Summary

干细胞衍生病毒Ag特异性T淋巴细胞抑制小鼠HBV复制

Published: September 25, 2019
doi:

Summary

这里介绍了一个协议,通过利用干细胞衍生病毒抗原(Ag)特异性T淋巴细胞的采用细胞转移(ACT),有效抑制小鼠乙型肝炎病毒(HBV)复制。这个程序可以适应潜在的基于ACT的HBV感染免疫治疗。

Abstract

乙型肝炎病毒(HBV)感染是一个全球性的健康问题。全世界有超过3.5亿人感染乙肝病毒,乙肝病毒感染仍然是肝癌的主要原因。这是一个重大关切,特别是在发展中国家。免疫系统未能对乙肝病毒进行有效反应,导致慢性感染。虽然乙肝疫苗存在,新的抗病毒药物正在研制中,但消灭病毒储存细胞仍然是一个主要的健康主题。本文介绍的一种用于生成病毒抗原 (Ag) – 特异性 CD8细胞毒性 T 淋巴细胞 (CTLs) 的方法,该细胞来自诱导多能干细胞 (iPSC)(即 iPSC-CTLs),能够抑制 HBV 复制。通过将HBV表达质粒(pAAV/HBV1.2)水动力注射到肝脏,在小鼠中有效诱导HBV复制。然后,HBV表面Ag特异性小鼠iPSC-CTLs被采用转移,这极大地抑制了乙肝病毒在肝脏和血液中的复制,并防止了肝细胞中HBV表面Ag表达。该方法在水动力注射后证明小鼠HBV复制,干细胞衍生的病毒Ag特异性CTL可以抑制HBV复制。该协议为HBV免疫治疗提供了一种有用的方法。

Introduction

急性感染后,适应性免疫系统(即体液和细胞免疫)控制与HBV相关的急性肝炎的大部分。然而,在乙肝病毒流行地区的一些人无法消除病毒,随后转化为慢性个体。全球超过25%的慢性病患者(超过2.5亿人)发展为渐进性肝病,导致肝硬化和/或肝细胞癌(HCC)1。因此,尽管现有疫苗2和许多抗病毒药物正在研制中,但根除持续感染的细胞仍然是一个普遍的健康问题。HBV感染的标准治疗包括IFN-α、核苷和核苷酸类似物。这些制剂具有直接的抗病毒活性和免疫调节能力。然而,HBe抗原(Ag)的血清转化+具有抗HBe抗体(Ab)的载体和血清HBV脱氧核糖核酸(DNA)的流失在大约20%的治疗患者中单独出现,病毒的整个免疫控制经核实,HBsAg的剥夺不超过5%3。此外,对治疗的反应往往不持久。使用重组HBs Ag进行预防性疫苗接种在预防感染方面非常有效,但治疗性HBs Ag疫苗接种无效。显然,T细胞介导的免疫反应在控制HBV感染和肝损伤方面起着关键作用;然而,在慢性肝炎患者中,HBV反应性T细胞经常被删除、功能失调或转换耗尽4,5,6。因此,在持续性乙肝病毒感染者中,没有试图通过抗病毒疗法、免疫调节细胞因子或治疗性免疫恢复HBV特异性免疫(即T细胞基免疫)的努力取得了成功。

HBV AG特异性T细胞的收养细胞转移(ACT)是一种有效的治疗方法,旨在最终根除剩余的肝细胞wih HBV7,8。HBV特异性CTP在HBV感染小鼠中已被证明会导致暂时性、轻度肝炎,肝细胞核糖核酸(RNA)转录本的显著下降。在这些研究中,CTL没有抑制HBV基因的转录,但提高了HBV录9的降解。HBV特异性CTL对预防病毒感染和调解HBV10、11的清除非常重要。对于基于ACT的补救措施,HBV特异性T细胞在体外扩张,具有高反应性,用于体内再定居,建议其为理想的方法12、13、14;然而,目前的方法在产生、分离和从患者身上产生、分离和生长适当数量的HBV特异性T细胞的能力方面受到限制,以进行潜在的治疗。

尽管临床试验通过针对受HBV病毒感染的肝细胞的工程T细胞提供细胞治疗的安全性、实用性和前瞻性治疗活性,但人们担心这种不良影响由于错误配对T细胞受体(TCR)15、16、非特异性TCR17的离目标Ag识别和由嵌合Ag受体(CAR)对靶向脱毒性的交叉反应,自体免疫反应发生18,19与健康组织。目前,转基因T细胞,只有短期的持久性在体内,通常是中间或后期效应T细胞。迄今为止,多能干细胞(PSCs)是唯一可用于产生大量天真的单型Ag特异性T细胞20、21、22、23的来源。诱导PSCs(iPSCs)通过使用多个转录因子的基因转导,从患者的体细胞中转换。因此,iPSC具有与胚胎干细胞(ESCs)24相似的特性。由于具有无限自我更新能力的灵活性和可能性,除了组织替代外,基于iPSC的治疗可以广泛应用于再生医学。此外,iPSC的底层军团可能会显著改善目前的基于细胞的疗法。

该方法的总体目标是从 iPSC(即 iPSC-CTL)生成大量 HBV 特异性 CTL,用于基于 ACT 的免疫治疗。与替代技术的优点是,HBV 特异性 iPSC-CTL 具有单型 TCR 和天真的表型,这导致在 ACT 之后产生更多的记忆 T 细胞发育。证明HBV特异性iPSC-CTL的ACT可增加肝功能CD8+T细胞的迁移,减少被管理小鼠肝脏和血液中的HBV复制。这种方法揭示了病毒Ag特异性iPSC-CTLs在HBV免疫治疗中的潜在用途,并可能用于生成用于病毒免疫治疗的其他病毒Ag特异性iPSC-T细胞。

Protocol

所有动物实验均获得德克萨斯A&M大学动物护理委员会(IACUC;#2018-0006)的批准,并符合实验室动物护理评估和认证协会的指导方针。小鼠在6-9周大期间使用。 1. 从 iPSC 生成病毒 Ag 特异性 CD8+ T 细胞(iPSC-CD8+ T 细胞) 逆转录病毒结构的创建注:TCR +和β基因与2A自结序列相关。逆转录病毒载体 MSCV-IRES-DsRed (MiDR) 是 DsRed= 23。<…

Representative Results

如此处所示,HBV病毒Ag特异性iPSC-CD8+ T细胞由体外培养系统生成。在这些病毒Ag特异性iPSC-CD8+ T细胞的ACT后,严重抑制HBV复制在鼠模型中(补充文件1)。小鼠 iPSC 与 MIDR 抗逆转录病毒结构进行转导,编码人-小鼠混合 HBV TCR 基因(HBs183-191-特异性,s183),然后基因转导 iPSC 与表达 Notch 配体(DL1 和 DL4)的 OP9-DL1/DL4 细胞共同培养分子在rFlt3L和rIL-7的存在。在体外共?…

Discussion

该协议提供了一种生成病毒性Ag特异性iPSC-CTLs的方法,用作ACT,以抑制小鼠模型中的HBV复制。在慢性HBV感染中,病毒基因组形成一个稳定的迷你染色体,即共价闭合的圆形DNA(cccDNA),可以在整个肝细胞的生命周期内持续。靶向病毒小染色体的清除可能导致慢性HBV感染的治愈。目前的抗病毒治疗针对病毒逆转录酶,但很少建立由cccDNA驱动的HBV复制的免疫控制。HBV 特异性 CD8 CTL 可以调解?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢多伦多总医院研究所的Adam J Gehring博士为HBs183-91(S183)(FLLTRILTI)提供cDNA-特定A2限制人-鼠杂交TCR基因,以及台湾国立大学的陈佩杰博士提供pAAV/HBV 1.2 结构。这项工作得到国家卫生补助机构R01AI121180、R01CA221867和R21AI109239到J.S的支持。

Materials

HHD mice Institut Pasteur, Paris, France H-2 class I knockout, HLA-A2.1-transgenic (HHD) mice
iPS-MEF-Ng-20D-17 RIKEN Cell Bank APS0001
SNL76/7 ATCC SCRC-1049
OP9 ATCC CRL-2749
pAAV/HBV1.2 plasmid Dr. Dr. Pei-Jer Chen (National Taiwan University Hospital, Taiwan) HBV DNA construct
HBs183-91(s183) (FLLTRILTI)-specific TCR genes Dr. Adam J Gehring (Toronto General Hospital Research Institute, Toronto, Canada) FLLTRILTI-specific A2-restricted human-murine hybrid TCR genes (Vα34 and Vβ28)
OVA257–264-specific TCR genes Dr. Dario A. Vignali (University of Pittsburgh, PA) SIINFEKL-specific H-2Kb-restricted TCR genes
Anti-CD3 (17A2) antibody Biolegend 100236
Anti-CD44 (IM7) antibody BD Pharmingen 103012
Anti-CD4 (GK1.5) antibody Biolegend 100408
Anti-CD8 (53-6.7) antibody Biolegend 100732
Anti-IFN-γ (XMG1.2) antibody Biolegend 505810
Anti-TNF-a (MP6-XT22) antibody Biolegend 506306
α-MEM Invitrogen A10490-01
Anti-HBs antibody Thermo Fisher MA5-13059
ACK Lysis buffer Lonza 10-548E
Brefeldin A Sigma B7651
DMEM Invitrogen ABCD1234
FBS Hyclone SH3007.01
FACSAria Fusion cell sorter BD 656700
Gelatin MilliporeSigma G9391
GeneJammer Agilent 204130
HLA-A201-HBs183-91-PE pentamer Proimmune F027-4A – 27
HRP Anti-Mouse Secondary Antibody Invitrogen A27025
mFlt-3L Peprotech 250-31L
mIL-7 Peprotech 217-17
Nuclease S7 Roche 10107921001
Paraformaldehyde MilliporeSigma P6148-500G Caution: Allergenic, Carcenogenic, Toxic
Permeabilization buffer Biolegend 421002
Polybrene MilliporeSigma 107689
ProLong™ Gold Antifade Mountant with DAPI Invitrogen P36931
QIAamp MinElute Virus Spin Kit Qiagen 57704

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Cite This Article
Xiong, X., Lei, F., Haque, M., Song, J. Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice. J. Vis. Exp. (151), e60043, doi:10.3791/60043 (2019).

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