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. 2021 Jan 12;2(1):100264.
doi: 10.1016/j.xpro.2020.100264. eCollection 2021 Mar 19.

V体育安卓版 - An optimized protocol for the generation of HBV viral antigen-specific T lymphocytes from pluripotent stem cells

Mohammad Haque  1 , Xiaofang Xiong  1 , Fengyang Lei  2 , Jugal Kishore Das  1 , Jianxun Song  1
Affiliations

An optimized protocol for the generation of HBV viral antigen-specific T lymphocytes from pluripotent stem cells

Mohammad Haque (V体育官网入口) et al. STAR Protoc. .

Abstract

In T cell-based cancer immunotherapy, tumor antigen (Ag)-specific CD8+ cytotoxic T lymphocytes (CTLs) can specifically target tumor Ags on malignant cells. This promising approach drove us to adopt this strategy of T cell transfer (ACT)-based immunotherapy for chronic viral infections VSports手机版. Here, we describe the generation of hepatitis B virus (HBV) Ag-specific CTLs from induced pluripotent stem cells (iPSCs), i. e. , iPSC-CTLs. Ag-specific iPSC-CTLs can target HBV Ag+ cells and infiltrate into the liver to suppress HBV replication in a murine model. For complete details on the use and execution of this protocol, please refer to Haque et al. (2020). .

Keywords: Immunology; Stem Cells. V体育安卓版.

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Conflict of interest statement

The authors declare no competing interests.

"VSports注册入口" Figures

None
Graphical abstract
Figure 1
Figure 1
How to generate HBV viral Ag-specific iPSC-CTLs Transduce mouse iPSCs with the following retroviral constructs: HBs183-91 TCR (MiDR-HBV s183 TCR) or OVA257–264 TCR (MiDR-OVA TCR), and co-culture the transduced iPSCs with OP9-DL1/DL4 stromal cells for T lineage differentiation. (A) Present the schematic representation of the retrovirus constructs expressing HBV s183 TCR. Ψ, packaging signal; 2A, picornavirus self-cleaving 2A sequence; LTR, long terminal repeats. (B) Visualize the HBV TCR-transduced iPSCs by a fluorescence microscope. (C) Transduce GFP+ iPSCs (left and middle, no transduction) with the retroviral construct MiDR or MiDR with HBV s183 TCR, and analyze and sort the GFP+ DsRed+ iPSCs by flow cytometry (right) and by a high-speed cell sorter. (D) Analyze HBV s183 TCR for Vβ28 gene expression by PCR. (E) Show morphology of T cell differentiation on various days. (F) Show flow cytometric analysis of the iPSC-derived cells on day 28, gating on CD3+CD8+ cells, and analyze for the expression of CD8 and TCRVβ28. Data shown are representative of three individual experiments.
Figure 2
Figure 2
Perform functional analysis of HBV viral Ag-specific iPSC-CTLs On day 28 of in vitro co-culture, sort the SP CD8+s183 TCR pentamer+ iPSC-T cells. Stimulate the iPSC-T cells and CD8+ T cells transduced with MiDR-s183 TCR with s183 peptide (FLLTRILTI)-pulsed T-depleted splenocytes (APCs) from HHD mice. (A) Show intracellular staining of IL-2 and IFN-γ after 7 h (gated on CD8+ cells) (T/APCs = 1:4). (B) Present ELISA of IL-2 and IFN-γ after 40 h. (C) Measure T cell cytotoxicity after co-culture for 6 h using the 7-AAD/CFSE cell-mediated cytotoxicity assay kit. Data shown are representative of three individual experiments.
Figure 3
Figure 3
Induce HBV replication in HHD mice by hydrodynamic injection Perform hydrodynamic tail vein injection of HBV plasmid on HHD mice by injecting 10 μg of plasmid in 8% body weight of PBS. (A) Show serum HBV copies. On indicated time points after injection, isolate the serum from the blood and extract DNA for RT-PCR. (B) Present serum HBs Ag. (C) Show serum HBe Ag. (D) Show serum ALT. On indicated time points after injection, isolate the serum from the blood and determine protein expression by ELISA. (E) Present liver tissue histology. Euthanize mice on day 8 after HBV infection. Isolate and stain liver samples for histologic examination. (F) Show quantitation of HBs Ag+ cells on various days. Data shown are representative of three individual experiments (n = 5).
Figure 4
Figure 4
Show accumulation of HBV viral Ag-specific iPSC-CTLs in the liver with HBV replication Euthanize mice at day 8 after the adoptive transfer of T cells. Harvest the spleens or livers and isolate the intra-hepatic lymphocytes by enzymatic digestion. (A) Analyze accumulation of HBV-specific T cells by flow cytometry. Make single cell suspension from the intra-hepatic lymphocytes and stain cells with fluorochrome-conjugated antibodies. Gate on CD8+ T cells to analyze HBV-specific TCR. (B) Show immunohistochemistry. Stain liver slides with both fluorochrome-conjugated CD8 antibody and s183 TCR pentamer, and examine under a fluorescent microscope. Green color indicates CD8 and red color indicates HBV-specific TCR. Data are representative of five mice per group of three independent experiments.
Figure 5
Figure 5
Demonstrate in vivo reduction of HBV replication by viral Ag-specific iPSC-CTLs (A) Show serum HBV copies. At the indicated time points after the adoptive transfer of T cells, isolate the serum from the blood and extract DNA for RT-PCR analysis. (B) Present liver tissue histology. Euthanize mice on day 8 after the adoptive transfer of T cells. Isolate liver samples and stain for histologic examination. The upper panel shows HBs Ag protein expression (↑) in infected mice (IHC staining) and the lower panel shows the inflammatory cell infiltration (H&E staining). (C) Present quantitation of HBs Ag positive cells at various days. Data shown are three individual experiments (n = 5).
Figure 6
Figure 6
Identify the critical role of IFN-γ and TNF-α by viral Ag-specific iPSC-CTLs in the reduction of HBV replication (A) Show IFN-γ or TNF-α producing CD8+ T cells analyzed by intracellular staining and flow cytometric analysis. (B) Present immunohistochemistry. Stain liver slides with fluorescence-conjugated CD8 and IFN-γ or TNF-α antibodies, and examine under a fluorescent microscope (red, CD8+; green, IFN-γ+ or TNF-α+). (C) Show percentage of IFN-γ or TNF-α producing CD8+ T cells. (D) Show IFN-γ and TNF-α producing CD8+ T cells analyzed by intracellular staining and flow cytometric analysis, gating on CD8+ populations. Data are representative of five mice per group of three independent experiments.
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References

    1. Benechet A.P., De Simone G., Di Lucia P., Cilenti F., Barbiera G., Le Bert N., Fumagalli V., Lusito E., Moalli F., Bianchessi V. Dynamics and genomic landscape of CD8(+) T cells undergoing hepatic priming. Nature. 2019;574:200–205. - PMC - PubMed
    1. Benechet A.P., Iannacone M. Determinants of hepatic effector CD8(+) T cell dynamics. J. Hepatol. 2017;66:228–233. - PubMed
    1. Gehring A.J., Xue S.A., Ho Z.Z., Teoh D., Ruedl C., Chia A., Koh S., Lim S.G., Maini M.K., Stauss H., Bertoletti A. Engineering virus-specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines. J. Hepatol. 2011;55:103–110. - PubMed
    1. Haque M., Lei F., Xiong X., Ren Y., Kumar A., Das J.K., Ren X., Fang D., de Figueiredo P., Yang J.M., Song J. Stem Cell-Derived Viral Antigen-Specific T Cells Suppress HBV Replication through Production of IFN-γ and TNF-⍺. iScience. 2020;23:101333. - PMC - PubMed
    1. Isogawa M., Chung J., Murata Y., Kakimi K., Chisari F.V. CD40 activation rescues antiviral CD8(+) T cells from PD-1-mediated exhaustion. PLoS Pathog. 2013;9:e1003490. - PMC - PubMed

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