【作者】 刘宇；

【导师】 吴雄文； 翁秀芳；

【作者基本信息】 华中科技大学 ， 免疫学， 2020， 博士

【摘要】 MAIT细胞是人体中相对富集的固有免疫样T细胞,主要分布于肝脏、肺脏、小肠固有层及外周血中。MAIT细胞TCR由半恒定Vα链和有限的Vβ链组成,识别由主要组织相容性复合物I相关分子(MHC-I related molecular-1,MR1)提呈的核黄素代谢产物5-OP-RU(5-(2-oxopropylideneamino)-6-d-ribitylaminouracil)。乙型肝炎病毒(hepatitis B virus,HBV)是嗜肝DNA病毒,全球感染人数约2.57亿,年死亡率超过78万。宿主免疫系统缺乏有效免疫应答是抗病毒治疗后持续病毒存在和病毒反弹的主要原因之一。因此,更好地了解慢性乙型肝炎病毒感染免疫机制,设计新的免疫治疗方法,以促进有效抗病毒免疫反应十分必要。MAIT细胞的先天性表型及活化后反应特征,决定其在抗病毒免疫中可能发挥重要作用。然而,MAIT细胞在慢性HBV感染中的研究较少且结论不一。本研究目的是探究慢性HBV感染患者MAIT细胞数量与功能动态变化及相关机制,明确MAIT细胞抗病毒效应,体内功能状态改变及可能的干预靶点,为MAIT细胞在抗HBV感染方面的临床应用提供实验基础。论文共分为三个部分:一、慢性HBV感染患者MAIT细胞数量、功能变化及MAIT细胞抗病毒潜能研究本研究共收集234例健康个体,237例慢性HBV感染患者外周血标本,并收集20例HBV~-肝血管瘤和32例HBV~+HCC患者非肿瘤肝组织标本,分析患者外周血中及肝内MAIT细胞频率变化。发现慢性HBV感染患者外周血中及肝内MAIT细胞频率均显著减少。我们利用5-OP-RU/MR1四聚体及抗CD28抗体包被beads成功建立人工抗原提呈细胞(artificial antigen presenting cell,a APC),即5-OP-RU/MR1 a APCs,对健康个体外周血单个核细胞(peripheral blood mononuclear cells,PBMC)进行刺激,成功建立MAIT细胞体外扩增方法,并经磁珠分选获得高纯度MAIT细胞。在此基础上,通过经典细胞杀伤实验,证明MAIT细胞对病毒质粒转染靶细胞具有显著细胞毒效应,且这种效应可被MR1中和抗体部分抑制,提示MAIT细胞TCR依赖性抗病毒潜能。另外,慢性HBV感染患者外周血MATI细胞功能检测结果显示,与健康对照相比,患者外周血中MAIT细胞IFN-γ及TNF-α分泌潜能显著降低,提示患者外周血中MAIT细胞抗病毒潜能受限。二、慢性HBV感染患者MAIT细胞数量及功能变化相关因素研究通过相关分析,我们发现慢性HBV感染患者体内MAIT细胞频率与血清HBV-DNA水平无相关性,HBe Ag血清状态对MAIT细胞频率并无影响。对不同病程患者外周血MAIT细胞频率进行比较,发现具有进行性肝损伤及肝脏活动性炎症的慢性乙型肝炎患者(chronic hepatitis B,CHB)外周血中MAIT细胞频率较病毒携带者/免疫耐受期患者显著减少,提示肝损伤和/或炎症相关机制参与MAIT细胞频率降低。然而,外周血中MAIT细胞频率与血清AST/ALT水平仅呈较弱相关,而与肝脏炎症纤维化指数APRI(AST-to-platelet ratio index)及FIB-4(fibrosis-4 index)呈显著负相关,说明肝脏炎症水平,而非单纯病毒或肝损伤与MAIT细胞频率降低密切相关。为了进一步分析影响MAIT细胞频率减少相关因素,我们选取MAIT细胞频率较低(HBV MAIT low,HBVL)及频率较高(HBV MAIT high,HBVH)肝细胞性肝癌患者(hepatocellular carcinoma,HCC)非肿瘤肝组织标本及HBV~-肝血管瘤非肿瘤肝组织标本(对照标本)进行转录组及非靶代谢组检测。结果发现:HBVL组炎症相关细胞因子及趋化因子m RNA水平增高,并且胆红素相关代谢物表达量增高。这些结果不仅验证了炎症因素与MAIT细胞频率降低相关,同时提示胆红素可能成为MAIT细胞频率降低的另一独立因素。进一步相关分析发现,患者体内MAIT细胞频率与血清总胆红素(total bilirubin,TBIL)水平,尤其是直接胆红素(direct bilirubin,DBIL)水平呈负相关,并且高胆红素患者组(DBIL>10ULN)外周血中MAIT细胞频率减少最为显著且细胞凋亡水平最高。另外,血清DBIL水平与患者外周血中MAIT细胞IFN-γ及TNF-α分泌能力呈显著负相关。因此,患者体内DBIL水平增高,可能是MAIT细胞数量减少与功能失调的关键因素。三、结合胆红素对MAIT细胞频率及功能的影响体外实验证明,高浓度DBIL可直接刺激MAIT细胞活化并诱导MAIT细胞发生凋亡,且随DBIL浓度增加,MAIT细胞活化与凋亡水平递增;另外,在高浓度DBIL存在的条件下,MAIT细胞TCR依赖性扩增及增殖能力受到显著抑制,但IFN-γ分泌能力未表现出明显缺陷。进一步分析HBV感染患者MAIT细胞体外扩增能力发现,高胆红素患者组(DBIL>10ULN)外周血中MAIT细胞TCR依赖性扩增能力显著降低,且可被IL-2部分修复。因此,HBV感染患者经治疗DBIL恢复后,IL-2有望可以促进MAIT细胞数量及功能的恢复。本研究结论和意义:1.本研究揭示了MAIT细胞TCR依赖性抗病毒潜能,而HBV感染患者体内MAIT细胞频率减少以及IFN-γ及TNF-α分泌潜能缺陷,提示患者体内MAIT细胞抗病毒能力受限。2.本研究发现慢性HBV感染患者体内MAIT细胞频率减少与肝脏炎症及血清胆红素水平密切相关。其中,高胆红素患者组(DBIL>10ULN)体内MAIT细胞数量减少与功能障碍严重,说明高水平DBIL可能是患者体内MAIT细胞缺陷的关键因素及干预靶点。3.体外实验证明DBIL可直接促进MAIT细胞活化及凋亡,并可抑制其TCR依赖性细胞扩增及增殖,IL-2有望在DBIL恢复正常水平后促进MAIT细胞频率及功能的修复。本研究的创新点1.本研究揭示MAIT细胞TCR依赖性抗HBV潜能,并从相对大样本的分析中阐析了其在慢性乙肝患者体内的数量与功能变化及相关机制。2.本研究发现DBIL是慢性乙肝患者体内MAIT细胞数量及功能缺陷的主要因素之一,为体内建立基于MAIT细胞抗病毒策略提供了新的干预靶点。更多还原

【Abstract】 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 Vα chain and a limited Vβ 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-γ and TNF–α 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-γ and TNF-α 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-γ 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-γ and TNF-α 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.更多还原