A Study On The Role Of TCRγδT Cells In The Immunopathogenesis Of Chronic Hepatitis B

Objective: Chronic hepatitis B(CHB) is one of the common chronic infectious diseases in our country, which seriously endangers people's health. The immune response in our body plays the important role in the clearance of hepatitis B virus(HBV). Till now, there have been few studies concerning the relations between theγδT cells and CHB. So in this study, we are intent to explore whether there is some difference in the proportion ofγδT or other immune cells in peripheral blood sample or liver puncture specimen between the health blood donors and the patients with chronic HBV infection, and analyze the relationship between theγδT and the other immune cells or the clinical parameters, and further investigate the immune function in vitro of theγδT cells separated and activated from the peripheral blood. The results might offer some experimental evidence to explain the role ofγδT cells in the immunopathogenisis mechanism of chronic hepatitis B.Methods:1. Study on the relationship between the proportion ofγδT in peripheral blood sample and the immune state or the clinical condition: Flow cytometry was used to detect the cells proportion ofγδT/T,δ2T/γδT, CD45RO+γδT/γδT, CD45RO+δ2T/δ2T, CD45RO+T/T, CD4+/T, CD8+/T, or NK/lymphocyte in peripheral blood from 40 asymptomatic HBV carriers(AsC), 30 CHB patients in low grade, 50 CHB patients in moderate or severe grade, and 24 health blood donors. All the data was analyzed by the SPSS 13.0 software for the difference in the cells proportion or the relationship between the cells or the cells and the clinical parameters.2. Detection and analysis ofγδT in the liver puncture slice from patients with chronic HBV infection: Immunohistochemistry method was used to detect theγδT cells in the slice from 5 patients.γδT was counted in 10 sights under high power objective. The relationship ofγδT amount in slice and peripheral blood was analyzed by SPSS13.0.3. Separation, activation and expansion ofγδT from peripheral blood: TheγδT cells from 6 patients and 3 health donors were separated by density gradient centrifugating, cultured plastic-adherence, and flow cytometry negative sorting. The purity or activity was tested afer theγδT sorting. TheγδT was activated and expansion with 0.2,0.5,1,2μg/mL anti-Vγδand 50 U/ mL rhIL-2 for 14 days, and the optimum condition was determined.4. Study on the immune function ofγδT from peripheral blood in vitro4.1 Obtaining the LO2-mock,LO2-HBV cells model: The mock control plasmid was built by digesting pEcob6 plasmid with EcoRⅠat the HBV-DNA site and ligating the fragment without HBV-DNA. The LO2 cells were transfected with pEcob6 or mock plasmid by lipofectamine 2000. The expression of HBsAg, HBcAg/HBeAg, or HBV-DNA was detected by immunofluorescence assay, ELISA, AXSYM(Abbott), or FQ-PCR. The expression of MICA/B was determined by flow cytometry.4.2 Study on immune function ofγδT in vitro: TNF-αor IFN-γsecretion was detected in the supernatant ofγδT cells after stimulation by ELISA. LDH release experiment was used to detect the cytotoxity ofγδT on LO2, LO2-mock, or LO2-HBV. The difference of the immune function ofγδT between 6 patients and 3 controls was analyzed by SPSS13.0.Results:1. It was showed from the H test that the difference inγδT proportion or CD45RO+γδT proportion wasn't significant(P>0.05), butδ2T(P<0.01) or CD45RO+δ2T(P<0.05) was. The difference inδ2T proportion between normal controls and other groups was significant(P<0.01), or between CHB patients in moderate or severe grade and other groups was also significant (P<0.01). The difference in CD45RO+δ2T or CD45RO+T proportion between normal controls and AsC was significant(P<0.05). The difference in NK proportion between normal controls and patients in low grade was significant (P<0.05).2. The results of Spearman correlation test showed thatδ2T proportion had positive correlation withγδT(P<0.05), but negative correlation with CD45RO+γδT or CD45RO+δ2T proportion(P<0.01), and there was a tight correlation between CD45RO+γδT, CD45RO+δ2T and CD45ROT proportion(P<0.01). Andδ2T proportion had the negative correlation with ALT, AST and TB(P<0.01).3. Only 2 to 3γδT cells could be seen in one sight under high power objective. There was no relationship between theγδT amount in blood and the liver slice(P>0.05).4. The purity ofγδT was improved from <23% to >71% by the cytometry negative sorting, and the activity was greater than 90%. 106γδT cells could be obtained from 20ml peripheral blood. TheγδT cells were stimulated optimally in the medium with 0.2μg/mL anti-γδand rhIL-2 for 9 days.5. By LSCM, we could observe that HBsAg expressed in cytoplasm brightly but HBcAg in cytoplasm or cytonucleu darkly in LO2-HBV cells, but not in LO2-mock cells. The HBsAg or HBeAg was positive or the quantity of HBV-DNA was 9.67×104 copies/mL in supernatant of LO2-HBV but not in LO2-mock or LO2 cells. The MFI of MICA/B was 13.3±0.7, 7.4±0.9, or 4.2±0.6 on LO2-HBV, LO2-mock, or LO2 respectively, and the difference was significant by F test(P<0.01).6. IFN-γor TNF-αwas 140.8±82.9pg/mL or 129.8±71.5pg/mL in the supernatant ofγδT cells from patients, and 440.7±172.8pg/mL or 233.4±218.0pg/mL from normal control. The difference of IFN-γwas significant(P<0.01) but TNF-αwas not(P>0.05). 7. At E/T=30:1, the cytotoxicity of Vγδ+T from patients on LO2,LO2-mock, or LO2-HBV was 9.98±1.2%, 11.05±3.5%, or 18.1±6.0%, but 6.6±1.04%, 18.7±0.98% or 31.5±4.16% from controls. These difference between the two groups were significant(P<0.05). The difference of cytotoxicity between LO2 and LO2-HBV, or LO2-mock and LO2-HBV was significant(P<0.05).Conclusion:1. Although chronic HBV infection could makeδ2T cells proportion decreased,δ2T was the preponderant subpopulation ofγδT in blood. CD45ROδ2T cells were the major component of CD45ROγδT.2. The results that there was some negative correlation between CD4 andγδT but no correlation betweenγδT and other immune cells showedγδT had relationship with the helper T immune response. The stronger immne response in liver was accompanied with the decreacedδ2T. The HBV-DNA load had no effect on theγδT proportion.3. The method of cytometry negative sorting could meet the demand of the experiment to obtain theγδT from the blood. And LO2-mock or LO2-HBV cells were got as the target cells for the cytotoxicity experiment.4. The ability of IFN-γsecretion or cytotoxicity on target cells ofγδT from patients was weaker compared to the normal controls. This might hint IFN-γwas a very important factor in the cytotoxicity. 5. It was showed that the replication or transcription of HBV-DNA might improve the MICA/B expression on the LO2-HBV cells, which would raise the cytotoxicity effect ofγδT on the target cells.