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

Abstract

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

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) Creation of the retroviral constructs NOTE: T-cell receptor (TCR) α an…

Representative Results

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 transduced iPSCs are co-cultured with OP9-DL1/DL4 stromal cells for T lineage differentiation. (A) Schematic representation of the retroviral constr…

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