Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Summary

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

Abstract

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of liver cancer. This is a major concern, especially in developing countries. Failure of the immune system to mount an effective response against HBV leads to chronic infection. Although HBV vaccine is present and novel antiviral medicines are being created, eradication of virus-reservoir cells remains a major health topic. Described here is a method for the generation of viral antigen (Ag) -specific CD8⁺ cytotoxic T lymphocytes (CTLs) derived from induced pluripotent stem cells (iPSCs) (i.e., iPSC-CTLs), which have the ability to suppress HBV replication. HBV replication is efficiently induced in mice through hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver. Then, HBV surface Ag-specific mouse iPSC-CTLs are adoptively transferred, which greatly suppresses HBV replication in the liver and blood as well as prevents HBV surface Ag expression in hepatocytes. This method demonstrates HBV replication in mice after hydrodynamic injection and that stem cell-derived viral Ag-specific CTLs can suppress HBV replication. This protocol provides a useful method for HBV immunotherapy.

Introduction

Following acute infection, the adaptive immune system (i.e., humoral and cellular immunity) controls the bulk of acute HBV-related hepatitis. Still, a number of people in the HBV-endemic regions cannot eliminate the viruses and subsequently convert as chronic individuals. More than 25% of chronic patients (>250 million people) worldwide develop progressive liver disease, resulting in liver cirrhosis and/or hepatocellular carcinoma (HCC)¹. As a result, eradication of insistently infected cells remains a general healthiness problem, even though there is an available vaccine² and numerous antiviral medicines are under d....

Protocol

All animal experiments are approved by The Texas A&M University Animal Care Committee (IACUC; #2018-0006) and are conducted in compliance with the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care. Mice are used during 6–9 weeks of age.

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

1. Creation of the retroviral constructs
   NOTE: TCR α and β genes are linked with 2A self-cleaving sequence. The retroviral vector MSCV-IRES-DsRed (MiDR) is DsRed^{+ 23}.
   1. Sub-clone HBs

Results

As shown here, HBV viral Ag-specific iPSC-CD8⁺ T cells are generated by an in vitro culture system. After ACT of these viral Ag-specific iPSC-CD8⁺ T cells substantially suppress HBV replication in a murine model (Supplemental File 1). Mouse iPSC are transduced with the MIDR retroviral construct encoding a human-mouse hybrid HBV TCR gene (HBs_183-191-specific, s183), then the gene-transduced iPSCs are co-cultured with OP9-DL1/DL4 cells expressing Notch ligands (both DL1 and.......

Discussion

This protocol presents a method to generate the viral Ag-specific iPSC-CTLs for use as ACT to suppress HBV replication in a murine model. In chronic HBV infection, the viral genome forms a stable mini chromosome, the covalently closed circular DNA (cccDNA) that can persist throughout the lifespan of the hepatocyte. Targeting the clearance of the viral mini chromosome may result in a cure of chronic HBV infection. Current antiviral therapy targets the virus reverse transcriptase but rarely establishes immunological contro.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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