Tim-3在HBV慢性感染中的表达及作用研究
摘要
乙型肝炎病毒(Hepatitis B virus,HBV)感染肝细胞引发一系列的肝脏炎症性疾病,严重影响人类的健康。已有研究证实,HBV感染所致多种淋巴细胞功能失调不仅是乙肝患者持续感染的重要机制之一,而且在乙肝患者不同的预后、转归中发挥着不容忽视、甚至是决定性的作用。但HBV所致的免疫失调的具体机制尚未完全明确。
众多研究表明,多种免疫调节分子在HBV感染所致淋巴细胞功能失调中发挥着重要作用。Tim-3(T cell immunoglobulin- and mucin-domain-containingmolecule-3,Tim-3)是在2002年被发现的,特异性表达在分化终末期的Th1细胞上的新型免疫调节分子。Tim-3与其配体之一—galectin-9相互作用可负向调控IFN-γ的产生,并可影响T细胞耐受的形成。近来的研究结果显示,Tim-3除了在Th1细胞上表达外,在细胞毒性T细胞(Te1细胞)、树突状细胞和NK细胞上均有表达,并且通过影响这些细胞的功能,参与多种疾病的发生发展过程。我室在前期研究中发现,Tim-3mRNA在慢乙肝患者外周血单个核细胞上的表达升高,提示Tim-3很可能在HBV慢性感染中发挥着重要的作用。本课题利用临床研究、体外细胞实验和动物实验研究Tim-3在慢乙肝发生发展中的表达及功能,为进一步深入阐明HBV感染相关疾病的发病机制提供实验依据。
方法
1.Tim-3在慢乙肝患者体内的表达
1.1 Tim-3在慢乙肝患者体内不同淋巴细胞亚群中的表达
收集慢乙肝患者、健康志愿者和脂肪肝患者新鲜外周肝素抗凝血,流式细胞术检测Tim-3在外周血不同淋巴细胞亚群中的表达。收集慢乙肝患者和脂肪肝患者肝组织,免疫组化检测Tim-3在肝脏中的表达。
1.2慢乙肝患者外周血不同淋巴细胞亚群上Tim-3的表达与血清ALT水平,HBVDNA等的相关性分析
利用Prism GraphPad软件对慢乙肝患者外周血不同淋巴细胞亚群上Tim-3的表达与患者血清ALT水平,HBV DNA量进行Spearman相关性分析。
2.HBV感染对Tim-3表达的影响
2.1以人NK92细胞系为研究对象体外模拟研究HBV感染对Tim-3表达的影响
取对数生长期的人NK细胞系NK92细胞,利用电穿孔转染法将HBV表达质粒pcDNA3-1.1HBV转入细胞内,以含200μg/mL G418的培养基稳筛1～2周。同时以转染pcDNA3空载体的NK92细胞为对照细胞。利用RT-PCR和流式细胞术检测Tim-3的表达。
2.2以HBV转基因小鼠为研究模型研究HBV感染对Tim-3表达的影响
利用Percoll分离液密度梯度离心法分离HBV转基因小鼠肝脏内单个核细胞,并以正常Balb/c小鼠作为对照,流式细胞术检测小鼠肝脏内单个核细胞上Tim-3的表达。
3.Tim-3对NK细胞和CD8~6T细胞功能影响的研究
3.1 Tim-3对NK细胞功能影响的研究
3.1.1阻断Tim-3对NK92功能的影响
以NK92细胞为效应细胞,给予Tim-3抗体/Tim-3-Fc融合蛋白、同型抗体或PBS缓冲液作用后,按不同效靶比(1:1、5:1和25:1)与人肝癌细胞系HepG2或HepG2.2.15细胞进行共孵育4小时,CCK-8试剂盒检测细胞杀伤效率,ELISA检测细胞培养上清中IFN-γ分泌。
3.1.2阻断Tim-3对慢乙肝患者外周血淋巴细胞杀伤功能的影响
分离慢乙肝患者外周血单个核细胞为效应细胞,给予Tim-3抗体、同型抗体或PBS缓冲液后,研究效应细胞对靶细胞HepG2或HepG2.2.15细胞的杀伤效率和IFN-γ分泌。
3.2 Tim-3对肝炎模型小鼠肝脏CD8~+T细胞功能影响的研究
3.2.1制备小鼠HBV肝炎模型
利用尾静脉高压注射法将HBV表达质粒pcDNA3-1.1HBV注入小鼠体内制备小鼠HBV肝炎模型,同时以pcDNA3质粒制备小鼠模型对照。在不同时间点处死小鼠,实时免疫荧光检测(IFMA)试剂盒检测小鼠血清中表面抗原、表面抗体和e抗原的水平,免疫组化检测肝细胞内HBc的表达。实时定量PCR检测血清中HBV DNA的水平。谷丙转氨酶(ALT/GPT)检测试剂盒测定血清谷丙转氨酶活性。
3.2.2 Tim-3在模型小鼠肝脏内单个核细胞上的表达
分离模型小鼠和对照小鼠肝脏内单个核细胞,流式细胞术检测Tim-3在肝脏内单个核细胞上表达。
3.2.3构建特异针对Tim-3的shRNA表达载体
设计并合成针对Tim-3或无关序列的shRNA寡核苷酸,退火后与shRNA表达载体pmU6相连,转化大肠杆菌感受态细胞DH5α,经抗性筛选、酶切、DNA测序鉴定获得阳性重组子,并被分别命名为Tim-3shRNA-1,Tim-3shRNA-2和unshRNA。
3.2.4体外验证Tim-3 shRNA表达载体的抑制效果
利用脂质体转染法将shRNA表达质粒转染入小鼠巨噬细胞系RAW264.7细胞,24、48和72小时后收集细胞,RT-PCR和western blot检测Tim-3和β-actin表达,同时检测Tim家族另一成员—Tim-1的表达以验证Tim-3 shRNA抑制效果的特异性。
3.2.5 shRNA抑制Tim-3表达对小鼠肝炎模型肝脏内CD8~+T细胞功能的影响
利用尾静脉高压注射法将pcDNA3-1.1HBV与Tim-3shRNA或unshRNA表达载体注入小鼠体内,流式细胞术检测肝脏内单个核细胞上Tim-3的表达水平以验证Tim-3shRNA的体内抑制效果,同时检测细胞内IFN-γ表达研究Tim-3被抑制后对细胞功能的影响。
4.统计学分析
采用GraphPad Prism4软件对实验数据进行Mann-Whitney非参数U检验、Kruskal-Wallis非参数H检验和Spearman相关性分析。p＜0.05或p＜0.01有统计学意义。
结果
1.Tim-3在慢乙肝患者外周血单个核细胞和肝脏内单个核细胞上表达升高
1.1 Tim-3在慢乙肝患者外周血单个核细胞,尤其是NK细胞和CD8~+T细胞上表达升高
流式细胞术检测结果显示,Tim-3在慢乙肝患者外周血单个核细胞上的表达显著高于健康志愿者和脂肪肝患者对照(p＜0.05)。进一步的分析结果显示,Tim-3在NK细胞上高表达,而且慢乙肝患者外周血NK细胞上的Tim-3的表达水平显著高于健康志愿者和脂肪肝患者(p＜0.05)。此外,Tim-3在T细胞,尤其是CD8~+T细胞上的表达显著高于健康志愿者和脂肪肝患者(p＜0.0001)。
1.2 Tim-3在慢乙肝患者肝脏内单个核细胞上表达升高
为了进一步研究Tim-3在慢乙肝患者肝脏内的表达,我们收集慢乙肝患者和脂肪肝患者肝穿标本,利用免疫组化的方法研究Tim-3在肝脏内的表达。结果显示,慢乙肝患者肝组织中Tim-3的表达显著高于脂肪肝患者,而且从细胞形态上可以看出Tim-3主要在肝脏内浸润淋巴细胞上表达。
1.3慢乙肝患者外周血T细胞和CD8~+T细胞上Tim-3表达水平与ALT水平正相关
统计学分析发现,T细胞和CD8~+T细胞上Tim-3表达水平与患者血清ALT水平均呈正相关(r=0.41,p＜0.01),而与HBV DNA滴度并不相关,提示Tim-3很可能通过调节CD8~+T细胞的功能参与肝损伤过程。
2.HBV感染可上调Tim-3表达
2.1 HBV表达质粒转染NK92细胞后可上调Tim-3的表达
已有研究结果显示在慢乙肝患者外周血单个核细胞和NK细胞上可检测到HBV的肝外感染。为研究HBV感染是否可以上调细胞中Tim-3表达,本研究利用HBV表达质粒pcDNA3-1.1HBV转染的NK92细胞,作为HBV体外感染的细胞模型(空载体pcDNA3转染组为对照)。半定量RT-PCR的结果显示,在pcDNA3-1.1HBV转染的NK92细胞中检测到HBx和HBc mRNA表达,并且检测到Tim-3的表达上调,显著高于空载体对照;流式细胞术检测结果显示,Tim-3蛋白在HBV表达质粒转染的NK92细胞上的表达显著高于空载体对照,与RT-PCR结果相吻合。
2.2 HBV转基因小鼠肝脏内单个核细胞以及NK细胞上Tim-3的表达升高
HBV转基因小鼠模型是目前较为公认的HBV感染模型,本研究利用该模型进一步研究HBV感染对Tim-3表达的影响。流式细胞术分析结果显示:在HBV转基因小鼠肝脏内单个核细胞以及NK细胞上Tim-3的表达水平显著高于正常对照小鼠(p＜0.05)。
3 Tim-3抑制NK细胞和CD8~+T细胞的功能
3.1 Tim-3阻断后可上调NK细胞的杀伤功能
3.1.1 Tim-3阻断后可以上调NK92细胞的杀伤功能
为了研究Tim-3对NK细胞功能的影响,我们利用Tim-3的中和抗体或Tim-3-Fc融合蛋白阻断Tim-3的信号通路,研究Tim-3对NK92细胞杀伤和细胞因子分泌的影响。
1)细胞杀伤率:CCK-8检测结果显示,随着Tim-3中和抗体或Tim-3-Fc融合蛋白作用浓度的增加,其NK92细胞细胞杀伤效率呈现明显的剂量依赖性,当Tim-3中和抗体或Tim-3-Fe融合蛋白作用浓度为5μg/mL时,NK92细胞的杀伤率达到平台期,因此,将该浓度确定为随后实验的作用浓度。在不同效靶比条件下,Tim-3中和抗体或Tim-3-Fc融合蛋白处理组NK92细胞杀伤率显著高于同型抗体对照和PBS缓冲液对照作用的细胞(p＜0.05或p＜0.01),且该作用呈现明显的剂量依赖性。然而Tim-3抗体阻断后并不能上调NK92细胞对K562细胞的杀伤效率。半定量RT-PCR检测结果显示:galectin-9在HepG2和HepG2.2.15细胞上表达水平较高,而在K562细胞上表达水平较低。
2) IFN-γ的分泌:将NK92细胞与HepG2.2.15细胞(效靶比25:1)共同培养4小时后,分离细胞培养上清,ELISA检测IFN-γ的分泌。结果显示:与同型抗体对照和PBS缓冲液相比,Tim-3抗体阻断可显著上调NK92细胞在杀伤HepG2.2.15细胞时的IFN-γ分泌(同型对照抗体组421±19 pg/mL,PBS缓冲液对照402±32pg/mL,Tim-3抗体阻断组521±27 pg/mL)(p＜0.05)。
3.1.2 Tim-3抗体阻断后可上调慢乙肝患者外周血单个核细胞的杀伤效率
慢乙肝患者体内NK细胞上存在Tim-3的表达升高,提示Tim-3有可能通过影响NK细胞的功能参与HBV慢性感染过程。为了验证这种可能性,我们分离慢乙肝患者外周血单个核细胞,按不同效靶比(5:1、10:1和25:1),与HepG2或HepG2.2.15细胞共培养4小时后,CCK-8检测各组细胞杀伤率。结果显示,与同型抗体对照和PBS缓冲液对照相比,Tim-3抗体阻断后可上调慢乙肝患者外周血单个核细胞的杀伤效率(p＜0.05)。
3.2 shRNA抑制Tim-3表达可上调HBV小鼠肝炎模型肝脏内CD8~+T细胞功能
本研究发现,除NK外,慢乙肝患者外周血CD8~+T细胞的Tim-3表达明显上调,提示Tim-3可能影响CD8~+T细胞功能。由于临床分离慢乙肝患者CD8~+T细胞数量少、杀伤能力差、且靶细胞不易选择,本实验以小鼠肝炎模型研究Tim-3对CD8~+T细胞功能的影响。HBV转基因鼠对HBV完全耐受,不能完全反映慢乙肝患者体内情况。为此,本研究利用尾静脉高压注射法制备HBV小鼠肝炎模型,进一步研究HBV感染对Tim-3表达的影响。
3.2.1制备HBV小鼠肝炎模型
尾静脉高压注射法制备HBV小鼠肝炎模型是近年来建立并得到广泛认肯的肝炎模型。本实验利用该模型进一步验证HBV感染对Tim-3表达的上调作用。实时定量PCR检测发现模型制备第1天后可检测到小鼠血清HBV DNA,并于第7天达高峰,至第18天检测不到。免疫组化方法证实模型小鼠肝细胞内表达HBc。IFMA试剂盒检测结果显示随着抗原水平的降低,模型制备后第7天可以检测到血清中的表面抗体。模型小鼠和对照小鼠血清谷丙转氨酶水平于第1到3天升高,随后降低至正常水平,两者之间没有显著性差异。
3.2.2 Tim-3在HBV肝炎模型小鼠肝脏内CD8~+T细胞上表达升高
模型制备后在不同时间点处死小鼠,分离小鼠肝脏内单个核细胞,利用流式细胞术检测Tim-3表达。结果显示模型制备后第1天和第3天,模型小鼠和对照小鼠肝脏内T细胞上Tim-3的表达显著升高,但两组之间没有差异。但在第7天至第18天,模型小鼠肝脏内T细胞上Tim-3表达显著高于对照小鼠(p＜0.05或p＜0.01)。进一步分析发现主要是肝脏内CD8~+T细胞上Tim-3表达显著升高(第10天,p＜0.01)。在脾脏中并未检测到Tim-3表达升高。
3.2.3 HBV模型小鼠肝脏内CD8~+T细胞上Tim-3表达升高伴随血清中和抗体滴度升高
进一步的分析发现:在模型制备后第10天,HBV模型小鼠体内可以检测到较高水平的针对表面抗原的中和抗体,但小鼠之间抗体滴度水平的差异较大。将HBV模型小鼠以表面抗体水平50 mIU/mL(接近模型小鼠抗体水平的均值-58mIU/mL)为界分为两组:高抗体组(＞50 mIU/mL)和低抗体组(＜50 mIU/mL)。分析结果显示高抗体组模型小鼠肝脏内CD8~+T细胞上Tim-3的表达显著高于低抗体组(p＜0.05)。
3.2.4 Tim-3shRNA表达载体可有效抑制RAW264.7细胞上Tim-3的表达
为了进一步研究Tim-3对CD8~+T细胞功能的影响,我们成功构建Tim-3shRNA表达载体Tim-3shRNA-1和Tim-3shRNA-2,以及针对无关序列的unshRNA表达载体,并转染RAW264.7细胞,半定量RT-PCR和Western blot检测结果显示,Tim-3shRNA-1转染组细胞与其他组细胞相比,Tim-3 mRNA水平和蛋白质水平的表达均显著降低(p＜0.05),并且这种抑制效果是特异的,并不影响Tim家族另一成员Tim-1的表达。
3.2.5 shRNA抑制Tim-3表达可上调HBV小鼠肝炎模型肝脏内CD8~+T细胞功能
上述针对Tim-3的shRNA表达载体与HBV表达质粒同时以尾静脉高压注射法注入小鼠,体内研究Tim-3对模型小鼠CD8~+T细胞功能的影响。流式细胞术结果显示:于模型制备后第10天Tim-3shRNA-1处理组CD8~+T细胞上Tim-3的表达水平降低,显著低于无关对照unshRNA处理组Tim-3表达未受影响(p＜0.05),且Tim-3shRNA-1处理组CD8~+T细胞上IFN-γ表达水平显著高于后者(p＜0.05)。
3.2.6 shRNA抑制Tim-3表达可下调血清中和抗体的水平
进一步对小鼠血清中和抗体的检测发现:pcDNA3-1.1HBV+Tim-3shRNA-1组小鼠血清中和抗体水平显著低于pcDNA3-1.1HBV+unshRNA组(p＜0.05)。
结论
1.慢乙肝患者外周血单个核细胞,尤其是NK细胞和CD8~+T细胞,及肝脏内单个核细胞上Tim-3表达显著高于对照者。
2.HBV感染可上调NK92细胞和HBV转基因小鼠以及尾静脉高压法制备的肝炎模型小鼠肝脏内NK细胞或CD8~+T细胞上Tim-3的表达。
3.Tim-3途径阻断可上调NK92细胞和慢乙肝患者外周血单个核细胞的细胞杀伤效率和IFN-γ分泌。
4.shRNA表达载体抑制Tim-3表达,可上调模型小鼠肝脏内CD8~+T细胞IFN-γ表达,同时模型小鼠中和抗体水平降低。
创新点及意义
1.本研究发现慢乙肝患者外周血单个核细胞(尤其NK细胞和CD8~+T细胞)和肝脏内单个核细胞上Tim-3表达显著升高,并分别利用转染了HBV表达质粒的NK92细胞和HBV转基因小鼠为模型,首次证实HBV感染可上调Tim-3的表达,为Tim-3参与HBV慢性感染过程提出新的论点。
2.本研究利用NK92细胞系和慢乙肝患者外周血单个核细胞,证实阻断Tim-3途径可上调NK细胞的杀伤效率,提示Tim-3有可能作为慢乙肝治疗的新靶点。
3.本研究利用特异针对Tim-3的shRNA表达载体抑制小鼠肝炎模型肝内Tim-3表达,发现抑制Tim-3表达可上调CD8~+T细胞IFN-γ的表达,且降低模型小鼠血清中和抗体水平,提出Tim-3可抑制CD8~+T细胞的功能,并间接影响中和抗体产生的观点。
Hepatitis B virus(HBV) primarily infects hepatocytes,which causes a series of necroinflammatory liver diseases worldwide.Persistent carriers of HBV,however, show a severe dysfunction in HBV-specific immune response.However,the mechanisms have not been well-defined.
It has been shown that immune regulatory molecules play important roles in HBV-induced lymphocyte dysfunction.Tim-3 was identified to be specifically expressed on polarized Th1 cells in 2002,and might play great roles in immune regulation and tolerance induction.Engagement of Tim-3 by its ligand-galectin-9 negatively regulates IFN-γproduction and influences the ability to induce T cell tolerance.Recent studies have shown that Tim-3 is also expressed on Tcl cells, dendritic cells and natural killer cells,and may involve in the pathogenesis of a range of diseases by influencing these cell function.In our early studies,we demonstrated that Tim-3 mRNA is up-regulated on peripheral blood mononuclear cells(PBMCs) from patients with chronic hepatitis B(CHB),indicating Tim-3 might play an important role in the development of CHB.In this study,we investigated the expression and functional role of Tim-3 in CHB by clinical samples,in vitro HBV transfected cell lines and mouse model of HBV infection.
Methods
1.Tim-3 expression on lymphocytes from CHB patients.
1.1 Tim-3 expression on lymphocytes from CHB patients.
Fresh heparin-treated blood from CHB patients,healthy controls and patients with fatty liver disease(FLD) was collected to detect Tim-3 expression on PBMCs with flow cytometric analysis.Liver tissues from CHB patients and FLD patients were obtained to detect Tim-3 expression by immunohistochemical staining.
1.2 Correlation between Tim-3 expression on lymphocytes and levels of serum ALT activities and HBV DNA in CHB patients
Spearman correlation analysis was performed between Tim-3 expression on lymphocytes and serum alanine aminotransferase(ALT) activities or HBV DNA titers in CHB patients with Prism GraphPad software.
2.Effects of HBV infection on Tim-3 expression
2.1 Effects of HBV infection on Tim-3 expression in NK92 cells in vitro
Human NK cell line NK92 cells were electroporated with HBV expression plasmid,then grown in 200μg/ml of G418 for 1-2 weeks.Cells electroporated with pcDNA3 served as controls.Tim-3 expression was detected with RT-PCR and flow cytometric analysis.
2.2 The effects of HBV infection on Tim-3 expression in HBV-transgenic (HBV-Tg) mice
Hepatic mononuclear cells(HMCs) from HBV transgenic mice were isolated by metrizamide gradient centrifugation in Percoll,and cells from normal Balb/c mice served as control.Tim-3 expression on HMCs was detected with flow cytometric analysis.
3.The roles of Tim-3 in NK and CD8~+ T cell function
3.1 The roles of Tim-3 in NK cell function
3.1.1 The roles of Tim-3 in NK92 cytotoxicity and cytokine production
NK92 cells were treated with anti-Tim-3/Tim-3-Fc,isotype antibody or PBS respectively,then coincubated with HepG2 or HepG2.2.15 cells for 4h at different E/T ratio.Cytotoxicity was detected with Cell-counting kit-8 and IFN-γsecretion was assayed with ELISA kit
3.1.2 The roles of Tim-3 in PBMCs from CHB patients
PBMCs were obtained from CHB patients as effector cells,treated with anti-Tim-3,isotype antibody or PBS,and coincubated with HepG2 or HepG2.2.15 cells.Cytotoxicity and IFN-γsecretion were detected as above.
3.2 The roles of Tim-3 in hepatic CD8~+ T cell function in mouse model of HBV infection
3.2.1 The hydrodynamics-based mouse model of HBV infection
The mouse model of HBV infection was induced by hydrodynamic injection of HBV expression plasmid pcDNA3-1.1HBV,and mice treated with pcDNA3 served as control.Mice were sacrificed at indicated time point,and levels of HBs antigen,HBs antibody and HBe antigen in the sera were detected with time-resolved immunofluorometric assay(IFMA) kit.HBc expression was detected in hepatocytes with immunohistochemical staining.Serum HBV DNA titers were detected with real-time PCR.Serum ALT activities were measured using the ALT Infinity Reagent.
3.2.2 Tim-3 expression in mouse model of HBV infection
HMCs were obtained from model mice and control mice to detect Tim-3 expression with flow cytometric analysis.
3.2.3 Construction of Tim-3 specific shRNA expression plasmid
Tim-3 specific short hairpin RNAs(shRNAs) or unrelated shRNA oligos were designed and chemical synthesized.After annealing,shRNA oligos were cloned into pmU6 vector and transformed into E.coli DH5α.Positive recombinant plasmids identified by restriction enzyme digestion and DNA sequencing,were named as Tim-3shRNA-1,Tim-3shRNA-2 and unshRNA.
3.2.4 Inhibitory effects of Tim-3shRNA in vitro
shRNA expression plasmids were transfected into murine macrophage line RAW264.7 mediated by lipofectamine 2000.Cells were harvested at 24h,48h and 72h post the transfection to detect Tim-3 andβ-actin expression with RT-PCR and western blot.Expression of Tim-1,another member of Tim family,was detected for the specificity of Tim-3 shRNA.
3.2.5 Effects of Tim-3 shRNA expression plasmid on hepatic CD8~+ T cell function
HMCs were prepared from mice treated with hydrodynamic injection of pcDNA3-1.1HBV with Tim-3shRNA or unshRNA.Tim-3 expression and IFN-γproduction were analyzed by flow cytometry.
4.Statistical analyses
All data were analyzed using the GraphPad Prism 4.The Kruskal-Wallis nonparametric H test and Mann-Whitney nonparametric U test were used for comparison between groups.Spearman correlation analysis was performed between Tim-3 expression on CD8~+ T cells and serum ALT levels or HBV DNA.Value of p＜0.05 or p＜0.01 is considered as significant difference.
Results
1.Tim-3 expression was increased on PBMCs and HMCs from CHB patients
1.1 Tim-3 expression was augmented on PBMCs,especially on NK cells and CD8~+ T cells from CHB patients
Flow cytometric analysis showed that Tim-3 expression was significantly increased on PBMCs from CHB patients compared to healthy controls and FLD patients(p＜0.05).Further analysis showed that Tim-3 expression was abundant on NK cells,and was significantly increased on NK cells from CHB patients compared to healthy controls and FLD patients(p＜0.05).Besides,Tim-3 was significantly up-regulated on CD8~+ T cells from CHB patients compared to controls(p＜0.0001).
1.2 Tim-3 expression was increased in liver tissue from CHB patients
Immunohistochemical staining showed that CHB patients exhibited an increased number of Tim-3~+ cells compared to FLD controls.Morphologically,these cells appeared to be infiltrating lymphocytes.
1.3 Tim-3 expression on T cells or CD8~+ T cells was correlated with ALT levels in sera from CHB patients
Spearman correlation analysis showed that Tim-3 expression on T cells or CD8~+ T cells was positively correlated with serum ALT levels in CHB patients(p＜0.01). There was no correlation between Tim-3 expression and HBV DNA titers.
2.Up-regulation of Tim-3 by HBV infection
2.1 Up-regulation of Tim-3 on NK92 cells transfected with HBV expression plasmid
Semi-quantitative RT-PCR revealed that HBx and HBc mRNA could be detected in NK92 cells transfected with pcDNA3-1.1HBV(NK92-HBV) which showed higher Tim-3 expression than cells transfected with control plasmid.Consistent with RT-PCR, flow cytometric analysis showed that Tim-3 protein was significantly increased on NK92-HBV cells.
2.2 Up-regulation of Tim-3 on HMCs and NK cells from HBV transgenic mice.
Flow cytometric analysis showed that Tim-3 expression was significantly up-regulated on HMCs and NK cells from HBV transgenic mice compared to control mice(p＜0.05).
3.Regulatory roles of Tim-3 in NK and CD8~+ T cell function
3.1 Tim-3 blockade up-regulates cytotoxicity of NK cells
3.1.1 Tim-3 blockade up-regulates cytotoxicity of NK92 cells
1) Cytotoxicity:CCK-8 assay showed an increase in cytotoxicity in anti-Tim-3/Tim-3-Fc-treated NK92 cells against HepG2 or HepG2.2.15 cells at different E/T ratio,compared to isotype-and PBS-treated control(p＜0.05 or p＜0.01).However,Tim-3 blockade could not up-regulate the cytotoxicity of NK92 cells against K562 cells.Semi-quantitative RT-PCR was used to detect galectin-9 expression in HepG2,HepG2.2.15 and K562 cells.Our data showed that galectin-9 was abundant in HepG2 and HepG2.2.15 cells,and in low levels in K562 cells.
2) IFN-γproduction:After coincubation of NK92 cells and HepG2.2.15 cells for 4h,cell culture supernatant was collected to detect IFN-γproduction with ELISA kit.Our data showed that compared to isotype control and PBS control,Tim-3 blockade could significantly up-regulate IFN-γproduction from NK92 cells against HepG2.2.15 cells(p＜0.05).
3.1.2 Tim-3 blockade up-regulates cytotoxicity of PBMCs from CHB patients
PBMCs from CHB patients were obtained and incubated with HepG2 or HepG2.2.15 cells for 4h at different E/T ratio.CCK-8 reagent was used to detect cytotoxicity.Our data showed that compared to isotype control and PBS control, Tim-3 blocking antibody could up-regulate cytotoxicity of PBMCs from CHB patients(p＜0.05).
3.2 Tim-3 knockdown increased IFN-γproduction of hepatic CD8~+ T cells from mouse model of HBV infection
3.2.1 The mouse model of HBV-induced hepatitis
After hydrodynamic injection of pCDNA3-HBV1.1,HBV antigens(HBsAg, HBeAg) and antibody(anti-HBs) were detected in sera with IFMA kit.The production of neutralizing antibody against HBsAg(anti-HBs) became detectable from day 7,accompanying with the decline of HBsAg.Real-time PCR showed that HBV DNA appeared after day 1,peaked at day 7,and became undetectable at day 18 (＜10~3 copies/ml),indicating HBV replication in model mice.Immunohistochemical staining clearly showed HBcAg~+ hepatocytes in HBV model mice,illustrating HBV expression in hepatocytes.ALT activities were slightly increased in both HBV model mice and control mice at day 1 and 3,then declined to normal levels.
3.2.2 Increased Tim-3 expression on hepatic T cells,especially on CD8~+ T cells from HBV model mice
Flow cytometric analysis showed that Tim-3 was slightly induced on hepatic T cells from both HBV model mice and control mice at day 1 and 3.However,no difference in Tim-3 expression was found in two groups.In contrast,Tim-3 expression was dramatically increased in HBV model mice from day 7 to day 18(p＜0.05 at day 7 and 18,p＜0.01 at day 10 and 14).The increase in Tim-3 expression was prominent on CD8~+ T cells(p＜0.05 at day 10),especially on IFN-γ-producing CD8~+ T cells.However,we did not detect any changes in Tim-3 expression in the spleen.
3.2.3 Relationship between Tim-3 expression on CD8~+ T cells and serum neutralizing antibody in HBV model mice
At day 10 post the hydradynamic injection,certain amount of neutralizing antibody against HBsAg could be detected in HBV model mice although antibody titer differed in various mice.HBV model mice were divided into two groups based on the serum anti-HBs levels.A total of 50 mIU/ml was taken as the cutoff value of anti-HBs production since it closes to the average anti-HBs production(58 mIU/ml) of three independent experiments in the study.It was showed that mice with serum anti-HBs production＞50 mIU/ml(HBV model-anti-HBs~H) had a higher expression of Tim-3 on hepatic CD8~+ T cells than mice with serum anti-HBs production＜50 mIU/ml(HBV model-anti-HBs~L)(p＜0.05)
3.3.4 Tim-3shRNA expression plasmid effectively inhibits Tim-3 expression in vitro
Tim-3shRNA expression plasmids and unrelated shRNA control plasmid were successfully constructed and named as Tim-3shRNA-1,Tim-3shRNA-2 and unshRNA.Transfection of Tim-3shRNA-1 suppressed Tim-3 mRNA and protein expression in murine macrophage cell line RAW264.7(compared to control shRNA (unshRNA),p＜0.05).This suppression was specific because the transfection of Tim-3shRNA-1 had no inhibitory effect on Tim-1,another Tim family member.
3.3.5 Tim-3 knockdown up-regulates IFN-γproduction from hepatic CD8~+ T cells in vivo
Flow cytometric analysis showed that Tim-3 expression on hepatic CD8~+ T cells from HBV model mice treated with Tim-3shRNA-1 was significantly decreased than that from unshRNA-treated HBV model mice(p＜0.05).Further analysis showed that IFN-γproduction was also significantly increased in hepatic CD8~+ T cells from Tim-3shRNA-1-treated HBV model mice(p＜0.05).
3.3.6 Tim-3 knockdown could down-regulate the titer of serum neutralizing antibody
Our data showed that production of serum anti-HBs decreased in Tim-3shRNA-1-treated HBV model mice compared to that in unshRNA-treated HBV model mice(p＜0.05).
Conclusions
1.Compared to controls,Tim-3 expression on PBMCs(especially on NK cells and CD8~+ T cells) and HMCs was significantly increased in CHB patients.
2.Tim-3 was up-regulated on NK cells and CD8~+ T cells from HBV-Tg mice and hydrodynamics-based HBV model mice.Consistently,HBV transfection in NK92 cells leads to Tim-3 up-regulation.All indicates that HBV infection could induce increased Tim-3 expression.
3.Tim-3 blockade could up-regulate cytotoxicity and IFN-γsecretion from NK92 cells and PBMCs in CHB patients.
4.Tim-3 knockdown with shRNA expression plasmid could up-regulate IFN-γproduction from hepatic CD8~+ T cells,accompanied with decreased levels of serum neutralizing antibody.
Innovations and significance
1.In this study,we firstly reported the increased expression of Tim-3 on PBMCs (especially on NK cells and CD8~+ T cells) and HMCs from CHB patients,which might be a basic for further mechanism study and act as a new target for clinical therapy.
2.In this study three different models were involved:NK92 cells transfected with HBV expression plasmid(NK92-HBV),HBV-Tg mice and mouse model of HBV-induced hepatitis,to investigate the possibility that HBV infection could up-regulate Tim-3 expression,and to supply new data for the involvement of Tim-3 in HBV infection..
3.NK92 cells and PBMCs from hepatitis B patients were used to investigate the possibility that Tim-3 blockade might help to up-regulate the cytotoxicity of NK cells, indicating Tim-3 might be a new therapeutic target for chronic HBV infection.
4.With Tim-3 specific shRNA expression plasmid,we found that Tim-3 knockdown could up-regulate IFN-γfrom CD8~+ T cells in the mouse model of HBV-induced hepatitis.We also detected decreased levels of neutralizing antibody,indicating that in HBV infection Tim-3 may act as a potent regulator of hepatic CD8~+ T cells and indirectly affect neutralizing antibody production by regulating CD8~+ T cell function.
众多研究表明,多种免疫调节分子在HBV感染所致淋巴细胞功能失调中发挥着重要作用。Tim-3(T cell immunoglobulin- and mucin-domain-containingmolecule-3,Tim-3)是在2002年被发现的,特异性表达在分化终末期的Th1细胞上的新型免疫调节分子。Tim-3与其配体之一—galectin-9相互作用可负向调控IFN-γ的产生,并可影响T细胞耐受的形成。近来的研究结果显示,Tim-3除了在Th1细胞上表达外,在细胞毒性T细胞(Te1细胞)、树突状细胞和NK细胞上均有表达,并且通过影响这些细胞的功能,参与多种疾病的发生发展过程。我室在前期研究中发现,Tim-3mRNA在慢乙肝患者外周血单个核细胞上的表达升高,提示Tim-3很可能在HBV慢性感染中发挥着重要的作用。本课题利用临床研究、体外细胞实验和动物实验研究Tim-3在慢乙肝发生发展中的表达及功能,为进一步深入阐明HBV感染相关疾病的发病机制提供实验依据。
方法
1.Tim-3在慢乙肝患者体内的表达
1.1 Tim-3在慢乙肝患者体内不同淋巴细胞亚群中的表达
收集慢乙肝患者、健康志愿者和脂肪肝患者新鲜外周肝素抗凝血,流式细胞术检测Tim-3在外周血不同淋巴细胞亚群中的表达。收集慢乙肝患者和脂肪肝患者肝组织,免疫组化检测Tim-3在肝脏中的表达。
1.2慢乙肝患者外周血不同淋巴细胞亚群上Tim-3的表达与血清ALT水平,HBVDNA等的相关性分析
利用Prism GraphPad软件对慢乙肝患者外周血不同淋巴细胞亚群上Tim-3的表达与患者血清ALT水平,HBV DNA量进行Spearman相关性分析。
2.HBV感染对Tim-3表达的影响
2.1以人NK92细胞系为研究对象体外模拟研究HBV感染对Tim-3表达的影响
取对数生长期的人NK细胞系NK92细胞,利用电穿孔转染法将HBV表达质粒pcDNA3-1.1HBV转入细胞内,以含200μg/mL G418的培养基稳筛1～2周。同时以转染pcDNA3空载体的NK92细胞为对照细胞。利用RT-PCR和流式细胞术检测Tim-3的表达。
2.2以HBV转基因小鼠为研究模型研究HBV感染对Tim-3表达的影响
利用Percoll分离液密度梯度离心法分离HBV转基因小鼠肝脏内单个核细胞,并以正常Balb/c小鼠作为对照,流式细胞术检测小鼠肝脏内单个核细胞上Tim-3的表达。
3.Tim-3对NK细胞和CD8~6T细胞功能影响的研究
3.1 Tim-3对NK细胞功能影响的研究
3.1.1阻断Tim-3对NK92功能的影响
以NK92细胞为效应细胞,给予Tim-3抗体/Tim-3-Fc融合蛋白、同型抗体或PBS缓冲液作用后,按不同效靶比(1:1、5:1和25:1)与人肝癌细胞系HepG2或HepG2.2.15细胞进行共孵育4小时,CCK-8试剂盒检测细胞杀伤效率,ELISA检测细胞培养上清中IFN-γ分泌。
3.1.2阻断Tim-3对慢乙肝患者外周血淋巴细胞杀伤功能的影响
分离慢乙肝患者外周血单个核细胞为效应细胞,给予Tim-3抗体、同型抗体或PBS缓冲液后,研究效应细胞对靶细胞HepG2或HepG2.2.15细胞的杀伤效率和IFN-γ分泌。
3.2 Tim-3对肝炎模型小鼠肝脏CD8~+T细胞功能影响的研究
3.2.1制备小鼠HBV肝炎模型
利用尾静脉高压注射法将HBV表达质粒pcDNA3-1.1HBV注入小鼠体内制备小鼠HBV肝炎模型,同时以pcDNA3质粒制备小鼠模型对照。在不同时间点处死小鼠,实时免疫荧光检测(IFMA)试剂盒检测小鼠血清中表面抗原、表面抗体和e抗原的水平,免疫组化检测肝细胞内HBc的表达。实时定量PCR检测血清中HBV DNA的水平。谷丙转氨酶(ALT/GPT)检测试剂盒测定血清谷丙转氨酶活性。
3.2.2 Tim-3在模型小鼠肝脏内单个核细胞上的表达
分离模型小鼠和对照小鼠肝脏内单个核细胞,流式细胞术检测Tim-3在肝脏内单个核细胞上表达。
3.2.3构建特异针对Tim-3的shRNA表达载体
设计并合成针对Tim-3或无关序列的shRNA寡核苷酸,退火后与shRNA表达载体pmU6相连,转化大肠杆菌感受态细胞DH5α,经抗性筛选、酶切、DNA测序鉴定获得阳性重组子,并被分别命名为Tim-3shRNA-1,Tim-3shRNA-2和unshRNA。
3.2.4体外验证Tim-3 shRNA表达载体的抑制效果
利用脂质体转染法将shRNA表达质粒转染入小鼠巨噬细胞系RAW264.7细胞,24、48和72小时后收集细胞,RT-PCR和western blot检测Tim-3和β-actin表达,同时检测Tim家族另一成员—Tim-1的表达以验证Tim-3 shRNA抑制效果的特异性。
3.2.5 shRNA抑制Tim-3表达对小鼠肝炎模型肝脏内CD8~+T细胞功能的影响
利用尾静脉高压注射法将pcDNA3-1.1HBV与Tim-3shRNA或unshRNA表达载体注入小鼠体内,流式细胞术检测肝脏内单个核细胞上Tim-3的表达水平以验证Tim-3shRNA的体内抑制效果,同时检测细胞内IFN-γ表达研究Tim-3被抑制后对细胞功能的影响。
4.统计学分析
采用GraphPad Prism4软件对实验数据进行Mann-Whitney非参数U检验、Kruskal-Wallis非参数H检验和Spearman相关性分析。p＜0.05或p＜0.01有统计学意义。
结果
1.Tim-3在慢乙肝患者外周血单个核细胞和肝脏内单个核细胞上表达升高
1.1 Tim-3在慢乙肝患者外周血单个核细胞,尤其是NK细胞和CD8~+T细胞上表达升高
流式细胞术检测结果显示,Tim-3在慢乙肝患者外周血单个核细胞上的表达显著高于健康志愿者和脂肪肝患者对照(p＜0.05)。进一步的分析结果显示,Tim-3在NK细胞上高表达,而且慢乙肝患者外周血NK细胞上的Tim-3的表达水平显著高于健康志愿者和脂肪肝患者(p＜0.05)。此外,Tim-3在T细胞,尤其是CD8~+T细胞上的表达显著高于健康志愿者和脂肪肝患者(p＜0.0001)。
1.2 Tim-3在慢乙肝患者肝脏内单个核细胞上表达升高
为了进一步研究Tim-3在慢乙肝患者肝脏内的表达,我们收集慢乙肝患者和脂肪肝患者肝穿标本,利用免疫组化的方法研究Tim-3在肝脏内的表达。结果显示,慢乙肝患者肝组织中Tim-3的表达显著高于脂肪肝患者,而且从细胞形态上可以看出Tim-3主要在肝脏内浸润淋巴细胞上表达。
1.3慢乙肝患者外周血T细胞和CD8~+T细胞上Tim-3表达水平与ALT水平正相关
统计学分析发现,T细胞和CD8~+T细胞上Tim-3表达水平与患者血清ALT水平均呈正相关(r=0.41,p＜0.01),而与HBV DNA滴度并不相关,提示Tim-3很可能通过调节CD8~+T细胞的功能参与肝损伤过程。
2.HBV感染可上调Tim-3表达
2.1 HBV表达质粒转染NK92细胞后可上调Tim-3的表达
已有研究结果显示在慢乙肝患者外周血单个核细胞和NK细胞上可检测到HBV的肝外感染。为研究HBV感染是否可以上调细胞中Tim-3表达,本研究利用HBV表达质粒pcDNA3-1.1HBV转染的NK92细胞,作为HBV体外感染的细胞模型(空载体pcDNA3转染组为对照)。半定量RT-PCR的结果显示,在pcDNA3-1.1HBV转染的NK92细胞中检测到HBx和HBc mRNA表达,并且检测到Tim-3的表达上调,显著高于空载体对照;流式细胞术检测结果显示,Tim-3蛋白在HBV表达质粒转染的NK92细胞上的表达显著高于空载体对照,与RT-PCR结果相吻合。
2.2 HBV转基因小鼠肝脏内单个核细胞以及NK细胞上Tim-3的表达升高
HBV转基因小鼠模型是目前较为公认的HBV感染模型,本研究利用该模型进一步研究HBV感染对Tim-3表达的影响。流式细胞术分析结果显示:在HBV转基因小鼠肝脏内单个核细胞以及NK细胞上Tim-3的表达水平显著高于正常对照小鼠(p＜0.05)。
3 Tim-3抑制NK细胞和CD8~+T细胞的功能
3.1 Tim-3阻断后可上调NK细胞的杀伤功能
3.1.1 Tim-3阻断后可以上调NK92细胞的杀伤功能
为了研究Tim-3对NK细胞功能的影响,我们利用Tim-3的中和抗体或Tim-3-Fc融合蛋白阻断Tim-3的信号通路,研究Tim-3对NK92细胞杀伤和细胞因子分泌的影响。
1)细胞杀伤率:CCK-8检测结果显示,随着Tim-3中和抗体或Tim-3-Fc融合蛋白作用浓度的增加,其NK92细胞细胞杀伤效率呈现明显的剂量依赖性,当Tim-3中和抗体或Tim-3-Fe融合蛋白作用浓度为5μg/mL时,NK92细胞的杀伤率达到平台期,因此,将该浓度确定为随后实验的作用浓度。在不同效靶比条件下,Tim-3中和抗体或Tim-3-Fc融合蛋白处理组NK92细胞杀伤率显著高于同型抗体对照和PBS缓冲液对照作用的细胞(p＜0.05或p＜0.01),且该作用呈现明显的剂量依赖性。然而Tim-3抗体阻断后并不能上调NK92细胞对K562细胞的杀伤效率。半定量RT-PCR检测结果显示:galectin-9在HepG2和HepG2.2.15细胞上表达水平较高,而在K562细胞上表达水平较低。
2) IFN-γ的分泌:将NK92细胞与HepG2.2.15细胞(效靶比25:1)共同培养4小时后,分离细胞培养上清,ELISA检测IFN-γ的分泌。结果显示:与同型抗体对照和PBS缓冲液相比,Tim-3抗体阻断可显著上调NK92细胞在杀伤HepG2.2.15细胞时的IFN-γ分泌(同型对照抗体组421±19 pg/mL,PBS缓冲液对照402±32pg/mL,Tim-3抗体阻断组521±27 pg/mL)(p＜0.05)。
3.1.2 Tim-3抗体阻断后可上调慢乙肝患者外周血单个核细胞的杀伤效率
慢乙肝患者体内NK细胞上存在Tim-3的表达升高,提示Tim-3有可能通过影响NK细胞的功能参与HBV慢性感染过程。为了验证这种可能性,我们分离慢乙肝患者外周血单个核细胞,按不同效靶比(5:1、10:1和25:1),与HepG2或HepG2.2.15细胞共培养4小时后,CCK-8检测各组细胞杀伤率。结果显示,与同型抗体对照和PBS缓冲液对照相比,Tim-3抗体阻断后可上调慢乙肝患者外周血单个核细胞的杀伤效率(p＜0.05)。
3.2 shRNA抑制Tim-3表达可上调HBV小鼠肝炎模型肝脏内CD8~+T细胞功能
本研究发现,除NK外,慢乙肝患者外周血CD8~+T细胞的Tim-3表达明显上调,提示Tim-3可能影响CD8~+T细胞功能。由于临床分离慢乙肝患者CD8~+T细胞数量少、杀伤能力差、且靶细胞不易选择,本实验以小鼠肝炎模型研究Tim-3对CD8~+T细胞功能的影响。HBV转基因鼠对HBV完全耐受,不能完全反映慢乙肝患者体内情况。为此,本研究利用尾静脉高压注射法制备HBV小鼠肝炎模型,进一步研究HBV感染对Tim-3表达的影响。
3.2.1制备HBV小鼠肝炎模型
尾静脉高压注射法制备HBV小鼠肝炎模型是近年来建立并得到广泛认肯的肝炎模型。本实验利用该模型进一步验证HBV感染对Tim-3表达的上调作用。实时定量PCR检测发现模型制备第1天后可检测到小鼠血清HBV DNA,并于第7天达高峰,至第18天检测不到。免疫组化方法证实模型小鼠肝细胞内表达HBc。IFMA试剂盒检测结果显示随着抗原水平的降低,模型制备后第7天可以检测到血清中的表面抗体。模型小鼠和对照小鼠血清谷丙转氨酶水平于第1到3天升高,随后降低至正常水平,两者之间没有显著性差异。
3.2.2 Tim-3在HBV肝炎模型小鼠肝脏内CD8~+T细胞上表达升高
模型制备后在不同时间点处死小鼠,分离小鼠肝脏内单个核细胞,利用流式细胞术检测Tim-3表达。结果显示模型制备后第1天和第3天,模型小鼠和对照小鼠肝脏内T细胞上Tim-3的表达显著升高,但两组之间没有差异。但在第7天至第18天,模型小鼠肝脏内T细胞上Tim-3表达显著高于对照小鼠(p＜0.05或p＜0.01)。进一步分析发现主要是肝脏内CD8~+T细胞上Tim-3表达显著升高(第10天,p＜0.01)。在脾脏中并未检测到Tim-3表达升高。
3.2.3 HBV模型小鼠肝脏内CD8~+T细胞上Tim-3表达升高伴随血清中和抗体滴度升高
进一步的分析发现:在模型制备后第10天,HBV模型小鼠体内可以检测到较高水平的针对表面抗原的中和抗体,但小鼠之间抗体滴度水平的差异较大。将HBV模型小鼠以表面抗体水平50 mIU/mL(接近模型小鼠抗体水平的均值-58mIU/mL)为界分为两组:高抗体组(＞50 mIU/mL)和低抗体组(＜50 mIU/mL)。分析结果显示高抗体组模型小鼠肝脏内CD8~+T细胞上Tim-3的表达显著高于低抗体组(p＜0.05)。
3.2.4 Tim-3shRNA表达载体可有效抑制RAW264.7细胞上Tim-3的表达
为了进一步研究Tim-3对CD8~+T细胞功能的影响,我们成功构建Tim-3shRNA表达载体Tim-3shRNA-1和Tim-3shRNA-2,以及针对无关序列的unshRNA表达载体,并转染RAW264.7细胞,半定量RT-PCR和Western blot检测结果显示,Tim-3shRNA-1转染组细胞与其他组细胞相比,Tim-3 mRNA水平和蛋白质水平的表达均显著降低(p＜0.05),并且这种抑制效果是特异的,并不影响Tim家族另一成员Tim-1的表达。
3.2.5 shRNA抑制Tim-3表达可上调HBV小鼠肝炎模型肝脏内CD8~+T细胞功能
上述针对Tim-3的shRNA表达载体与HBV表达质粒同时以尾静脉高压注射法注入小鼠,体内研究Tim-3对模型小鼠CD8~+T细胞功能的影响。流式细胞术结果显示:于模型制备后第10天Tim-3shRNA-1处理组CD8~+T细胞上Tim-3的表达水平降低,显著低于无关对照unshRNA处理组Tim-3表达未受影响(p＜0.05),且Tim-3shRNA-1处理组CD8~+T细胞上IFN-γ表达水平显著高于后者(p＜0.05)。
3.2.6 shRNA抑制Tim-3表达可下调血清中和抗体的水平
进一步对小鼠血清中和抗体的检测发现:pcDNA3-1.1HBV+Tim-3shRNA-1组小鼠血清中和抗体水平显著低于pcDNA3-1.1HBV+unshRNA组(p＜0.05)。
结论
1.慢乙肝患者外周血单个核细胞,尤其是NK细胞和CD8~+T细胞,及肝脏内单个核细胞上Tim-3表达显著高于对照者。
2.HBV感染可上调NK92细胞和HBV转基因小鼠以及尾静脉高压法制备的肝炎模型小鼠肝脏内NK细胞或CD8~+T细胞上Tim-3的表达。
3.Tim-3途径阻断可上调NK92细胞和慢乙肝患者外周血单个核细胞的细胞杀伤效率和IFN-γ分泌。
4.shRNA表达载体抑制Tim-3表达,可上调模型小鼠肝脏内CD8~+T细胞IFN-γ表达,同时模型小鼠中和抗体水平降低。
创新点及意义
1.本研究发现慢乙肝患者外周血单个核细胞(尤其NK细胞和CD8~+T细胞)和肝脏内单个核细胞上Tim-3表达显著升高,并分别利用转染了HBV表达质粒的NK92细胞和HBV转基因小鼠为模型,首次证实HBV感染可上调Tim-3的表达,为Tim-3参与HBV慢性感染过程提出新的论点。
2.本研究利用NK92细胞系和慢乙肝患者外周血单个核细胞,证实阻断Tim-3途径可上调NK细胞的杀伤效率,提示Tim-3有可能作为慢乙肝治疗的新靶点。
3.本研究利用特异针对Tim-3的shRNA表达载体抑制小鼠肝炎模型肝内Tim-3表达,发现抑制Tim-3表达可上调CD8~+T细胞IFN-γ的表达,且降低模型小鼠血清中和抗体水平,提出Tim-3可抑制CD8~+T细胞的功能,并间接影响中和抗体产生的观点。
Hepatitis B virus(HBV) primarily infects hepatocytes,which causes a series of necroinflammatory liver diseases worldwide.Persistent carriers of HBV,however, show a severe dysfunction in HBV-specific immune response.However,the mechanisms have not been well-defined.
It has been shown that immune regulatory molecules play important roles in HBV-induced lymphocyte dysfunction.Tim-3 was identified to be specifically expressed on polarized Th1 cells in 2002,and might play great roles in immune regulation and tolerance induction.Engagement of Tim-3 by its ligand-galectin-9 negatively regulates IFN-γproduction and influences the ability to induce T cell tolerance.Recent studies have shown that Tim-3 is also expressed on Tcl cells, dendritic cells and natural killer cells,and may involve in the pathogenesis of a range of diseases by influencing these cell function.In our early studies,we demonstrated that Tim-3 mRNA is up-regulated on peripheral blood mononuclear cells(PBMCs) from patients with chronic hepatitis B(CHB),indicating Tim-3 might play an important role in the development of CHB.In this study,we investigated the expression and functional role of Tim-3 in CHB by clinical samples,in vitro HBV transfected cell lines and mouse model of HBV infection.
Methods
1.Tim-3 expression on lymphocytes from CHB patients.
1.1 Tim-3 expression on lymphocytes from CHB patients.
Fresh heparin-treated blood from CHB patients,healthy controls and patients with fatty liver disease(FLD) was collected to detect Tim-3 expression on PBMCs with flow cytometric analysis.Liver tissues from CHB patients and FLD patients were obtained to detect Tim-3 expression by immunohistochemical staining.
1.2 Correlation between Tim-3 expression on lymphocytes and levels of serum ALT activities and HBV DNA in CHB patients
Spearman correlation analysis was performed between Tim-3 expression on lymphocytes and serum alanine aminotransferase(ALT) activities or HBV DNA titers in CHB patients with Prism GraphPad software.
2.Effects of HBV infection on Tim-3 expression
2.1 Effects of HBV infection on Tim-3 expression in NK92 cells in vitro
Human NK cell line NK92 cells were electroporated with HBV expression plasmid,then grown in 200μg/ml of G418 for 1-2 weeks.Cells electroporated with pcDNA3 served as controls.Tim-3 expression was detected with RT-PCR and flow cytometric analysis.
2.2 The effects of HBV infection on Tim-3 expression in HBV-transgenic (HBV-Tg) mice
Hepatic mononuclear cells(HMCs) from HBV transgenic mice were isolated by metrizamide gradient centrifugation in Percoll,and cells from normal Balb/c mice served as control.Tim-3 expression on HMCs was detected with flow cytometric analysis.
3.The roles of Tim-3 in NK and CD8~+ T cell function
3.1 The roles of Tim-3 in NK cell function
3.1.1 The roles of Tim-3 in NK92 cytotoxicity and cytokine production
NK92 cells were treated with anti-Tim-3/Tim-3-Fc,isotype antibody or PBS respectively,then coincubated with HepG2 or HepG2.2.15 cells for 4h at different E/T ratio.Cytotoxicity was detected with Cell-counting kit-8 and IFN-γsecretion was assayed with ELISA kit
3.1.2 The roles of Tim-3 in PBMCs from CHB patients
PBMCs were obtained from CHB patients as effector cells,treated with anti-Tim-3,isotype antibody or PBS,and coincubated with HepG2 or HepG2.2.15 cells.Cytotoxicity and IFN-γsecretion were detected as above.
3.2 The roles of Tim-3 in hepatic CD8~+ T cell function in mouse model of HBV infection
3.2.1 The hydrodynamics-based mouse model of HBV infection
The mouse model of HBV infection was induced by hydrodynamic injection of HBV expression plasmid pcDNA3-1.1HBV,and mice treated with pcDNA3 served as control.Mice were sacrificed at indicated time point,and levels of HBs antigen,HBs antibody and HBe antigen in the sera were detected with time-resolved immunofluorometric assay(IFMA) kit.HBc expression was detected in hepatocytes with immunohistochemical staining.Serum HBV DNA titers were detected with real-time PCR.Serum ALT activities were measured using the ALT Infinity Reagent.
3.2.2 Tim-3 expression in mouse model of HBV infection
HMCs were obtained from model mice and control mice to detect Tim-3 expression with flow cytometric analysis.
3.2.3 Construction of Tim-3 specific shRNA expression plasmid
Tim-3 specific short hairpin RNAs(shRNAs) or unrelated shRNA oligos were designed and chemical synthesized.After annealing,shRNA oligos were cloned into pmU6 vector and transformed into E.coli DH5α.Positive recombinant plasmids identified by restriction enzyme digestion and DNA sequencing,were named as Tim-3shRNA-1,Tim-3shRNA-2 and unshRNA.
3.2.4 Inhibitory effects of Tim-3shRNA in vitro
shRNA expression plasmids were transfected into murine macrophage line RAW264.7 mediated by lipofectamine 2000.Cells were harvested at 24h,48h and 72h post the transfection to detect Tim-3 andβ-actin expression with RT-PCR and western blot.Expression of Tim-1,another member of Tim family,was detected for the specificity of Tim-3 shRNA.
3.2.5 Effects of Tim-3 shRNA expression plasmid on hepatic CD8~+ T cell function
HMCs were prepared from mice treated with hydrodynamic injection of pcDNA3-1.1HBV with Tim-3shRNA or unshRNA.Tim-3 expression and IFN-γproduction were analyzed by flow cytometry.
4.Statistical analyses
All data were analyzed using the GraphPad Prism 4.The Kruskal-Wallis nonparametric H test and Mann-Whitney nonparametric U test were used for comparison between groups.Spearman correlation analysis was performed between Tim-3 expression on CD8~+ T cells and serum ALT levels or HBV DNA.Value of p＜0.05 or p＜0.01 is considered as significant difference.
Results
1.Tim-3 expression was increased on PBMCs and HMCs from CHB patients
1.1 Tim-3 expression was augmented on PBMCs,especially on NK cells and CD8~+ T cells from CHB patients
Flow cytometric analysis showed that Tim-3 expression was significantly increased on PBMCs from CHB patients compared to healthy controls and FLD patients(p＜0.05).Further analysis showed that Tim-3 expression was abundant on NK cells,and was significantly increased on NK cells from CHB patients compared to healthy controls and FLD patients(p＜0.05).Besides,Tim-3 was significantly up-regulated on CD8~+ T cells from CHB patients compared to controls(p＜0.0001).
1.2 Tim-3 expression was increased in liver tissue from CHB patients
Immunohistochemical staining showed that CHB patients exhibited an increased number of Tim-3~+ cells compared to FLD controls.Morphologically,these cells appeared to be infiltrating lymphocytes.
1.3 Tim-3 expression on T cells or CD8~+ T cells was correlated with ALT levels in sera from CHB patients
Spearman correlation analysis showed that Tim-3 expression on T cells or CD8~+ T cells was positively correlated with serum ALT levels in CHB patients(p＜0.01). There was no correlation between Tim-3 expression and HBV DNA titers.
2.Up-regulation of Tim-3 by HBV infection
2.1 Up-regulation of Tim-3 on NK92 cells transfected with HBV expression plasmid
Semi-quantitative RT-PCR revealed that HBx and HBc mRNA could be detected in NK92 cells transfected with pcDNA3-1.1HBV(NK92-HBV) which showed higher Tim-3 expression than cells transfected with control plasmid.Consistent with RT-PCR, flow cytometric analysis showed that Tim-3 protein was significantly increased on NK92-HBV cells.
2.2 Up-regulation of Tim-3 on HMCs and NK cells from HBV transgenic mice.
Flow cytometric analysis showed that Tim-3 expression was significantly up-regulated on HMCs and NK cells from HBV transgenic mice compared to control mice(p＜0.05).
3.Regulatory roles of Tim-3 in NK and CD8~+ T cell function
3.1 Tim-3 blockade up-regulates cytotoxicity of NK cells
3.1.1 Tim-3 blockade up-regulates cytotoxicity of NK92 cells
1) Cytotoxicity:CCK-8 assay showed an increase in cytotoxicity in anti-Tim-3/Tim-3-Fc-treated NK92 cells against HepG2 or HepG2.2.15 cells at different E/T ratio,compared to isotype-and PBS-treated control(p＜0.05 or p＜0.01).However,Tim-3 blockade could not up-regulate the cytotoxicity of NK92 cells against K562 cells.Semi-quantitative RT-PCR was used to detect galectin-9 expression in HepG2,HepG2.2.15 and K562 cells.Our data showed that galectin-9 was abundant in HepG2 and HepG2.2.15 cells,and in low levels in K562 cells.
2) IFN-γproduction:After coincubation of NK92 cells and HepG2.2.15 cells for 4h,cell culture supernatant was collected to detect IFN-γproduction with ELISA kit.Our data showed that compared to isotype control and PBS control,Tim-3 blockade could significantly up-regulate IFN-γproduction from NK92 cells against HepG2.2.15 cells(p＜0.05).
3.1.2 Tim-3 blockade up-regulates cytotoxicity of PBMCs from CHB patients
PBMCs from CHB patients were obtained and incubated with HepG2 or HepG2.2.15 cells for 4h at different E/T ratio.CCK-8 reagent was used to detect cytotoxicity.Our data showed that compared to isotype control and PBS control, Tim-3 blocking antibody could up-regulate cytotoxicity of PBMCs from CHB patients(p＜0.05).
3.2 Tim-3 knockdown increased IFN-γproduction of hepatic CD8~+ T cells from mouse model of HBV infection
3.2.1 The mouse model of HBV-induced hepatitis
After hydrodynamic injection of pCDNA3-HBV1.1,HBV antigens(HBsAg, HBeAg) and antibody(anti-HBs) were detected in sera with IFMA kit.The production of neutralizing antibody against HBsAg(anti-HBs) became detectable from day 7,accompanying with the decline of HBsAg.Real-time PCR showed that HBV DNA appeared after day 1,peaked at day 7,and became undetectable at day 18 (＜10~3 copies/ml),indicating HBV replication in model mice.Immunohistochemical staining clearly showed HBcAg~+ hepatocytes in HBV model mice,illustrating HBV expression in hepatocytes.ALT activities were slightly increased in both HBV model mice and control mice at day 1 and 3,then declined to normal levels.
3.2.2 Increased Tim-3 expression on hepatic T cells,especially on CD8~+ T cells from HBV model mice
Flow cytometric analysis showed that Tim-3 was slightly induced on hepatic T cells from both HBV model mice and control mice at day 1 and 3.However,no difference in Tim-3 expression was found in two groups.In contrast,Tim-3 expression was dramatically increased in HBV model mice from day 7 to day 18(p＜0.05 at day 7 and 18,p＜0.01 at day 10 and 14).The increase in Tim-3 expression was prominent on CD8~+ T cells(p＜0.05 at day 10),especially on IFN-γ-producing CD8~+ T cells.However,we did not detect any changes in Tim-3 expression in the spleen.
3.2.3 Relationship between Tim-3 expression on CD8~+ T cells and serum neutralizing antibody in HBV model mice
At day 10 post the hydradynamic injection,certain amount of neutralizing antibody against HBsAg could be detected in HBV model mice although antibody titer differed in various mice.HBV model mice were divided into two groups based on the serum anti-HBs levels.A total of 50 mIU/ml was taken as the cutoff value of anti-HBs production since it closes to the average anti-HBs production(58 mIU/ml) of three independent experiments in the study.It was showed that mice with serum anti-HBs production＞50 mIU/ml(HBV model-anti-HBs~H) had a higher expression of Tim-3 on hepatic CD8~+ T cells than mice with serum anti-HBs production＜50 mIU/ml(HBV model-anti-HBs~L)(p＜0.05)
3.3.4 Tim-3shRNA expression plasmid effectively inhibits Tim-3 expression in vitro
Tim-3shRNA expression plasmids and unrelated shRNA control plasmid were successfully constructed and named as Tim-3shRNA-1,Tim-3shRNA-2 and unshRNA.Transfection of Tim-3shRNA-1 suppressed Tim-3 mRNA and protein expression in murine macrophage cell line RAW264.7(compared to control shRNA (unshRNA),p＜0.05).This suppression was specific because the transfection of Tim-3shRNA-1 had no inhibitory effect on Tim-1,another Tim family member.
3.3.5 Tim-3 knockdown up-regulates IFN-γproduction from hepatic CD8~+ T cells in vivo
Flow cytometric analysis showed that Tim-3 expression on hepatic CD8~+ T cells from HBV model mice treated with Tim-3shRNA-1 was significantly decreased than that from unshRNA-treated HBV model mice(p＜0.05).Further analysis showed that IFN-γproduction was also significantly increased in hepatic CD8~+ T cells from Tim-3shRNA-1-treated HBV model mice(p＜0.05).
3.3.6 Tim-3 knockdown could down-regulate the titer of serum neutralizing antibody
Our data showed that production of serum anti-HBs decreased in Tim-3shRNA-1-treated HBV model mice compared to that in unshRNA-treated HBV model mice(p＜0.05).
Conclusions
1.Compared to controls,Tim-3 expression on PBMCs(especially on NK cells and CD8~+ T cells) and HMCs was significantly increased in CHB patients.
2.Tim-3 was up-regulated on NK cells and CD8~+ T cells from HBV-Tg mice and hydrodynamics-based HBV model mice.Consistently,HBV transfection in NK92 cells leads to Tim-3 up-regulation.All indicates that HBV infection could induce increased Tim-3 expression.
3.Tim-3 blockade could up-regulate cytotoxicity and IFN-γsecretion from NK92 cells and PBMCs in CHB patients.
4.Tim-3 knockdown with shRNA expression plasmid could up-regulate IFN-γproduction from hepatic CD8~+ T cells,accompanied with decreased levels of serum neutralizing antibody.
Innovations and significance
1.In this study,we firstly reported the increased expression of Tim-3 on PBMCs (especially on NK cells and CD8~+ T cells) and HMCs from CHB patients,which might be a basic for further mechanism study and act as a new target for clinical therapy.
2.In this study three different models were involved:NK92 cells transfected with HBV expression plasmid(NK92-HBV),HBV-Tg mice and mouse model of HBV-induced hepatitis,to investigate the possibility that HBV infection could up-regulate Tim-3 expression,and to supply new data for the involvement of Tim-3 in HBV infection..
3.NK92 cells and PBMCs from hepatitis B patients were used to investigate the possibility that Tim-3 blockade might help to up-regulate the cytotoxicity of NK cells, indicating Tim-3 might be a new therapeutic target for chronic HBV infection.
4.With Tim-3 specific shRNA expression plasmid,we found that Tim-3 knockdown could up-regulate IFN-γfrom CD8~+ T cells in the mouse model of HBV-induced hepatitis.We also detected decreased levels of neutralizing antibody,indicating that in HBV infection Tim-3 may act as a potent regulator of hepatic CD8~+ T cells and indirectly affect neutralizing antibody production by regulating CD8~+ T cell function.
引文
1 Lee WM. Hepatitis B virus infection[J]. N Engl J Med. 1997;337:1733-1745.
2 Grob PJ. Hepatitis B: virus, pathogenesis and treatment[J]. Vaccine. 1998; 16: 11-16.
3 Jung MC, Pape GR. Immunology of hepatitis B infection[J]. Lancet Infect Dis. 2001 ;2: 43-50.
4 Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis[J]. Annu Rev Immunol. 1995;13:29-60.
5 Bertoletti A, Ferrari C, Fiaccadori F. Role of the cell-mediated immune response in the pathogenesis of hepatitis B virus infection: possible immune-therapeutic strategies[J]. Acta Biomed Ateneo Parmense. 1996;67(3-4):87-93.
6 Marcellin P, Castelnau C, Martinot-Peignoux M, et al. Natural history of hepatitis B[J]. Minerva Gastroenterol Dietol. 2005;51(1):63-75.
7 Chu CM. Natural history of chronic hepatitis B virus infection in adults with emphasis on the occurrence of cirrhosis and hepatocellular carcinoma[J]. J Gastroenterol Hepatol. 2000; 15 Suppl:E25-30.
8 Merican I, Guan R, Amarapuka D, et al. Chronic hepatitis B virus infection in Asian countries[J]. J Gastroenterol Hepatol. 2000;15(12):1356-1361.
9 Biron CA. Activation and function of natural killer cell responses during viral infections[J]. Curr Opin Immunol. 1997;9: 24-34.
10 Biron CA, Nguyen KB, Pien GC, et al. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol. 1999; 17:189-220.
11 Vivier E, Tomasello E, Baratin M, et al. Functions of natural killer cells. Nat Immunol. 2008;9(5):503-10.
12 Trinchieri G. Human natural killer cells. Biology of natural killer cells[J]. Adv Immunol. 198947:187-376.
13 Vivier, E, Nunes, JA, Vely, F. Natural killer cell signaling pathways[J]. Science. 2004;306:1517-1519.
14 Martz E, Howell DM, CTL: virus control cells first and cytolytic cells second[J]? Immunol Today. 1989; 10: 79-86.
15 Keefe D, Shi L, Feske S, et al. Perforin triggers a plasma membrane-repair response that facilitates CTL induction of apoptosis[J]. Immunity 2005; 23: 249-262
16 Guidotti LG, Ishikawa T, Hobbs MV, et al. Intracellular inactivation of the hepatitis B virus by cytotoxic T lymphocytes[J]. Immunity. 1996;4: 25-36.
17 Thimme R, Wieland S, Steiger C, et al. CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection[J]. J Virol. 2003; 77: 68-76.
18 Kakimi K, Guidotti LG, Koezuka Y, et al. Natural killer T cell activation inhibits hepatitis B virus replication in vivo[J]. J Exp Med. 2000;192: 921-930.
19 Kimura K, Kakimi K, Wieland S, et al. Activated intrahepatic antigen-presenting cells inhibit hepatitis B virus replication in the liver of transgenic mice[J]. J Immunol. 2002;169: 5188-5195.
20 Pasquetto V, Guidotti LG, Kakimi K, et al. Host-virus interaction during malaria infection in hepatitis B virus transgenic mice[J]. J Exp Med. 2000;192: 529-535.
21 Echevarria S, Casafont F, Miera M, et al. Interleukin-2 and natural killer activity in acute type B hepatitis[J]. Hepatogastroenterology. 1991;38:307-310.
22 Monsalve-De Castillo F, Romero TA, Estevez J, et al. Concentrations of cytokines, soluble interleukin-2 receptor, and soluble CD30 in sera of patients with hepatitis B virus infection during acute and convalescent phases[J]. Clin Diagn Lab Immunol. 2002;9:1372-1375.
23 Ono K, Yamanaga Y, Yamamoto K, et al. Natural killing activities in chronic liver diseases and hepatocellular carcinoma[J]. J Clin Immunol. 1996; 16:41-45.
24 Boni C, Fisicaro P, Valdatta C, et al. Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection[J]. J Virol. 2007; 81: 4215-4225.
25 Ferrari CA, Penna A, Bertoletti, A, et al. Cellular immune response to hepatitis B virus-encoded antigens in acute and chronic hepatitis B virus infection[J]. J Immunol. 1990; 145:3442-3449.
26 Jung ML, Hartmann B, Gerlach JT, et al. Virus-specific lymphokine production differs quantitatively but not qualitatively in acute and chronic hepatitis B infection[J]. Virology. 1999; 261:165-172.
27 Jung ML, Spengler U, Schraut W, et al. Hepatitis B virus antigen-specific T cell activation in patients with acute and chronic hepatitis B[J]. J Hepatol. 1991;13:310-317.
28 Penna A, Chisari FV, Bertoletti A, et al. Cytotoxic T lymphocytes recognize an HLA-A2-restricted epitope within the hepatitis B virus nucleocapsid antigen[J]. J ExpMed. 1991;174:1565-1570.
29 Sobao Y, Tomiyama H, Sugi K, et al. The role of hepatitis B virus-specific memory CD8 T cells in the control of viral replication[J]. J Hepatol. 2002;36:105-115.
30 Dong H, Zhu G, Tamada K, et al. B7-H1 determines accumulation and deletion of intrahepatic CD8(+) T lymphocytes[J]. Immunity. 2004; 20: 327-336.
31 Greenwald RJ, Freeman GJ, Sharpe AH. The B7 family revisited[J]. Annu Rev Immunol. 2005; 23: 515-548.
32 Maier H, Isogawa M, Freeman GJ, et al. PD-1:PD-L1 interactions contribute to the functional suppression of virus-specific CD8+ T lymphocytes in the liver[J]. J Immunol. 2007;178(5):2714-20.
33 Boni C, Fisicaro P, Valdatta C, et al. Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection[J]. J Virol. 2007;81(8):4215-25.
34 Peng G, Li S, Wu W, et al. PD-1 upregulation is associated with HBV-specific T cell dysfunction in chronic hepatitis B patients[J]. Mol Immunol. 2008;45(4):963-70.
35 Zhang Z, Zhang JY, Wherry EJ, et al. Dynamic programmed death 1 expression by virus-specific CD8 T cells correlates with the outcome of acute hepatitis B[J]. Gastroenterology. 2008; 134(7): 1938-49,
36 Cerwenka A, Lanier LL. Natural killer cells, viruses and cancer [J]. Nat Rev Immunol. 2001 ;1: 41-49.
37 Moser JM, Gibbs J, Jensen PE, et al. CD94-NKG2A receptors regulate antiviral CD8 + T cell responses[J]. Nat Immunol. 2002; 3: 189-195.
38 Bertoletti A, Gehring AJ.The immune response during hepatitis B virus infection[J]. J Gen Virol. 2006;87(Pt 6): 1439-49.
39 Lopes AR, Kellam P, Das A, et al. Bim-mediated deletion of antigen specific CD8 T cells in patients unable to control HBV infection[J]. J Clin Invest. 2008; 118: 1835-1845
40 Stoop JN, van der Molen RG, Baan CC, et al. Regulatory T cells contribute to the impaired immune response in patients with chronic hepatitis B virus infection[J]. Hepatology. 2005; 41: 771-778.
41 Stoop JN, van der Molen RG, Kuipers EJ, et al. Inhibition of viral replication reduces regulatory T cells and enhances the antiviral immune response in chronic hepatitis B. Virology. 2007;361(l):141-8.
42 Alatrakchi N, Koziel M. Regulatory T cells and viral liver disease. J Viral Hepat. 2009;16(4):223-9.
43 Monney L, Sabatos CA, Gaglia JL, et al. Th1-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease [J]. Nature. 2002; 415:536-541.
44 Frisancho-Kiss S, Nyland JF, Davis SE, et al.Cutting edge: T cell Ig mucin-3 reduces inflammatory heart disease by increasing CTLA-4 during innate immunity[J]. J Immunol. 2006;176(11):6411-5.
45 Frisancho-Kiss S, Davis SE, Nyland JF, et al. Cutting edge: cross-regulation by TLR4 and T cell Ig mucin-3 determines sex differences in inflammatory heart disease[J]. J Immunol. 2007;178(11):6710-4.
46 Sabatos CA, Chakravarti S, Cha E, et al. Interaction of Tim-3 and Tim-3 ligand regulates T helper type 1 responses and induction of peripheral tolerance[J]. Nat Immunol. 2003;4:1102 -1110.
47 Kuchroo VK, Umetsu DT, DeKruyff RH, et al. The TIM gene family: emerging roles in immunity and disease[J].Nat Rev Immunol.2003;3:454-462.
48 Sanchez-Fueyo A,Tian J,Picarella D,et al.Tim-3 inhibits T helper type 1-mediated auto- and alloimmune responses and promotes immunological tolerance[J].Nat Immunol 2003;4:1093-1101.
49 Khademi M,Illes Z,Gielen AW,et al.T Cell Ig- and mucin-domain-containing molecule-3(TIM-3) and TIM-1 molecules are differentially expressed on human Th1 and Th2 cells and in cerebrospinal fluid-derived mononuclear cells in multiple sclerosis[J].J Immunol 2004;172:7169-7176.
50 Mariat C,Sanchez-Fueyo A,Alexopoulos SP,et al.Regulation of T cell dependent immune responses by TIM family members[J].Philos Trans R Soc Lond B Biol Sci.2005;360(1461):1681-5.
51 Meyers JH,Sabatos CA,Chakravarti S,et al.The TIM gene family regulates autoimmune and allergic diseases[J].Trends Mol Med.2005;11(8):362-9.
52 Zhu C,Anderson AC,Schubart A,et al.The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity[J].Nat Immunol.2005;6:1245-1252.
53 Kuchroo VK,Meyers JH,Umetsu DT,et al.TIM family of genes in immunity and tolerance[J].Adv Immunol.2006;91:227-49.
54 Anderson AC,Anderson DE.TIM-3 in autoimmunity[J].Curr Opin Immunol.2006;18(6):665-9.
55 Oikawa T,Kamimura Y,Akiba H,et al.Preferential involvement of Tim-3 in the regulation of hepatic CD8+ T cells in murine acute graft-versus-host disease[J].J Immunol.2006;177:4281-4287.
56 Koguchi K,Anderson DE,Yang L,et al.Dysregulated T cell expression of TIM3in multiple sclerosis[J].J Exp Med 2006;203:1413-1418.
57 Degauque N,Mariat C,Kenny J,et al.Regulation of T-cell immunity by T-cell immunoglobulin and mucin domain proteins[J].Transplantation.2007;84(1Suppl):S12-6.
58 Yang L,Anderson DE,Kuchroo J,et al,Lack of TIM-3 immunoregulation in multiple sclerosis[J].J Immunol.2008;180(7):4409-14.
59 Su EW,Lin JY,Kane LP.TIM-1 and TIM-3 proteins in immune regulation[J]. Cytokine.2008;44(1):9-13.
60 Umetsu DT,Umetsu SE,Freeman GJ,et al.TIM gene family and their role in atopic diseases[J].Curr Top Microbiol Immunol.2008;321:201-15.
61 Hafler DA,Kuchroo V.TIMs:central regulators of immune responses[J].J Exp Med.2008;205(12):2699-701.
62 Fukushima A,Sumi T,Fukuda K,et al.Roles of galectin-9 in the development of experimental allergic conjunctivitis in mice[J].Int Arch Allergy Immunol.2008;146(1):36-43.
63 Gielen AW,Lobell A,Lidman O,,et al.Expression of T cell immunoglobulin- and mucin-domain-containing molecules-1 and -3(Tim-1 and -3) in the rat nervous and immune systems[J].J Neuroimmunol.2005;164:93-104.
64 Wang Y,Meng J,Wang X,et al.Expression of human TIM-1 and TIM-3 on lymphocytes from systemic lupus erythematosus patients[J].Scand J Immunol.2008;67:63-70.
65 Simmons WJ,Koneru M,Mohindru M,et al.Tim-3+ T-bet+ tumor-specific Th1cells colocalize with and inhibit development and growth of murine neoplasms[J].J Immunol.2005;174:1405-1415.
66 Fukushima A,Sumi T,Fukuda K,et al.Antibodies to T cell Ig and mucin domain-containing proteins(Tim)-1 and -3 suppress the induction and progression of murine allergic conjunctivitis[J].Biochem Biophys Res Commun.2007;353:211-216.
67 Kearley J,McMillan SJ,Lloyd CM.Th2-driven,allergen-induced airway inflammation is reduced after treatment with anti-Tim-3 antibody in vivo[J].J Exp Med.2007;204:1289-1294.
68 Anderson AC,Anderson DE,Bregoli L,et al.Promotion of tissue inflammation by the immune receptor Tim-3 expressed on innate immune cells[J].Science.2007;318:1141-1143.
69 Zhang J,Gu Y,Xu C,et al.Increased T cell immunoglobulin mucin-3 and its ligand in acquired aplastic anemia[J].Eur J Haematol.2008;81(2):130-9.
70 Nagahara K,Arikawa T,Oomizu S,et al.Galectin-9 increases Tim-3+ dendritic cells and CD8+ T cells and enhances antitumor immunity via galectin-9-Tim-3 interactions[J]. J Immunol. 2008; 181(11):7660-9.
71 Jones RB, Ndhlovu LC, Barbour JD, et al. Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection[J]. J Exp Med 2008;205:2763-2779.
72 Sehrawat S, Suryawanshi A, Hirashima M, et al. Role of Tim-3/galectin-9 inhibitory interaction in viral-induced immunopathology: shifting the balance toward regulators[J]. J Immunol. 2009; 182(5):3191-201.
73 Liang X, Liu Y, Zhang Q, et al. Hepatitis B virus sensitizes hepatocytes to TRAIL-induced apoptosis through Bax[J]. J Immunol. 2007;178:503-510.
74 Xiong Y, Jia Y, Wang H, et al. High-level hepatitis B virus expression in transgenic mice[J]. Infect Dis Info. 2000;13:164-165.
75 Trippler M, Meyer zum Buschenfelde KH, Gerken G. HBV viral load within subpopulations of peripheral blood mononuclear cells in HBV infection using limiting dilution PCR[J]. J Virol Methods.l999;78:129-147.
76 Chemin I, Vermot-Desroches C, Baginski I, et al. Selective detection of human hepatitis B virus surface and core antigens in peripheral blood mononuclear cell subsets by flow cytometry[J]. J Viral Hepat.1994;1:39-44.
77 Chisari FV. Hepatitis B virus transgenic mice: insights into the virus and the disease[J]. Hepatology. 1995;22:1316-1325.
78 Chisari FV. Hepatitis B virus transgenic mice: models of viral immunobiology and pathogenesis[J]. Curr Top Microbiol Immunol. 1996;206:149-73.
79 Akbar SK, Onji M. Hepatitis B virus (HBV)-transgenic mice as an investigative tool to study immunopathology during HBV infection[J]. Int J Exp Pathol. 1998;79(5):279-91.
80 Weber OF. Animal models in virology[J]. Contrib Microbiol. 2001;9:15-30.
81 Kekule AS, Lauer U, Weiss L, et al. Trans-activation by hepatitis B virus X protein is mediated via a tumour promoter pathway [J]. Arch Virol Suppl. 1992;4:63-64.
82 Chung TW, Lee YC, Kim CH. Hepatitis B viral HBx induces matrix metalloproteinase-9 gene expression through activation of ERK and PI-3K/AKT pathways: involvement of invasive potential[J]. FASEB J. 2004;18:1123-1125.
83 Lee DK, Park SH, Yi Y, et al. The hepatitis B virus encoded oncoprotein pX amplifies TGF-beta family signaling through direct interaction with Smad4: potential mechanism of hepatitis B virus-induced liver fibrosis[J]. Genes Dev. 2001; 15:455-466.
84 Zou Z, Li B, Xu D, et al. Imbalanced intrahepatic cytokine expression of interferon-gamma, tumor necrosis factor-alpha, and interleukin-10 in patients with acute-on-chronic liver failure associated with hepatitis B virus infection[J]. J Clin Gastroenterol. 2009;43(2): 182-90.
85 Chang JJ, Thompson AJ, Visvanathan K, et al. The phenotype of hepatitis B virus-specific T cells differ in the liver and blood in chronic hepatitis B virus infection[J]. Hepatology 2007;46:1332-1340.
86 Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA[J]. Gene Therapy. 1999;6:1258-1266.
87 Yang PL, Althage A, Chung J, Chisari FV. Hydrodynamic injection of viral DNA: A mouse model of acute hepatitis B virus infection[J]. Proc Natl Acad Sci USA.2002;99:13528-13530
88 Giladi H, Ketzinel-Gilad M, Rivkin L, et al. Small interfering RNA inhibits hepatitis B virus replication in mice[J]. Mol Ther. 2003;8(5):769-76.
89 Ketzinel-Gilad M, Zauberman A, Nussbaum O, et al. The use of hydrodynamic HBV animal model to study HBV biology and antiviral therapy [J]. Hepatol Res.2006;34:228-237.
90 Carmona S, Ely A, Crowther C, et al. Effective inhibition of HBV replication in vivo by anti-HBx short hairpin RNAs[J]. Mol Ther. 2006;13(2):411-21.
91 Matsumoto R, Matsumoto H, Seki M, et al. Human ecalectin,a variant of human galectin-9,is a novel eosinophil chemoattractant produced by T lymphocytes[J]. J Biol Chem 1998;273:16976-16984.
92 Wada J, Kanwar YS. Identification and Characterization of galectin-9, a novel beta-galactoside-binding mammalian lectin[J].J Biol Chem.l997;272:6078-6086.
93 Whitmire JK, Tan JT, Whitton JL. Interferon-gamma acts directly on CD8+T cells to increase their abundance during virus infection[J]. J Exp Med. 2005;201:1053-1059.
94 Battegay M, Moskophidis D, Waldner H, et al. Impairment and delay of neutralizing antiviral antibody responses by virus-specific cytotoxic T cells[J]. J Immunol. 1993;151:5408-5415.
95 Ciurea A, Klenerman P, Hunziker L, et al. Viral persistence in vivo through selection of neutralizing antibodyescape variants[J]. Proc Natl Acad Sci USA. 2000;97:2749-2754.
96 Rasmussen RA, Hofinann-Lehmann R, Li PL, et al. Neutralizing antibodies as a potential secondary protective mechanism during chronic SHIV infection in CD8+ T-cell-depleted macaques[J]. AIDS. 2002; 16:829-838.
97 Recher M, Lang KS, Hunziker L, et al. Deliberate removal of T cell help improves virus-neutralizing antibody production[J]. Nat Immunol. 2004;5:934-942.
98 McIntire JJ, Umetsu S E, Akbari O, et al. Identification of Tapr(an airway hyperreactivity regulatory locus)and the linked Tim gene family[J] . Nat Immunol.2001;2:1109-1116.
99 Butcher EC, Picker LJ. Lymphocyte homing and homeostasis[J]. Science.1996;272:60-66.
100 Nakae S, Iwakura Y, Suto H, et al. Phenotypic differences between Th1 and Th17 cells and negative regulation of Th1 cell differentiation by IL-17[J]. J Leukoc Biol.2007;81:1258-1268.
101 Kageshita T, Kashio Y, Yamauchi A, et al. Possible role of galectin-9 in cell aggregation and apoptosis of human melanoma cell lines and its clinical significance[J]. Int J Cancer. 2002;99: 809 -816.
102 Irie A, Yamauchi A, Kontani K, et al. Galectin-9 as a prognostic factor with antimetastatic potential in breast cancer[J]. Clin Cancer Res.2005;11:2962 -2968.
103 Lahm H, Andre S, Hoeflich A, et al. Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures[J].J Cancer Res Clin Oncol.2001;127:375-386.
2 Grob PJ. Hepatitis B: virus, pathogenesis and treatment[J]. Vaccine. 1998; 16: 11-16.
3 Jung MC, Pape GR. Immunology of hepatitis B infection[J]. Lancet Infect Dis. 2001 ;2: 43-50.
4 Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis[J]. Annu Rev Immunol. 1995;13:29-60.
5 Bertoletti A, Ferrari C, Fiaccadori F. Role of the cell-mediated immune response in the pathogenesis of hepatitis B virus infection: possible immune-therapeutic strategies[J]. Acta Biomed Ateneo Parmense. 1996;67(3-4):87-93.
6 Marcellin P, Castelnau C, Martinot-Peignoux M, et al. Natural history of hepatitis B[J]. Minerva Gastroenterol Dietol. 2005;51(1):63-75.
7 Chu CM. Natural history of chronic hepatitis B virus infection in adults with emphasis on the occurrence of cirrhosis and hepatocellular carcinoma[J]. J Gastroenterol Hepatol. 2000; 15 Suppl:E25-30.
8 Merican I, Guan R, Amarapuka D, et al. Chronic hepatitis B virus infection in Asian countries[J]. J Gastroenterol Hepatol. 2000;15(12):1356-1361.
9 Biron CA. Activation and function of natural killer cell responses during viral infections[J]. Curr Opin Immunol. 1997;9: 24-34.
10 Biron CA, Nguyen KB, Pien GC, et al. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu Rev Immunol. 1999; 17:189-220.
11 Vivier E, Tomasello E, Baratin M, et al. Functions of natural killer cells. Nat Immunol. 2008;9(5):503-10.
12 Trinchieri G. Human natural killer cells. Biology of natural killer cells[J]. Adv Immunol. 198947:187-376.
13 Vivier, E, Nunes, JA, Vely, F. Natural killer cell signaling pathways[J]. Science. 2004;306:1517-1519.
14 Martz E, Howell DM, CTL: virus control cells first and cytolytic cells second[J]? Immunol Today. 1989; 10: 79-86.
15 Keefe D, Shi L, Feske S, et al. Perforin triggers a plasma membrane-repair response that facilitates CTL induction of apoptosis[J]. Immunity 2005; 23: 249-262
16 Guidotti LG, Ishikawa T, Hobbs MV, et al. Intracellular inactivation of the hepatitis B virus by cytotoxic T lymphocytes[J]. Immunity. 1996;4: 25-36.
17 Thimme R, Wieland S, Steiger C, et al. CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection[J]. J Virol. 2003; 77: 68-76.
18 Kakimi K, Guidotti LG, Koezuka Y, et al. Natural killer T cell activation inhibits hepatitis B virus replication in vivo[J]. J Exp Med. 2000;192: 921-930.
19 Kimura K, Kakimi K, Wieland S, et al. Activated intrahepatic antigen-presenting cells inhibit hepatitis B virus replication in the liver of transgenic mice[J]. J Immunol. 2002;169: 5188-5195.
20 Pasquetto V, Guidotti LG, Kakimi K, et al. Host-virus interaction during malaria infection in hepatitis B virus transgenic mice[J]. J Exp Med. 2000;192: 529-535.
21 Echevarria S, Casafont F, Miera M, et al. Interleukin-2 and natural killer activity in acute type B hepatitis[J]. Hepatogastroenterology. 1991;38:307-310.
22 Monsalve-De Castillo F, Romero TA, Estevez J, et al. Concentrations of cytokines, soluble interleukin-2 receptor, and soluble CD30 in sera of patients with hepatitis B virus infection during acute and convalescent phases[J]. Clin Diagn Lab Immunol. 2002;9:1372-1375.
23 Ono K, Yamanaga Y, Yamamoto K, et al. Natural killing activities in chronic liver diseases and hepatocellular carcinoma[J]. J Clin Immunol. 1996; 16:41-45.
24 Boni C, Fisicaro P, Valdatta C, et al. Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection[J]. J Virol. 2007; 81: 4215-4225.
25 Ferrari CA, Penna A, Bertoletti, A, et al. Cellular immune response to hepatitis B virus-encoded antigens in acute and chronic hepatitis B virus infection[J]. J Immunol. 1990; 145:3442-3449.
26 Jung ML, Hartmann B, Gerlach JT, et al. Virus-specific lymphokine production differs quantitatively but not qualitatively in acute and chronic hepatitis B infection[J]. Virology. 1999; 261:165-172.
27 Jung ML, Spengler U, Schraut W, et al. Hepatitis B virus antigen-specific T cell activation in patients with acute and chronic hepatitis B[J]. J Hepatol. 1991;13:310-317.
28 Penna A, Chisari FV, Bertoletti A, et al. Cytotoxic T lymphocytes recognize an HLA-A2-restricted epitope within the hepatitis B virus nucleocapsid antigen[J]. J ExpMed. 1991;174:1565-1570.
29 Sobao Y, Tomiyama H, Sugi K, et al. The role of hepatitis B virus-specific memory CD8 T cells in the control of viral replication[J]. J Hepatol. 2002;36:105-115.
30 Dong H, Zhu G, Tamada K, et al. B7-H1 determines accumulation and deletion of intrahepatic CD8(+) T lymphocytes[J]. Immunity. 2004; 20: 327-336.
31 Greenwald RJ, Freeman GJ, Sharpe AH. The B7 family revisited[J]. Annu Rev Immunol. 2005; 23: 515-548.
32 Maier H, Isogawa M, Freeman GJ, et al. PD-1:PD-L1 interactions contribute to the functional suppression of virus-specific CD8+ T lymphocytes in the liver[J]. J Immunol. 2007;178(5):2714-20.
33 Boni C, Fisicaro P, Valdatta C, et al. Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection[J]. J Virol. 2007;81(8):4215-25.
34 Peng G, Li S, Wu W, et al. PD-1 upregulation is associated with HBV-specific T cell dysfunction in chronic hepatitis B patients[J]. Mol Immunol. 2008;45(4):963-70.
35 Zhang Z, Zhang JY, Wherry EJ, et al. Dynamic programmed death 1 expression by virus-specific CD8 T cells correlates with the outcome of acute hepatitis B[J]. Gastroenterology. 2008; 134(7): 1938-49,
36 Cerwenka A, Lanier LL. Natural killer cells, viruses and cancer [J]. Nat Rev Immunol. 2001 ;1: 41-49.
37 Moser JM, Gibbs J, Jensen PE, et al. CD94-NKG2A receptors regulate antiviral CD8 + T cell responses[J]. Nat Immunol. 2002; 3: 189-195.
38 Bertoletti A, Gehring AJ.The immune response during hepatitis B virus infection[J]. J Gen Virol. 2006;87(Pt 6): 1439-49.
39 Lopes AR, Kellam P, Das A, et al. Bim-mediated deletion of antigen specific CD8 T cells in patients unable to control HBV infection[J]. J Clin Invest. 2008; 118: 1835-1845
40 Stoop JN, van der Molen RG, Baan CC, et al. Regulatory T cells contribute to the impaired immune response in patients with chronic hepatitis B virus infection[J]. Hepatology. 2005; 41: 771-778.
41 Stoop JN, van der Molen RG, Kuipers EJ, et al. Inhibition of viral replication reduces regulatory T cells and enhances the antiviral immune response in chronic hepatitis B. Virology. 2007;361(l):141-8.
42 Alatrakchi N, Koziel M. Regulatory T cells and viral liver disease. J Viral Hepat. 2009;16(4):223-9.
43 Monney L, Sabatos CA, Gaglia JL, et al. Th1-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease [J]. Nature. 2002; 415:536-541.
44 Frisancho-Kiss S, Nyland JF, Davis SE, et al.Cutting edge: T cell Ig mucin-3 reduces inflammatory heart disease by increasing CTLA-4 during innate immunity[J]. J Immunol. 2006;176(11):6411-5.
45 Frisancho-Kiss S, Davis SE, Nyland JF, et al. Cutting edge: cross-regulation by TLR4 and T cell Ig mucin-3 determines sex differences in inflammatory heart disease[J]. J Immunol. 2007;178(11):6710-4.
46 Sabatos CA, Chakravarti S, Cha E, et al. Interaction of Tim-3 and Tim-3 ligand regulates T helper type 1 responses and induction of peripheral tolerance[J]. Nat Immunol. 2003;4:1102 -1110.
47 Kuchroo VK, Umetsu DT, DeKruyff RH, et al. The TIM gene family: emerging roles in immunity and disease[J].Nat Rev Immunol.2003;3:454-462.
48 Sanchez-Fueyo A,Tian J,Picarella D,et al.Tim-3 inhibits T helper type 1-mediated auto- and alloimmune responses and promotes immunological tolerance[J].Nat Immunol 2003;4:1093-1101.
49 Khademi M,Illes Z,Gielen AW,et al.T Cell Ig- and mucin-domain-containing molecule-3(TIM-3) and TIM-1 molecules are differentially expressed on human Th1 and Th2 cells and in cerebrospinal fluid-derived mononuclear cells in multiple sclerosis[J].J Immunol 2004;172:7169-7176.
50 Mariat C,Sanchez-Fueyo A,Alexopoulos SP,et al.Regulation of T cell dependent immune responses by TIM family members[J].Philos Trans R Soc Lond B Biol Sci.2005;360(1461):1681-5.
51 Meyers JH,Sabatos CA,Chakravarti S,et al.The TIM gene family regulates autoimmune and allergic diseases[J].Trends Mol Med.2005;11(8):362-9.
52 Zhu C,Anderson AC,Schubart A,et al.The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity[J].Nat Immunol.2005;6:1245-1252.
53 Kuchroo VK,Meyers JH,Umetsu DT,et al.TIM family of genes in immunity and tolerance[J].Adv Immunol.2006;91:227-49.
54 Anderson AC,Anderson DE.TIM-3 in autoimmunity[J].Curr Opin Immunol.2006;18(6):665-9.
55 Oikawa T,Kamimura Y,Akiba H,et al.Preferential involvement of Tim-3 in the regulation of hepatic CD8+ T cells in murine acute graft-versus-host disease[J].J Immunol.2006;177:4281-4287.
56 Koguchi K,Anderson DE,Yang L,et al.Dysregulated T cell expression of TIM3in multiple sclerosis[J].J Exp Med 2006;203:1413-1418.
57 Degauque N,Mariat C,Kenny J,et al.Regulation of T-cell immunity by T-cell immunoglobulin and mucin domain proteins[J].Transplantation.2007;84(1Suppl):S12-6.
58 Yang L,Anderson DE,Kuchroo J,et al,Lack of TIM-3 immunoregulation in multiple sclerosis[J].J Immunol.2008;180(7):4409-14.
59 Su EW,Lin JY,Kane LP.TIM-1 and TIM-3 proteins in immune regulation[J]. Cytokine.2008;44(1):9-13.
60 Umetsu DT,Umetsu SE,Freeman GJ,et al.TIM gene family and their role in atopic diseases[J].Curr Top Microbiol Immunol.2008;321:201-15.
61 Hafler DA,Kuchroo V.TIMs:central regulators of immune responses[J].J Exp Med.2008;205(12):2699-701.
62 Fukushima A,Sumi T,Fukuda K,et al.Roles of galectin-9 in the development of experimental allergic conjunctivitis in mice[J].Int Arch Allergy Immunol.2008;146(1):36-43.
63 Gielen AW,Lobell A,Lidman O,,et al.Expression of T cell immunoglobulin- and mucin-domain-containing molecules-1 and -3(Tim-1 and -3) in the rat nervous and immune systems[J].J Neuroimmunol.2005;164:93-104.
64 Wang Y,Meng J,Wang X,et al.Expression of human TIM-1 and TIM-3 on lymphocytes from systemic lupus erythematosus patients[J].Scand J Immunol.2008;67:63-70.
65 Simmons WJ,Koneru M,Mohindru M,et al.Tim-3+ T-bet+ tumor-specific Th1cells colocalize with and inhibit development and growth of murine neoplasms[J].J Immunol.2005;174:1405-1415.
66 Fukushima A,Sumi T,Fukuda K,et al.Antibodies to T cell Ig and mucin domain-containing proteins(Tim)-1 and -3 suppress the induction and progression of murine allergic conjunctivitis[J].Biochem Biophys Res Commun.2007;353:211-216.
67 Kearley J,McMillan SJ,Lloyd CM.Th2-driven,allergen-induced airway inflammation is reduced after treatment with anti-Tim-3 antibody in vivo[J].J Exp Med.2007;204:1289-1294.
68 Anderson AC,Anderson DE,Bregoli L,et al.Promotion of tissue inflammation by the immune receptor Tim-3 expressed on innate immune cells[J].Science.2007;318:1141-1143.
69 Zhang J,Gu Y,Xu C,et al.Increased T cell immunoglobulin mucin-3 and its ligand in acquired aplastic anemia[J].Eur J Haematol.2008;81(2):130-9.
70 Nagahara K,Arikawa T,Oomizu S,et al.Galectin-9 increases Tim-3+ dendritic cells and CD8+ T cells and enhances antitumor immunity via galectin-9-Tim-3 interactions[J]. J Immunol. 2008; 181(11):7660-9.
71 Jones RB, Ndhlovu LC, Barbour JD, et al. Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection[J]. J Exp Med 2008;205:2763-2779.
72 Sehrawat S, Suryawanshi A, Hirashima M, et al. Role of Tim-3/galectin-9 inhibitory interaction in viral-induced immunopathology: shifting the balance toward regulators[J]. J Immunol. 2009; 182(5):3191-201.
73 Liang X, Liu Y, Zhang Q, et al. Hepatitis B virus sensitizes hepatocytes to TRAIL-induced apoptosis through Bax[J]. J Immunol. 2007;178:503-510.
74 Xiong Y, Jia Y, Wang H, et al. High-level hepatitis B virus expression in transgenic mice[J]. Infect Dis Info. 2000;13:164-165.
75 Trippler M, Meyer zum Buschenfelde KH, Gerken G. HBV viral load within subpopulations of peripheral blood mononuclear cells in HBV infection using limiting dilution PCR[J]. J Virol Methods.l999;78:129-147.
76 Chemin I, Vermot-Desroches C, Baginski I, et al. Selective detection of human hepatitis B virus surface and core antigens in peripheral blood mononuclear cell subsets by flow cytometry[J]. J Viral Hepat.1994;1:39-44.
77 Chisari FV. Hepatitis B virus transgenic mice: insights into the virus and the disease[J]. Hepatology. 1995;22:1316-1325.
78 Chisari FV. Hepatitis B virus transgenic mice: models of viral immunobiology and pathogenesis[J]. Curr Top Microbiol Immunol. 1996;206:149-73.
79 Akbar SK, Onji M. Hepatitis B virus (HBV)-transgenic mice as an investigative tool to study immunopathology during HBV infection[J]. Int J Exp Pathol. 1998;79(5):279-91.
80 Weber OF. Animal models in virology[J]. Contrib Microbiol. 2001;9:15-30.
81 Kekule AS, Lauer U, Weiss L, et al. Trans-activation by hepatitis B virus X protein is mediated via a tumour promoter pathway [J]. Arch Virol Suppl. 1992;4:63-64.
82 Chung TW, Lee YC, Kim CH. Hepatitis B viral HBx induces matrix metalloproteinase-9 gene expression through activation of ERK and PI-3K/AKT pathways: involvement of invasive potential[J]. FASEB J. 2004;18:1123-1125.
83 Lee DK, Park SH, Yi Y, et al. The hepatitis B virus encoded oncoprotein pX amplifies TGF-beta family signaling through direct interaction with Smad4: potential mechanism of hepatitis B virus-induced liver fibrosis[J]. Genes Dev. 2001; 15:455-466.
84 Zou Z, Li B, Xu D, et al. Imbalanced intrahepatic cytokine expression of interferon-gamma, tumor necrosis factor-alpha, and interleukin-10 in patients with acute-on-chronic liver failure associated with hepatitis B virus infection[J]. J Clin Gastroenterol. 2009;43(2): 182-90.
85 Chang JJ, Thompson AJ, Visvanathan K, et al. The phenotype of hepatitis B virus-specific T cells differ in the liver and blood in chronic hepatitis B virus infection[J]. Hepatology 2007;46:1332-1340.
86 Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA[J]. Gene Therapy. 1999;6:1258-1266.
87 Yang PL, Althage A, Chung J, Chisari FV. Hydrodynamic injection of viral DNA: A mouse model of acute hepatitis B virus infection[J]. Proc Natl Acad Sci USA.2002;99:13528-13530
88 Giladi H, Ketzinel-Gilad M, Rivkin L, et al. Small interfering RNA inhibits hepatitis B virus replication in mice[J]. Mol Ther. 2003;8(5):769-76.
89 Ketzinel-Gilad M, Zauberman A, Nussbaum O, et al. The use of hydrodynamic HBV animal model to study HBV biology and antiviral therapy [J]. Hepatol Res.2006;34:228-237.
90 Carmona S, Ely A, Crowther C, et al. Effective inhibition of HBV replication in vivo by anti-HBx short hairpin RNAs[J]. Mol Ther. 2006;13(2):411-21.
91 Matsumoto R, Matsumoto H, Seki M, et al. Human ecalectin,a variant of human galectin-9,is a novel eosinophil chemoattractant produced by T lymphocytes[J]. J Biol Chem 1998;273:16976-16984.
92 Wada J, Kanwar YS. Identification and Characterization of galectin-9, a novel beta-galactoside-binding mammalian lectin[J].J Biol Chem.l997;272:6078-6086.
93 Whitmire JK, Tan JT, Whitton JL. Interferon-gamma acts directly on CD8+T cells to increase their abundance during virus infection[J]. J Exp Med. 2005;201:1053-1059.
94 Battegay M, Moskophidis D, Waldner H, et al. Impairment and delay of neutralizing antiviral antibody responses by virus-specific cytotoxic T cells[J]. J Immunol. 1993;151:5408-5415.
95 Ciurea A, Klenerman P, Hunziker L, et al. Viral persistence in vivo through selection of neutralizing antibodyescape variants[J]. Proc Natl Acad Sci USA. 2000;97:2749-2754.
96 Rasmussen RA, Hofinann-Lehmann R, Li PL, et al. Neutralizing antibodies as a potential secondary protective mechanism during chronic SHIV infection in CD8+ T-cell-depleted macaques[J]. AIDS. 2002; 16:829-838.
97 Recher M, Lang KS, Hunziker L, et al. Deliberate removal of T cell help improves virus-neutralizing antibody production[J]. Nat Immunol. 2004;5:934-942.
98 McIntire JJ, Umetsu S E, Akbari O, et al. Identification of Tapr(an airway hyperreactivity regulatory locus)and the linked Tim gene family[J] . Nat Immunol.2001;2:1109-1116.
99 Butcher EC, Picker LJ. Lymphocyte homing and homeostasis[J]. Science.1996;272:60-66.
100 Nakae S, Iwakura Y, Suto H, et al. Phenotypic differences between Th1 and Th17 cells and negative regulation of Th1 cell differentiation by IL-17[J]. J Leukoc Biol.2007;81:1258-1268.
101 Kageshita T, Kashio Y, Yamauchi A, et al. Possible role of galectin-9 in cell aggregation and apoptosis of human melanoma cell lines and its clinical significance[J]. Int J Cancer. 2002;99: 809 -816.
102 Irie A, Yamauchi A, Kontani K, et al. Galectin-9 as a prognostic factor with antimetastatic potential in breast cancer[J]. Clin Cancer Res.2005;11:2962 -2968.
103 Lahm H, Andre S, Hoeflich A, et al. Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures[J].J Cancer Res Clin Oncol.2001;127:375-386.