| MAIT cells are relatively abundant innate immune like T cells in human,mainly distributed in liver,lung,intestinal lamina propria and peripheral blood.MAIT cells' TCR consists of an invariant V? chain and a limited V? chain.It recognizes the metabolite 5-OP-RU(5-(2-oxopropylideneamino)-6-d-riboflavin)presented by the major histocompatibility complex I related molecule-1(MR1).Hepatitis B virus(HBV)is a kind of hepatophilic DNA virus,which infects about 257 million people worldwide and has an annual mortality rate of over 780000.The lack of effective immune response of host immune system is one of the main reasons for the persistence of virus and virus rebound after antiviral treatment.Therefore,it is necessary to understand the immune mechanism of chronic hepatitis B virus infection and design new immunotherapy methods to promote effective antiviral immune response.The innate phenotype and post activation response of MAIT cells suggest that it may play an important role in antiviral immunity.However,the study of MAIT cells in chronic HBV infection is rare and the conclusions are controversial.The purpose of this study is to explore the dynamic changes in the frequency and function of MAIT cells in chronic HBV infected patients and the related mechanisms,to clarify the antiviral effect of MAIT cells,changes in functional state and possible intervention targets,and to provide experimental basis for the clinical application of MAIT cells in anti-HBV infection.The thesis is divided into three parts:1.Study on the frequency,function and antiviral potential of MAIT cells in chronic HBV-infected patientsIn this study,peripheral blood samples of 234 healthy donors,237 chronic HBVinfected patients,and the non-neoplastic liver tissue of 20 HBV-hemangioma patients and 32 HBV+ HCC patients were collected,and the frequency of MAIT cells in peripheral blood and liver was analyzed.It was found that the frequency of MAIT cells in the peripheral blood and liver of HBV-infected patients decreased significantly.Using 5-OP-RU/ MR1 tetramer and anti-CD28 antibody coated beads,we successfully established the artificial antigen presenting cell(a APC),namely 5-OP-RU / MR1 a APC.We successfully established the method of MAIT cell TCR-dependent expansion by stimulating peripheral blood mononuclear cells(PBMCs)of healthy donors with 5-OPRU / MR1 a APCs in vitro,and obtained high-purity MAIT cells by magnetic bead sorting(MACS).On this basis,through the classical cell killing experiments,it was proved that MAIT cells had significant cytotoxic effect on the target cells transfected with HBV viral plasmid,and this effect could be partially inhibited by MR1 neutralizing antibody,suggesting TCR dependent antiviral potential of MAIT cells.Compared with the healthy controls,the capacity of IFN-? and TNF–? secretion of circulating MAIT cells in patients was significantly reduced,suggesting the antiviral potential of MAIT cells was limited.2.Study on the related factors of MAIT cells frequency and function in chronic HBV-infected patientsThrough correlation analysis,we found that the frequency of circulating and hepatic MAIT cells were not related to the HBV-DNA level,and there was no difference of circulating MAIT cells ratio between HBe Ag+ and HBe Ag-patients.It was found that the frequency of MAIT cells in the peripheral blood of patients with chronic hepatitis B(CHB)with progressive liver injury and liver active inflammation was significantly lower than that of virus carriers / immunotolerant patients,suggesting that the mechanism of liver injury and / or inflammation was involved in the decrease of MAIT cell frequency.However,the frequency of MAIT cells in the peripheral blood was only weakly correlated with the serum AST / ALT level,while significantly negatively correlated with the APRI(AST-to-platelet ratio index)and FIB-4(fibrosis-4 index),indicating that the level of liver inflammation,rather than virus infection or liver injury,was closely related to the decrease of the ratio of MAIT cells.In order to further analyze the relevant factors affecting the frequency of MAIT cells,we selected the nonneoplastic liver tissue samples of HBV+HCC patients with low MAIT cells frequency(HBV MAIT low,HBVL)or high MAIT cells frequency(HBV MAIT high,HBVH)and non-neoplastic liver tissue samples of HBV-hemangioma(control samples)for transcriptomics and non-target metabonomics tests.The results showed that in HBVL group,the m RNA levels of inflammatory cytokines and chemokines increased,and the expression of bilirubin related metabolites increased.These results not only confirmed the correlation between inflammatory factors and the decrease of MAIT cell frequency,but also suggested that bilirubin might be another independent factor of the decrease of MAIT cell frequency.Further correlation analysis showed that the frequency of MAIT cells was negatively correlated with the level of total bilirubin(TBIL),especially direct bilirubin(DBIL),and the circulating MAIT cells in HBV-infected patients with high bilirubin level(DBIL > 10ULN)decreased most significantly and suffered highest level of apoptosis.In addition,there was a significant negative correlation between the serum DBIL level and the secretion capacity of IFN-? and TNF-? of circulating MAIT cells in patients.Hence,the increased level of DBIL may be the key factor for the decrease and dysfunciton of MAIT cells in HBV-infected patients.3.The effect of conjugated bilirubin on the frequency and function of MAIT cellsIn vitro experiments showed that high concentration of DBIL could directly stimulate the activation of MAIT cells and induce the apoptosis of MAIT cells,and with the increase of DBIL concentration,the level of activation and apoptosis of MAIT cells increased.In addition,in the presence of high concentration of DBIL,TCR dependent amplification and proliferation of MAIT cells were significantly inhibited,but IFN-? secretion capacity did not show any defect.TCR dependent expansion ability of circulating MAIT cells in chronic HBV-infected patients with high DBIL level(DBIL > 10ULN)was significantly reduced and could be partially repaired by IL-2.Therefore,IL-2 is expected to promote the recovery of the frequency and function of MAIT cells in HBV-infected patients with DBIL back to normal levels after treatment.Conclusion and significance of this study:1.We revealed the TCR dependent antiviral potential of MAIT cells,while the decrease of MAIT cell frequency and the deficiency of IFN-? and TNF-? secretion potential in HBV infected patients indicated that the antiviral capacity of MAIT cells in patients was limited.2.We found that the decrease of MAIT cell frequency in chronic HBV-infected patients was closely related to liver inflammation and serum bilirubin level.MAIT cells in patients with high DBIL level(DBIL>10ULN)decreased most significantly and the dysfunction was the most serious,indicating that high level of DBIL may be the key factor and intervention target of MAIT cell defects in patients.3.In vitro experiments showed that DBIL could directly promote the activation and apoptosis of MAIT cells,and inhibit TCR dependent cell expansion and proliferation.IL-2 is expected to promote the repair of MAIT cell frequency and function after the recovery of DBIL level after treatment.Original Pionts in this study1.We revealed the TCR dependent anti-HBV potential of MAIT cells,and analyzed the frequency and function of MAIT cells in HBV-infected patients and its related mechanisms from the analysis of a relatively large sample.2.We found that DBIL was one of the main factors that affected the frequency and function of MAIT cells in chronic HBV-infected patients,which provided a new intervention target for the establishment of antiviral strategy based on MAIT cells. |
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