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.
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)
2. Induction of HBV replication through hydrodynamic delivery of HBV plasmid
NOTE: pAAV/HBV1.2 construct was generated as described previously. The HBV 1.2 complete DNA is incorporated in the vector pAAV.
3. Reduction of HBV replication by ACT of viral Ag-specific iPSC-CD8+ T cells
Table 1: PCR reaction volume
DNA template | 2 ml |
DNA master hybridization mixture ((Taq DNA polymerase, PCR reaction buffer, 10 mM MgCl2, and dNTP mixture) | 1 ml |
25 mM MgCl2 | 0.8 ml |
0.3 μM the probe | 3 ml |
5 μM of each primer | 3.2 ml |
Total | 10 ml |
Table 2: PCR program
Denaturation | Temperature 95 °C |
Time 5 s |
Annealing | 53 °C | 10 s |
Extension | 72 °C | 20 s |
5 °C |
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 construct MiDR-s183 TCR expressing s183-specific TCR. Ψ = packaging signal; 2A = picornavirus self-cleaving 2A sequence; LTR = long terminal repeats. (B) Morphology of T cell differentiation on days 0, 7, 14, and 22. (C) Flow cytometric analysis for the iPSC-derived cells on day 28. CD3+CD8+ cells (left) are gated as indicated and analyzed for the expression of CD8 and TCRVβ28 (right). Data shown are representative of three individual experiments. The values represent mean ± SD (**p < 0.01; paired t-tests).
Figure 2: Functional analysis of the HBV viral Ag-specific iPSC-CD8+ T cells.
On day 28 of in vitro co-culture, the SP CD8+s183 TCR pentamer+ iPSC-T cells are sorted. The iPSC-T cells and CD8+ T cells transduced with MiDR-s183 TCR are stimulated by T-depleted splenocytes (APCs) from HHD mice and pulsed with s183 peptide (FLLTRILTI). (A) Intracellular staining of IFN-ϒ after 7 h (gated on CD8+ cells) (T/APCs = 1:4). (B) ELISA of IFN-γ after 40 h. Data shown are representative of three individual experiments. The values represent mean ± SD (n.s., p > 0.05; paired t-tests).
Figure 3: Induction of HBV replication in HHD mice by hydrodynamic injection.
HHD mice are i.v. administrated with HBV plasmid via hydrodynamic tail vein injection. 10 μg of the plasmid is injected with 8% of total body mass PBS. On indicated time points after injection, the serum is isolated from the blood and DNA is extracted for real-time PCR. Data shown are representative of three individual experiments. The values represent mean ± SD. Data are representative of five mice per group of three independent experiments.
The authors have nothing to disclose.
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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