HBV感染者中抗原特异性CTL免疫应答状况的研究
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摘要
目的
1．建立体外分析抗原特异性T细胞的检测方法--四聚体染色技术，为深入探讨表位特异性CTL的生物学功能以及更全面理解HBV特异CTL在HBV感染中的致病机理奠定实验基础；
2．应用四聚体染色技术和流式染色分析抗原特异性CTL在自限性和慢性HBV感染中频率和生物学功能；
3．分析HBV慢性感染者抗原特异性CTL细胞功能耗竭可能机制，为慢性感染免疫调节治疗提供可能途径和新的治疗靶点。
方法
1．应用基因工程技术原核高效表达HLA-A~*0201-BSP、HLA-A~*2402-BSP、HLA-A~*1101-BSP和β2m蛋白后，分别与常见的HBV抗原肽在体外复性折叠成可溶性的抗原肽复合物单体；经BirA酶作用并通过凝胶过滤层析法纯化复合物单体；分别将复合物单体与藻红蛋白标记的链霉亲和素按一定比例(5：1)耦合构建成四聚体。
2．Dot blotting方法检测HLA-A~*0201-β2m-抗原肽、HLA-A~*2402-p2m-抗原肽、HLA-A~*1101-β2m-抗原肽复合物单体正确构象和生物素化；四聚体染色流式检测四聚体特异性。
3．采用淋巴细胞分离液分离抗凝血的外周血单个核细胞(PBMC)；序列特异性引物聚合酶链反应(Sequence Specific Primer Polymerase Chain Reaction，SSP-PCR)分型技术确定HLA分型。
4．入选病例用ELISA检测乙肝五项指标，Realtime-PCR定量检测HBV DNA。ELISA检测排除入选病例HAV、HCV、HDV、HEV、HIV病毒感染。
5．应用四聚体染色技术检测表位特异性CTL的频率，借助流式细胞仪检测多肽体外刺激淋巴细胞产生IFN-γ的能力；流式染色检测细胞表面CD分子的表达；结合四聚体染色和细胞内细胞因子染色检测细胞因子(IL-10、TGF-β)的分泌。
6．Sigmaplot软件作图，SPSS13．0对数据进行统计分析。
结果
1．构建了HLA-A~*0201 core18-27、pol575-583、env335-343；HLA-A~*2402 core117-125、pol756-764；HLA-A~*1101 core88-96、preS1 10-17抗原肽四聚体，所构建的七种四聚体都具有较好的特异性。
2．抗原特异性CTL在自限性和慢性HBV感染者中频率和生物学功能：
(1)自限性HBV感染患者外周循环中抗原特异性CTL的频率、增殖能力和分泌IFN-γ的能力明显高于慢性HBV感染者；自限性HBV感染者外周血中core特异性CTL的频率高于pol、env特异性CTL频率。
(2)在慢性HBV感染者中只有一小部分低病毒滴度的患者抗原特异性CTL能够增殖和分泌IFN-γ，高病毒滴度的患者几乎丧失了抗原特异性CTL细胞增殖和分泌IFN-γ的能力；统计学分析结果显示：慢性HBV感染者外周血中HBV特异性CTL的频率同HBV DNA及ALT并没有显著相关性(p＞0．05)。
(3)HLA-A~*0201／HLA-A~*2402，HLA-A~*0201／HLA-A~*1101，HLA-A~*1101／HLA-A~*2402限制性自限性HBV感染患者外周循环中抗原特异性CTL的频率均高于慢性HBV感染者，慢性HBV感染者外周血抗原特异性CTL频率很低，而且和HLA型别没有显著相关性。
3．自限性和慢性HBV感染者CD8~+T细胞上CD127的表达频率没有显著性差异，而在慢性HBV感染者特异性CTL上CD127的表达要高于自限性感染组，统计学分析有显著性差异(p＜0．05)。
4．自限性、慢性HBV感染者和未感染HBV者外周血CD3~+CD4~+T细胞、CD3~+CD8~+T细胞的频率和CD4~+／CD8~+的比值统计学分析没有显著性差异(p＞0．05)。
5．外周血中CD4~+T细胞、CD8~+T细胞分泌IL-10、TGF-β、IFN-γ能力在自限性或慢性HBV感染中均无显著性差异(p＞0．05)。
6．在自限性和慢性HBV感染中，阻断IL-10／IL-10R或TGF-β／TGF-βR相互作用后，抗原特异性CTL的频率、分泌IFN-γ的能力均增加，特别是阻断TGF-β／TGF-βR相互作用后，抗原特异性CTL的频率、分泌IFN-γ的能力增加较强。
结论
1．构建的七种四聚体都具有较好的特异性，能够检测到表位特异性CTL。
2．在自限性HBV感染者外周血中这七种抗原特异性CTL的频率、体外抗原肽刺激后的增殖能力及分泌IFN-γ的能力均较慢性感染者高；慢性HBV感染患者外周血中抗原特异性CTL频率一般较低，甚至检测不到；只有一小部分低病毒滴度的患者外周血中抗原特异性CD8~+T能够增殖和分泌IFN-γ，高病毒滴度的患者几乎丧失了抗原特异性CD8~+T细胞增殖和分泌IFN-γ的能力。
3．细胞因子IL-10和TGF-β在HBV感染者T细胞应答低下、抗原特异性CTL功能耗竭中起作用。体外阻断IL-10／IL-10R或TGF-β／TGF-βR相互作用可以提高抗原特异性CTL的频率、分泌IFN-γ的能力。
本研究的创新点
1．应用现有文献报道的表位，结合四聚体技术对不同HLA限制性表位特异CTL在中国自限性和慢性HBV感染者中频率和生物学功能进行了初步研究，特别对在中国具有较高频率HLA-A~*2402和HLA-A~*1101限制性CTL的研究。并分析了HLA-A~*0201／HLA-A~*2402，HLA-A~*0201／HLA-A~*1101，HLA-A~*1101／HLA-A~*2402限制性自限性和慢性HBV感染者体内抗原特异CTL频率和优势表位。
2．对IL-10、TGF-β等抑制性细胞因子在HBV特异性CTL功能耗竭机制进行了初步研究，并通过体外实验阻断IL-10／IL-10R或TGF-β／TGF-βR相互作用可以提高病毒特异性CTL的频率和功能，为慢性感染免疫治疗提供新的治疗靶点。
本研究的意义
1．应用四聚体染色技术系统分析了不同HLA限制性不同表位肽特异性CTL在自限性和慢性HBV感染者的频率及生物学功能，有助于我们更全面理解HBV特异CTL在HBV致病机理中的作用，也有利于HBV治疗性疫苗的研制。
2．对HBV感染中抗原特异性T细胞功能失能可能机制研究，为慢性感染免疫治疗提供新的治疗靶点。
Objects
1.To develop a method to detect specific CTL-HLA classⅠ/peptide tetrameficcomplexes, and provide the experiment grounding of investigating to epitope-specific CTLeffect in vivo and understanding the role of HBV-specific CTL in the immunopathogenesisof HBV infection.
2.To investigate the frequencies and the effects of epitope-specific CTLs inself-limited and chronic HBV infections by tetrameric complexes.
3.To analyse the mechanism of HBV-specific T-cell exhaustion in chronic HBVpatients, and develop the immune-based therapies.
Methods
1.Proteins HLA-A~*0201-BSP, HLA-A~*2402-BSP, HLA-A~*1101-BSP andβ2m wereobtained by effective prokanyotical expression.After being purified, the proteins wererefolded into complexes in the presence of common antigenic peptides by dilution method.The complexes then were biotinylated by BirA enzyme and purified via the gel-filtrationchromatography.Tetramers were generated by mixing the complex with PE-Streptavidin ata molar ration of 5:1.
2.The biotinylated HLA-A~*0201-, HLA-A~*2402- and HLA-A~*1101-β2m-peptidecomplexes were identified by Western blot and were shown to have the naturalconformation by Dot-ELISA.The specificity of the tetramers was tested by flow cytometricanalysis.
3.PBMCs were isolated from fresh heparinized venous blood by Ficoll-Hypaquedensity gradient centrifugation and HLA allele was performed by PCR-SSP.
4.Hepatitis B surface antigen (HBsAg), anti-HBs, total and IgM anti-HBc, hepatitis Be antigen (HBeAg), anti-HBe, anti-HCV, and anti-HIV were determined by commercialenzyme immunoassay kits.HBV DNA was quantified by the Roche Amplicor Monitorsassay.
5.The analysis of stained PBMCs using the tetramers and other antibodies wasperformed by flow cytometry.
6.Data are analysed by Statistical Package for Social Sciences (SPSS) version 13.0.
Results
1.Constructing the HLA-A~*0201 core18-27, po1575-583, env335-343; HLA-A~*2402core117-125, po1756-764; HLA-A~*1101 core88-96, preS1 10-17 tetramers.Using thesetetramers can detect the specific CTLs.
2.The frequencies and characterization of HBV-specific cytotoxic T lymphocytes inself-limited and chronic HBV infection in Chinese population:
(1) The virus-specific CD8~+T cell responses during chronic HBV infection are weakor absent whereas in resolved infection these responses are generally vigorous andmultispecific.In self-limited HBV infected individuals the frequencies and expansionability of core-specific CD8~+T cells is higher than that of pol- and env-specific CD8~+Tcells.
(2) In patients with chronic infection the frequencies of HBV-specific CD8~+cells afterin vitro expansion is inversely proportional to the level of HBV DNA.A lower level of expansion and IFN-γ-production is present in patients with HBV DNA levels of <10~7copies/mL, and no expansion and IFN-γ-production is present in patients with HBV DNAlevels of>10~7 copies/mL.
(3) The frequencies of HBV-specific CD8~+T cells are higher in heterozygote patientswith self-limited infection than in patients with chronic infection, and the frequencies ofHBV-specific CD8~+T cells in patients with chronic infection are low, furthermore, nocorrelation is detected between the frequencies and the HLA allele.
3.The expression of CD127 on CD8~+T cells in self-limited and chronic HBV patientsis no difference, but the expression of CD127 on HBV-specific CD8~+T cells in chronicpatients is higher than that in self-limited patients.
4.In self-limited, chronic, uninfected HBV patients, no significant correlation is foundamong the frequencies of CD3~+CD4~+T cells, CD3~+CD8~+T cells and CD4~+/CD8~+ in thesethree groups.
5.There is no significant correlation in the frequencies of IL-10-, TGF-β-, IFN-γ-producingCD4~+, CD8~+T cells in self-limited or chronic HBV patients.
6.Either the IL-1 OR blockade or the TGF-βblockade can increase the frequencies andcharacterization of HBV-specific CTLs in self-limited and chronic HBV patients, especially,the function of the TGF-βblockade is stronger.
Conclusions
1.Using these constructed tetramers can detect the specific CTLs.
2.The frequencies, the ablity of expansion and IFN-γ-production of HBV-specificCD8~+T cells were higher in self-limited HBV patients than in chronic patients; thefrequencies of HBV-specific CD8~+T cells in chronic HBV patients were low, only a lowerlevel of expansion and IFN-γ-production is present in patients with low HBV DNA levels,and no expansion and IFN-γ-production is present in patients with high HBV DNA levels.
3.IL-10 and TGF-βsuppress cellular immune responses in HBV infection, so theblockade of the receptor for IL-10 or TGF-βcan restore the antiviral immune response andresult in viral clearance.
1．建立体外分析抗原特异性T细胞的检测方法--四聚体染色技术，为深入探讨表位特异性CTL的生物学功能以及更全面理解HBV特异CTL在HBV感染中的致病机理奠定实验基础；
2．应用四聚体染色技术和流式染色分析抗原特异性CTL在自限性和慢性HBV感染中频率和生物学功能；
3．分析HBV慢性感染者抗原特异性CTL细胞功能耗竭可能机制，为慢性感染免疫调节治疗提供可能途径和新的治疗靶点。
方法
1．应用基因工程技术原核高效表达HLA-A~*0201-BSP、HLA-A~*2402-BSP、HLA-A~*1101-BSP和β2m蛋白后，分别与常见的HBV抗原肽在体外复性折叠成可溶性的抗原肽复合物单体；经BirA酶作用并通过凝胶过滤层析法纯化复合物单体；分别将复合物单体与藻红蛋白标记的链霉亲和素按一定比例(5：1)耦合构建成四聚体。
2．Dot blotting方法检测HLA-A~*0201-β2m-抗原肽、HLA-A~*2402-p2m-抗原肽、HLA-A~*1101-β2m-抗原肽复合物单体正确构象和生物素化；四聚体染色流式检测四聚体特异性。
3．采用淋巴细胞分离液分离抗凝血的外周血单个核细胞(PBMC)；序列特异性引物聚合酶链反应(Sequence Specific Primer Polymerase Chain Reaction，SSP-PCR)分型技术确定HLA分型。
4．入选病例用ELISA检测乙肝五项指标，Realtime-PCR定量检测HBV DNA。ELISA检测排除入选病例HAV、HCV、HDV、HEV、HIV病毒感染。
5．应用四聚体染色技术检测表位特异性CTL的频率，借助流式细胞仪检测多肽体外刺激淋巴细胞产生IFN-γ的能力；流式染色检测细胞表面CD分子的表达；结合四聚体染色和细胞内细胞因子染色检测细胞因子(IL-10、TGF-β)的分泌。
6．Sigmaplot软件作图，SPSS13．0对数据进行统计分析。
结果
1．构建了HLA-A~*0201 core18-27、pol575-583、env335-343；HLA-A~*2402 core117-125、pol756-764；HLA-A~*1101 core88-96、preS1 10-17抗原肽四聚体，所构建的七种四聚体都具有较好的特异性。
2．抗原特异性CTL在自限性和慢性HBV感染者中频率和生物学功能：
(1)自限性HBV感染患者外周循环中抗原特异性CTL的频率、增殖能力和分泌IFN-γ的能力明显高于慢性HBV感染者；自限性HBV感染者外周血中core特异性CTL的频率高于pol、env特异性CTL频率。
(2)在慢性HBV感染者中只有一小部分低病毒滴度的患者抗原特异性CTL能够增殖和分泌IFN-γ，高病毒滴度的患者几乎丧失了抗原特异性CTL细胞增殖和分泌IFN-γ的能力；统计学分析结果显示：慢性HBV感染者外周血中HBV特异性CTL的频率同HBV DNA及ALT并没有显著相关性(p＞0．05)。
(3)HLA-A~*0201／HLA-A~*2402，HLA-A~*0201／HLA-A~*1101，HLA-A~*1101／HLA-A~*2402限制性自限性HBV感染患者外周循环中抗原特异性CTL的频率均高于慢性HBV感染者，慢性HBV感染者外周血抗原特异性CTL频率很低，而且和HLA型别没有显著相关性。
3．自限性和慢性HBV感染者CD8~+T细胞上CD127的表达频率没有显著性差异，而在慢性HBV感染者特异性CTL上CD127的表达要高于自限性感染组，统计学分析有显著性差异(p＜0．05)。
4．自限性、慢性HBV感染者和未感染HBV者外周血CD3~+CD4~+T细胞、CD3~+CD8~+T细胞的频率和CD4~+／CD8~+的比值统计学分析没有显著性差异(p＞0．05)。
5．外周血中CD4~+T细胞、CD8~+T细胞分泌IL-10、TGF-β、IFN-γ能力在自限性或慢性HBV感染中均无显著性差异(p＞0．05)。
6．在自限性和慢性HBV感染中，阻断IL-10／IL-10R或TGF-β／TGF-βR相互作用后，抗原特异性CTL的频率、分泌IFN-γ的能力均增加，特别是阻断TGF-β／TGF-βR相互作用后，抗原特异性CTL的频率、分泌IFN-γ的能力增加较强。
结论
1．构建的七种四聚体都具有较好的特异性，能够检测到表位特异性CTL。
2．在自限性HBV感染者外周血中这七种抗原特异性CTL的频率、体外抗原肽刺激后的增殖能力及分泌IFN-γ的能力均较慢性感染者高；慢性HBV感染患者外周血中抗原特异性CTL频率一般较低，甚至检测不到；只有一小部分低病毒滴度的患者外周血中抗原特异性CD8~+T能够增殖和分泌IFN-γ，高病毒滴度的患者几乎丧失了抗原特异性CD8~+T细胞增殖和分泌IFN-γ的能力。
3．细胞因子IL-10和TGF-β在HBV感染者T细胞应答低下、抗原特异性CTL功能耗竭中起作用。体外阻断IL-10／IL-10R或TGF-β／TGF-βR相互作用可以提高抗原特异性CTL的频率、分泌IFN-γ的能力。
本研究的创新点
1．应用现有文献报道的表位，结合四聚体技术对不同HLA限制性表位特异CTL在中国自限性和慢性HBV感染者中频率和生物学功能进行了初步研究，特别对在中国具有较高频率HLA-A~*2402和HLA-A~*1101限制性CTL的研究。并分析了HLA-A~*0201／HLA-A~*2402，HLA-A~*0201／HLA-A~*1101，HLA-A~*1101／HLA-A~*2402限制性自限性和慢性HBV感染者体内抗原特异CTL频率和优势表位。
2．对IL-10、TGF-β等抑制性细胞因子在HBV特异性CTL功能耗竭机制进行了初步研究，并通过体外实验阻断IL-10／IL-10R或TGF-β／TGF-βR相互作用可以提高病毒特异性CTL的频率和功能，为慢性感染免疫治疗提供新的治疗靶点。
本研究的意义
1．应用四聚体染色技术系统分析了不同HLA限制性不同表位肽特异性CTL在自限性和慢性HBV感染者的频率及生物学功能，有助于我们更全面理解HBV特异CTL在HBV致病机理中的作用，也有利于HBV治疗性疫苗的研制。
2．对HBV感染中抗原特异性T细胞功能失能可能机制研究，为慢性感染免疫治疗提供新的治疗靶点。
Objects
1.To develop a method to detect specific CTL-HLA classⅠ/peptide tetrameficcomplexes, and provide the experiment grounding of investigating to epitope-specific CTLeffect in vivo and understanding the role of HBV-specific CTL in the immunopathogenesisof HBV infection.
2.To investigate the frequencies and the effects of epitope-specific CTLs inself-limited and chronic HBV infections by tetrameric complexes.
3.To analyse the mechanism of HBV-specific T-cell exhaustion in chronic HBVpatients, and develop the immune-based therapies.
Methods
1.Proteins HLA-A~*0201-BSP, HLA-A~*2402-BSP, HLA-A~*1101-BSP andβ2m wereobtained by effective prokanyotical expression.After being purified, the proteins wererefolded into complexes in the presence of common antigenic peptides by dilution method.The complexes then were biotinylated by BirA enzyme and purified via the gel-filtrationchromatography.Tetramers were generated by mixing the complex with PE-Streptavidin ata molar ration of 5:1.
2.The biotinylated HLA-A~*0201-, HLA-A~*2402- and HLA-A~*1101-β2m-peptidecomplexes were identified by Western blot and were shown to have the naturalconformation by Dot-ELISA.The specificity of the tetramers was tested by flow cytometricanalysis.
3.PBMCs were isolated from fresh heparinized venous blood by Ficoll-Hypaquedensity gradient centrifugation and HLA allele was performed by PCR-SSP.
4.Hepatitis B surface antigen (HBsAg), anti-HBs, total and IgM anti-HBc, hepatitis Be antigen (HBeAg), anti-HBe, anti-HCV, and anti-HIV were determined by commercialenzyme immunoassay kits.HBV DNA was quantified by the Roche Amplicor Monitorsassay.
5.The analysis of stained PBMCs using the tetramers and other antibodies wasperformed by flow cytometry.
6.Data are analysed by Statistical Package for Social Sciences (SPSS) version 13.0.
Results
1.Constructing the HLA-A~*0201 core18-27, po1575-583, env335-343; HLA-A~*2402core117-125, po1756-764; HLA-A~*1101 core88-96, preS1 10-17 tetramers.Using thesetetramers can detect the specific CTLs.
2.The frequencies and characterization of HBV-specific cytotoxic T lymphocytes inself-limited and chronic HBV infection in Chinese population:
(1) The virus-specific CD8~+T cell responses during chronic HBV infection are weakor absent whereas in resolved infection these responses are generally vigorous andmultispecific.In self-limited HBV infected individuals the frequencies and expansionability of core-specific CD8~+T cells is higher than that of pol- and env-specific CD8~+Tcells.
(2) In patients with chronic infection the frequencies of HBV-specific CD8~+cells afterin vitro expansion is inversely proportional to the level of HBV DNA.A lower level of expansion and IFN-γ-production is present in patients with HBV DNA levels of <10~7copies/mL, and no expansion and IFN-γ-production is present in patients with HBV DNAlevels of>10~7 copies/mL.
(3) The frequencies of HBV-specific CD8~+T cells are higher in heterozygote patientswith self-limited infection than in patients with chronic infection, and the frequencies ofHBV-specific CD8~+T cells in patients with chronic infection are low, furthermore, nocorrelation is detected between the frequencies and the HLA allele.
3.The expression of CD127 on CD8~+T cells in self-limited and chronic HBV patientsis no difference, but the expression of CD127 on HBV-specific CD8~+T cells in chronicpatients is higher than that in self-limited patients.
4.In self-limited, chronic, uninfected HBV patients, no significant correlation is foundamong the frequencies of CD3~+CD4~+T cells, CD3~+CD8~+T cells and CD4~+/CD8~+ in thesethree groups.
5.There is no significant correlation in the frequencies of IL-10-, TGF-β-, IFN-γ-producingCD4~+, CD8~+T cells in self-limited or chronic HBV patients.
6.Either the IL-1 OR blockade or the TGF-βblockade can increase the frequencies andcharacterization of HBV-specific CTLs in self-limited and chronic HBV patients, especially,the function of the TGF-βblockade is stronger.
Conclusions
1.Using these constructed tetramers can detect the specific CTLs.
2.The frequencies, the ablity of expansion and IFN-γ-production of HBV-specificCD8~+T cells were higher in self-limited HBV patients than in chronic patients; thefrequencies of HBV-specific CD8~+T cells in chronic HBV patients were low, only a lowerlevel of expansion and IFN-γ-production is present in patients with low HBV DNA levels,and no expansion and IFN-γ-production is present in patients with high HBV DNA levels.
3.IL-10 and TGF-βsuppress cellular immune responses in HBV infection, so theblockade of the receptor for IL-10 or TGF-βcan restore the antiviral immune response andresult in viral clearance.
引文
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17. Manigold T, Racanelli V. T-cell regulation by CD4 regulatory T cells during hepatitis B and C virus infections: facts and controversies. Lancet Infect. Dis. 2007; 7: 804-13
18. Maier H, Isogawa M, Freeman GJ, Chisari FV. PD-1: PD-L1 interactions contribute to the functional suppression of 19 virus-specific CD8~+T lymphocytes in the liver. J. Immunol. 2007; 178: 2714-20.
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24. Brooks, D.G. et al. Interleukin-10 determines viral clearance or persistence in vivo. Nat. Med.2006: 12: 1301-1309
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2．中华医学会肝病学分会，感染病学分会。慢性乙型肝炎防治指南。中华肝脏病杂志；2005；13：881-890。
3. Takano S, Yokosuka O, Imazeki F, et al. Incidence of hepatocellular carcinoma in chronic hepatitis B and C: a prospective study of 251 patients[J]. Hepatology, 1995, 21(3): 650-655.
4. Chang JJ, Lewin SR, Immunopathegenesis of Hepatitis B virus infection. Immunol Cell Bio; 2007; 85: 16-23.
5. Thimme R, Wieland S, Steiger C, et al. CD8+ T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection. J Virol; 2003; 77: 68-76.
6. Guidotti LG, Chiari FV. Non-cytolytic control of viral infections by the innate and adaptive immune response. Annu Rev Immunol; 2001; 19: 65-91.
7. 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 Exp Med, 1991, 174: 1565-1570.
8. Maini MK, Boni C, Ogg GS, et al. Direct ex vivo analysis of hepatitis B virus-specific CD8(+) T cells associated with the control of infection. Gastroenterology, 1999, 117: 13 85-1395.
9. Yasunari N, Shuichi K, Hisao T, et al. Analysis of the CD8-positive T cell response in Japanese patients with chronic hepatitis c using HLA-A*2402 peptide tetramers[J]. Medical Virology, 2003, 70(1): 51-61.
10．肖露露，陈洪涛，叶欣等。HLA多态性在广东汉族人群分布的特殊性。中华微生物学和免疫学杂志；1999；19：302-305。
11. Alp N, Sissons JGP, Borysiewicz LK. Automation of limiting dilution cytotoxic assays. J Immuno Methods. 1990, 129: 269.
12. Jung T, Schauer U, Heusser C. et al. Detection of intracellular cytokines by flow cytometry. J Immunol Methods. 1993, 159: 197-207.
13. Lalvani AR, Brookes S. Rapid effector function in CD8+ memory T cells. J. Exp. Med. 1997,186:859-865.
14. Altman JD, Moss PAH, Goulder PJR, et al. Phenotypic analysis of antigen-specific T lymphocytes. Science; 1996; 274:94-96.
15. Greten TF, Slansky JE, Kubota R et al. Direct visualization of antigen-specific T cells: HTLV-1 Tax11-19-specific CD8+ T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients. Proc Natl Acad Sci USA 1998,95:7568-7573.
16. McMichael AJ, O' Callaghan CA: A new look at T cell. J Exp Med. 1998, 187: 1367-1371.
17. Guillaume P, Legler DF, Boucheron N et al. Soluble major histocompatibility complex -peptide octamers with impaired CD8 binding selectively induce Fas-dependent apoptosis. J Biol Chem. 2003, 278: 4500-4509.
18. Marrack P, Kappler J. Subversion of the immune system by pathogens. Cell, 1994, 76: 323-332.
19. Baron JL, Gardiner L, Nishimura S, et al. Activation of a nonclassical NKT cell subset in a transgenic mouse model of hepatitis B virus infection. Immunity, 2002, 16: 583-594.
20. Kakimi K, Guidotti LG, Koezuka Y, et al. Natural killer T cell activation inhibits hepatitis B virus replication in vivo. J Exp Med, 2000, 192: 921-930.
21. Bertoletti A, Maini M, Williams R. Role of hepatitis B virus specific cytotoxic T cells in liver damage and viral control. Antiviral Res. 2003 Oct; 60(2):61-6.
22. Lau GICK, Suri D, Liang R, et al. Resolution of Chronic Hepatitis B and Anti-HBs Seroconversion in Human by Adoptive Transfer of Immunity to Hepatitis B Core Antigen. Gastroerterology. 2002, 122:614-624.
23. Webster GJ, Reignat S, Brown D, Ogg GS, Jones L, Seneviratne SL, Williams R, Dusheiko G, Bertoletti A. Longitudinal analysis of CD8+ T cells specific for structural and nonstructural hepatitis B virus proteins in patients with chronic hepatitis B: implications for immunotherapy. J Virol. 2004 Jun; 78(11):5707-19.
24. Bertoletti A, Gehring AJ. The immune response during hepatitis B virus infection. J Gen Virol. 2006 Jun; 87(Pt 6): 1439-49.
25. Maini M, Bertoletti A. How can the cellular immune response control hepatitis B virus replication? J viral Hepat; 2000;7:321-326.
26. Jung MC, Page GR. Immunology of hepatitis B infection. Lancet Infectious Disease; 2001; 2:43-50.
27. Wherry EJ, Blattman JN, Murali-Krishna K, et al. Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J Virol 2003; 77:4911-4927
28. Anderson CF, Oukka M, Kuchroo VJ, Sacks D. CD4(+)CD25(-)Foxp3(-) Th1 cells are the source of IL-10-mediated immune suppression in chronic cutaneous leishmaniasis. J Exp Med 2007; 204:285-297
29. Muhlbauer M, Fleck M, Schutz C, et al. PD-L1 is induced in hepatocytes by viral infection and by interferon-alpha and -gamma and mediates T cell apoptosis. J Hepatol 2006; 45: 520-528
30. Ejmaes M, Filippi CM, Martinic MM, et al. Resolution of a chronic viral infection after interleukin-10 receptor blockade. J Exp Med 2006; 203:2461-2472
31. Sato Y, Sahara H, Tsukahara T, et al. Improved generation of HLA class Ⅰ peptide tetramers. J Immunol Methods; 2002; 271:177-184.
32. Ocallaghan CA, Byford MF, Wyer JR , et al . BirA enzyme: production and application in the study of membrane receptor 2 ligand interactions by site2 specific biotinylation. Anal Biochem; 1999;266: 9-15.
33. Bunce M. PCR-sequence-specific primer typing of HLA class Ⅰ and class Ⅱ alleles. Methods Mol Biol; 1999; 210:143-171.
34. Gehring AJ, Sun D, Kennedy PT, Nolte-'t Hoen E, Lim SG, Wasser S, Selden C, Maini MK, Davis DM, Nassal M, Bertoletti A. The level of viral antigen presented by hepatocytes influences CDS T-cell function. J Virol 2007; 81: 2940-2949.
35. Yuzhang WU, Jingbo Zhang, Shiyuan Chen, et al. Frequencies of epitope-specific cytotoxic T lymphocytes in active chronic viral hepatitis B infection by using MHC class Ⅰ peptide tetramers. Immunol Lett;2004; 92:253-258.
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