Method Article

Suppressing Hepatitis B Virus Replication Using Viral Antigen-Specific T Cells

July 8th, 2025

In This Article

Abstract

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Source: Xiong, X., et al. Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice. J. Vis. Exp. (2019).

The video demonstrates an assay using human induced pluripotent stem cell (iPSC)-derived CD8+ T cells to inhibit hepatitis B virus (HBV) replication in mice. These T cells, engineered with HBV-specific receptors, are transferred into transgenic mice. Replication-competent HBV genome plasmids are then injected directly into the liver, enabling viral DNA uptake. Once viral antigens are expressed and presented by hybrid MHC-I molecules, the engineered T cells recognize and bind to these complexes. This triggers the release of cytotoxic molecules, resulting in the destruction of HBV-infected liver cells and suppression of viral replication.

Protocol

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All procedures involving animal models have been reviewed by the local
institutional animal care committee and the JoVE veterinary review board. All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Generation of viral Ag-specific CD8+ T cells from iPSCs (iPSC-CD8+ T cells)

  1. Creation of the retroviral constructs
    NOTE: T-cell receptor (TCR) α and β genes are linked with 2A self-cleaving sequence. The retroviral vector MSCV-IRES-DsRed (MiDR) is DsRed+.
    1. Sub-clone HBs183-191 (FLLTRI....

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Results

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TCR gene construct diagram, cell development timeline, flow cytometry analysis for T cell markers.
Figure 1: Generation of HBV viral Ag-specific iPSC-CD8+ T cells.

Mouse iPSCs are transduced with the following retroviral constructs: HBs183-91 TCR (MiDR-s183 TCR) or OVA257–264 TCR (MiDR-OVA TCR), and the tran.......

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Disclosures

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No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
HHD miceInstitut Pasteur, Paris, FranceH-2 class I knockout, HLA-A2.1-transgenic (HHD) mice
iPS-MEF-Ng-20D-17RIKEN Cell BankAPS0001
SNL76/7ATCCSCRC-1049
OP9ATCCCRL-2749
pAAV/HBV1.2 plasmidDr. Dr. Pei-Jer Chen (National Taiwan University Hospital, Taiwan)HBV DNA construct
HBs183-91(s183) (FLLTRILTI)-specific TCR genesDr. 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 genesDr. Dario A. Vignali (University of Pittsburgh, PA)SIINFEKL-specific H-2Kb-restricted TCR genes
Anti-CD3 (17A2) antibodyBiolegend100236
Anti-CD44 (IM7) antibodyBD Pharmingen103012
Anti-CD4 (GK1.5) antibodyBiolegend100408
Anti-CD8 (53-6.7) antibodyBiolegend100732
Anti-IFN-γ (XMG1.2) antibodyBiolegend505810
Anti-TNF-a (MP6-XT22) antibodyBiolegend506306
α-MEMInvitrogenA10490-01
Anti-HBs antibodyThermo FisherMA5-13059
ACK Lysis bufferLonza10-548E
Brefeldin ASigmaB7651
DMEMInvitrogenABCD1234
FBSHycloneSH3007.01
FACSAria Fusion cell sorterBD656700
GelatinMilliporeSigmaG9391
GeneJammerAgilent204130
HLA-A201-HBs183-91-PE pentamerProimmuneF027-4A - 27
HRP Anti-Mouse Secondary AntibodyInvitrogenA27025
mFlt-3LPeprotech250-31L
mIL-7Peprotech217-17
Nuclease S7Roche10107921001
ParaformaldehydeMilliporeSigmaP6148-500GCaution: Allergenic, Carcenogenic, Toxic
Permeabilization bufferBiolegend421002
PolybreneMilliporeSigma107689
ProLong™ Gold Antifade Mountant with DAPIInvitrogenP36931
QIAamp MinElute Virus Spin KitQiagen57704

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Tags

Hepatitis B VirusViral Antigen Specific T CellsiPSC Derived CD8 T CellsHydrodynamic Plasmid DeliveryHBV Genome PlasmidsTail Vein InjectionAntigen Presenting MoleculesCytotoxic Molecule ReleaseHBV Infected Liver CellsViral Replication Suppression

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