetection by Flowjo and statistically analyze the statistics by GraphPad Prism5.0.Results: The expression negativity costimulatory molecule of PD-1in CHB Treg showed outstanding down-regulation, and the expression level indicated conspicuouspositive correlation to Treg ratio and serum markers of ALT and AST. BTLA andHVEM revealed conspicuous down-regulation compared to healthy individuals. BTLAshowed positive correlation to HVEM expression and distinct positive correlation toTreg ratio, PD-1, PD-L1and serum markers of ALT and AST, however, no notablecorrelation to HBV DNA.Conclusion: down-regulation of PD-L1probably contributed to Treg proliferation.the expression of BTLA and HVEM is down-regulated cogradiently, which has thepossibility to contribute to Treg amplification in CHB patients, and in coordination withPD-1negative signal may be involved in the regulation of Treg, and the immunologicalmechanism of their correlation to indicators of liver injury remains to be investigated.In conclusion, this study approved primarily that the increased percentage of Tregin CD4~+T cells from CHB patients peripheral blood. the expressions of negativecostimulatory molecules PD-1、BTLA/HVEM on Treg surface were down-regulatedcompared to healthy individuals, but gradually increased along with the increasednumber of Treg, and showed positive correlation to indicators of liver injury. Theexpression of PD-L1in CHB patients was up-regulated, and positive correlation to Tregratio and indicators of liver injury. Thereby indicated that negative costimulatorymolecules PD-1、BTLA/HVEM have the possibility to participate in the regulation ofTreg itself, and PD-L1may constitute one of the inhibitory function molecules on Tregand have significant effect on the development of CHB.