DC 欄位 | 值 | 語言 |
dc.contributor | 指導教授:許秉寧 | - |
dc.contributor | 臺灣大學:免疫學研究所 | zh_TW |
dc.contributor.author | 曾鴻泰 | zh_TW |
dc.contributor.author | Tzeng, Horng-Tay | en |
dc.creator | 曾鴻泰 | zh_TW |
dc.creator | Tzeng, Horng-Tay | en |
dc.date | 2014 | - |
dc.date.accessioned | 2014-11-26T08:52:33Z | - |
dc.date.accessioned | 2018-07-09T01:52:31Z | - |
dc.date.available | 2014-11-26T08:52:33Z | - |
dc.date.available | 2018-07-09T01:52:31Z | - |
dc.date.issued | 2014 | - |
dc.identifier.uri | http://ntur.lib.ntu.edu.tw//handle/246246/262025 | - |
dc.description.abstract | B型肝炎持續性感染會造成慢性肝炎,肝纖維化和肝癌。近來在動物模式中研究病毒感染的報告指出淋巴球上的抑制性受體 (PD-1) 和配體 (PD-L1) 間的相互作用對於抑制T細胞的活性扮演重要角色。我們利用最近發展的B型肝炎小鼠模式來探討T細胞exhaustion現象及阻斷PD-1/PD-L1作用後對於肝浸潤T細胞的效應。在此小鼠模式中,我們證明了B型肝炎的持續性和肝浸潤T細胞的PD-1表現成正相關,在帶原小鼠中肝浸潤CD8 T細胞表現較高的PD-1和較低的CD127 exhaustion現象,並且利用單株抗體阻斷PD-1/PD-L1作用可增加病毒核心蛋白專一性的IFN-γ產生,而且可以反轉T淋巴球的exhaustion及增加小鼠對病毒的清除能力。我們的結果指出,在小鼠模式中阻斷PD-1的途徑可恢復對B肝病毒的免疫反應及增進對病毒的清除。
先天與後天免疫反應的調和才能有效的控制病毒感染,為了瞭解對抗B肝病毒的先天免疫反應,我們使用了一系列與先天免疫反應相關受體之基因缺陷小鼠來測試,結果發現除了腫瘤壞死因子缺失的小鼠外,其餘包括IFN-α/β receptor, RIG-I, MDA5, MYD88, NLRP3, ASC, 和 IL-1R基因缺失小鼠都與控制組相當。我們進一步發現,給予小鼠腫瘤壞死因子受體阻斷劑可促使CD8 T細胞PD-1表現、肝臟中病毒複製、核心抗原和表面抗原及血清病毒量增加;純化後的病毒核心蛋白可在小鼠引發腫瘤壞死因子及IL-6表現,但IFN-β則否。我們也發現病毒核心蛋白造成肝臟中白血球引發的腫瘤壞死因子是清除病毒的重要途徑,這些結果提供了腫瘤壞死因子相關的先天免疫反應對抗病毒的機制,也解釋了臨床上使用腫瘤壞死因子阻斷劑造成B肝病毒再活化的機制。 | zh_TW |
dc.description.abstract | Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Recent studies in animal models of viral infection indicate that the interaction between the inhibitory receptor, programmed death (PD)-1, on lymphocytes and its ligand (PD-L1) play a critical role in T-cell exhaustion by inducing T-cell inactivation. We studied T-cell exhaustion and the effects of PD-1 and PD-L1 blockade on intrahepatic infiltrating T-cells in our recently developed mouse model of HBV persistence. In this mouse animal model, we demonstrated that the chronicity of hepatitis B virus infection was associated with PD-1 expression on intrahepatic lymphocytes. The Intrahepatic CD8+ T-cells expressed higher levels of PD-1 and lower levels of CD127 in carrier mice. Blockade of PD-1/PD-L1 interactions by an anti-PD-1 monoclonal antibody (mAb) increased HBcAg-specific interferon (IFN)-γ production in intrahepatic T lymphocytes and reversed the exhausted phenotype in intrahepatic T lymphocytes and viral persistence to clearance of HBV in vivo. Our results indicated that PD-1 blockage reverses immune dysfunction and viral persistence of HBV infection in a mouse animal model, suggesting that the anti-PD-1 mAb might be a good therapeutic candidate for chronic HBV infection.
An efficient control of virus infections requires the coordinated actions of both innate and adaptive immune responses. In order to define the role of innate immunity effectors against HBV, viral clearance was studied in a panel of immunodeficient mouse strains. Our results demonstrate that HBV viral clearance is not changed in IFN-α/β receptor (IFNAR), RIG-I, MDA5, MYD88, NLRP3, ASC, and IL-1R knockout mice. In contrast, HBV persists in the absence of tumor necrosis factor-alpha (TNF-α) or in mice treated with the soluble TNF receptor blocker, Etanercept. In these mice, there was an increase in PD-1-expressing CD8+ T-cells and an increase of serum HBV DNA, HBV core, and surface antigen expression as well as viral replication within the liver. Furthermore, the induction of TNF-α in clearing HBV is dependent on the HBV core, and TNF blockage eliminated HBV core-induced viral clearance effects. Finally, the intra-hepatic leukocytes (IHLs), but not the hepatocytes, are the cell source responsible for TNF-α production induced by HBcAg. These results provide evidences for TNF-α mediated innate immune mechanisms in HBV clearance and explain the mechanism of HBV reactivation during therapy with TNF blockage agents. | en |
dc.description.tableofcontents | 中文摘要 - - - - - - - - - - - - - - - - - - - - - - - - - - - i
Abstract - - - - - - - - - - - - - - - - - - - - - - - - - - - ii
Table of Contents- - - - - - - - - - - - - - - - - - - - - - - iv
Abbreviations- - - - - - - - - - - - - - - - - - - - - - - - - x
Chapter I: Introduction- - - - - - - - - - - - - - - - - - - - 1
1.1 Virology of Hepatitis B virus - - - - - - - - - - - - - - - - - 2
1.2 Natural history of Hepatitis B virus infection - - - - - - - - - - 3
1.3 Immune-mediated pathogenesis of Hepatitis B- - - - - - - - - - -5
1.3.1 Adaptive immunity against HBV - - - - - - - - - - - - - - - - 5
1.3.2 Innate immunity against HBV- - - - - - - - - - - - - - - - - -8
1.4 Animal models for exploring immune mechanism of hepatitis B - - - 9
1.4.1 Hydrodynamic-based transfection of HBV mouse model - - - - - - 11
1.4.2 HBV mouse model generated by delivery of adenovirus- - - - - - - 14
1.4.3 Adeno-associated viral vectors (AAV)-based HBV mouse model- - - -16
1.4.4 Comparison of the advantages and disadvantages in each model- - - - 16
Chapter II: Materials and Methods - - - - - - - - - - - - - - - 19
2.1 Animals and HBV constructs- - - - - - - - - - - - - - - - - - 20
2.2 Hydrodynamic injection- - - - - - - - - - - - - - - - - - - - 20
2.3 Isolation of intrahepatic leukocytes- - - - - - - - - - - - - - - -21
2.4 Isolation of primary hepatocytes - - - - - - - - - - - - - - - - 22
2.5 Detection of the HBV antigen, antibody (Ab), and DNA- - - - - - -22
2.6 Liver tissue preparation and immunoblotting- - - - - - - - - - - 23
2.7 Immunohistochemistry - - - - - - - - - - - - - - - - - - - - 24
2.8 Interferon (IFN)-γ enzyme-linked immunospot (ELISpot) assay - - - 25
2.9 Flow cytometry- - - - - - - - - - - - - - - - - - - - - - - -25
2.10 Co-culture assay- - - - - - - - - - - - - - - - - - - - - - -26
2.11 In vivo treatment of mice with the anti-PD-1 mAb- - - - - - - - -26
2.12 Purification of monoclonal antibody from hybridoma - - - - - - -27
Chapter III: Results - - - - - - - - - - - - - - - - - - - - - -28
3.1 Inhibitory receptor PD-1 in chronic viral infection - - - - - - - - -29
3.2 Increased PD-1-expressing CD8+ and CD4+ T-cells in
liver-infiltrating lymphocytes from mice with HBV persistence- - - -30
3.3 Liver-infiltrating CD8+ lymphocytes in carrier mice displayed the PD-1hiCD127low-exhausted phenotype - - - - - - - - - - - - - - 32
3.4 Liver-infiltrating PD-1+CD4+ T-cells in mice with HBV persistence exhibit the phenotype of regulatory T-cells (Tregs) - - - - - - - - - - - - --33
3.5 Blockade of the PD-1 pathway by an anti-PD-1 mAb reduced the HBV persistence rate in this mouse animal model- - - - - - - - - 34
3.6 Induction of host innate immune response by HBV - - - - - - - - 36
3.7 The TNF-α rather than IFN-mediated pathway is critical in HBV clearance- - - - - - - - - - - - - - - - - - - - - - - - - - 38
3.8 TNF-α deficiency leads to cytotoxic T lymphocyte dysfunction against HBV- - - - - - - - - - - - - - - - - - - - - - - - -40
3.9 TNF-α blockage-induced elevation of serum HBV viral loads and maintained HBV viral gene expression within the liver - - - - - - - - - - - -41
3.10 Lack of TNF-α eliminates HBcAg-induced HBsAg clearance- - - - 43
3.11 Intra-hepatic leukocytes, in contrasting with HBcAg containing hepatocytes, were responsible for HBcAg-induced TNF-α production- - - - - - -44
Chapter IV: Discussion - - - - - - - - - - - - - - - - - - - - -47
4.1 Immune suppression of intrahepatic T lymphocytes in
hydrodynamic-based transfection of HBV persistence mouse model- - 48
4.2 Antiviral response of intrahepatic T-cell can be restored by blocking PD-1 pathway in vivo - - - - - - - - - - - - - - - - - - - - - - - 51
4.3 TNF-α contributes to induce an effective T cell response and lead to HBV clearance - - - - - - - - - - - - - - - - - - - - - - - - - - 54
4.4 Intact structure of HBcAg is critical for triggering innate immune response
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -57
4.5 IFN-mediated pathways are not required for HBV clearance in vivo- - 61
4.6 Working hypothesis of HBV-induced immunity in this mouse model - -62
References - - - - - - - - - - - - - - - - - - - - - - - - - - - 65
List of Figures - - - - - - - - - - - - - - - - - - - - - - - - - -82
Figure 1. Increased programmed death (PD)-1-expressing CD8+ and CD4+ T-cells in liver-infiltrating lymphocytes from mice with hepatitis B viral (HBV) persistence- - - - - - - - - - - - - - - - - - - - - - - - - - - -84
Figure 2. Hepatitis B viral (HBV) core mutants induced upregulation of programmed death (PD)-1 expression in intrahepatic T lymphocytes - - -86
Figure 3. Liver-infiltrating CD8+ lymphocytes in carrier mice displayed the PD-1hiCD127low-exhausted phenotype- - - - - - - - - - - - - - - - 87
Figure 4. Liver-infiltrating PD-1+CD4+ T-cells in mice with hepatitis B viral (HBV) persistence exhibited the phenotype of regulatory T-cells- - - - - - - - 88
Figure 5. Impaired HBcAg-specific interferon (IFN)-γ production in C57BL/6 mice with hepatitis B viral (HBV) persistence was reversed by treatment of mice with anti-PD-1 mAb- - - - - - - - - - - - - - - - - - - - - - - - - -90
Figure 6. Blockade of the programmed death (PD)-1 pathway by an anti-PD-1 monoclonal antibody (mAb) reduced the hepatitis B viral (HBV) persistence rate in a mouse animal model- - - - - - - - - - - - - - - - - - - - - 91
Figure 7. Increased CD127 expression and reversed PD-1hiCD127low-exhausted CD8+ T cells phenotype in mice treated with an anti-PD-1 mAb- - - - - 92
Figure 8. Prolonged HBV persistence in TNF-α knockout mice - - - - - 93
Figure 9. Delayed HBsAg clearance in mice with TNF deficiency - - - - 95
Figure 10. Liver-infiltrating CD8+ lymphocytes in Etanercept-treated and TNF-α knockout mice displayed the PD-1hiCD127low-exhausted phenotype and impaired HBcAg-specific IFN-γ T cell response - - - - - - - - - - - - - - - -96
Figure 11. Delayed clearance of serum HBV DNA in mice with TNF deficiency
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -98
Figure 12. Decreased HBV replication and viral gene expression in Etanercept-treated and TNF-α knockout mice after pAAV/HBV1.2 injection- - - - - - - - - - - - - - - - - - - - - -99
Frigure 13. Hepatitis B viral core capsid triggered innate cytokines production in vivo - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 101
Figure 14. Intact HBV core capsid structure was required for innate cytokines induction - - - - - - - - - - - - - - - - - - - - - - - - - - - -102
Figure 15. A lack of TNF-α abolished HBcAg-induced HBsAg clearance in mice sera - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 103
Figure 16. Single dose administration of Enbrel at day 3 postinjection prolonged HBV persistence effectively- - - - - - - - - - - - - - - - - - - - -105
Figure 17. Intra hepatic leukocytes were responsible for HBcAg-induced TNF-α production in an ex vivo culture system- - - - - - - - - - - - - - - -106
Figure 18. Working hypothesis of HBV-induced immunity in this mouse model
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 108
Table 1 Characteristics of mice animal model for studying immune responses toward HBV- - - - - - - - - - - - - - - - - - - - - - - - - - - 109
Appendix - - - - - - - - - - - - - - - - - - - - - - - - - - - - 110
Figure A1. No significant difference in HBsAg clearance between wildtype C57BL/6 and IFNAR knockout mice- - - - - - - - - - - - - - - - - 111
Figure A2. Analysis of the induction kinetics of HBV-responsive genes- - 112
Figure A3. RIG-I-like receptor was critical for HBV core protein-induced IRF3 activation - - - - - - - - - - - - - - - - - - - - - - - - - - - - 113
Figure A4. Association of MDA5 and HBV core antigen was dependent on viral capsid structure- - - - - - - - - - - - - - - - - - - - - - - - - -115 | zh_TW |
dc.format.extent | 2489548 bytes | - |
dc.format.mimetype | application/pdf | - |
dc.language | en_US | - |
dc.rights | 論文公開時間:2017/03/17 | - |
dc.rights | 論文使用權限:同意無償授權 | - |
dc.subject | B型肝炎病毒 | zh_TW |
dc.subject | PD-1阻斷 | zh_TW |
dc.subject | 病毒持續性 | zh_TW |
dc.subject | 先天免疫力 | zh_TW |
dc.subject | 腫瘤壞死因子 | zh_TW |
dc.subject.classification | [SDGs]SDG3 | - |
dc.title | 以B型肝炎病毒小鼠模式探討核心蛋白引發免疫反應之機制 | zh_TW |
dc.title | Induction of Immune Response by Hepatitis B virus Core Protein in a Mouse Model | en |
dc.type | thesis | en |
dc.identifier.uri.fulltext | http://ntur.lib.ntu.edu.tw/bitstream/246246/262025/1/ntu-103-D96449003-1.pdf | - |
item.fulltext | with fulltext | - |
item.openairetype | thesis | - |
item.openairecristype | http://purl.org/coar/resource_type/c_46ec | - |
item.grantfulltext | open | - |
item.cerifentitytype | Publications | - |
顯示於: | 免疫學研究所
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