Name: stem cell-derived viral ag-specific t lymphocytes suppress hbv replication in mice
Uploaded: 2019-09-25T13:30:00
Description: Watch this Scientific Journal Video about Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice at JoVE.com

The authors thank Dr. Adam J Gehring from Toronto General Hospital Research Institute for providing cDNA for HBs183-91 (s183) (FLLTRILTI)- specific A2-restricted human-murine hybrid TCR genes, and Dr. Pei-Jer Chen from National Taiwan University for providing pAAV/HBV 1.2 construct. This work is supported by the National Institute of Health Grant R01AI121180, R01CA221867 and R21AI109239 to J. S.

All animal experiments are approved by The Texas A&#38;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&#8211;9 weeks of age.
1. Generation of viral Ag-specific CD8+ T cells from iPSCs (iPSC-CD8+ T cells)
<ol>
	<li>Creation of the retroviral constructs 
	NOTE: TCR &#945; and &#946; genes are linked with ...

<ol>
	<li>Scaglione, S. J., Lok, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+hepatitis+B+treatment+in+clinical+practice.">Effectiveness of hepatitis B treatment in clinical practice.</a> Gastroenterology. 142 (6), 1360-1368 (2012).</li><li>Osiowy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+infancy+and+beyond...+ensuring+a+lifetime+of+hepatitis+B+virus+(HBV)+vaccine-induced+immunity.">From infancy and beyond... ensuring a lifetime of hepatitis B virus (HBV) vaccine-induced immunity.</a> Human Vaccines &amp; Immunotherapeutics. 14 (8), 2093-2097 (2018).</li><li>Gish, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+HBsAg+antigen+during+treatment+with+entecavir+or+lamivudine+in+nucleoside-naive+HBeAg-positive+patients+with+chronic+hepatitis+B.">Loss of HBsAg antigen during treatment with entecavir or lamivudine in nucleoside-naive HBeAg-positive patients with chronic hepatitis B.</a> Journal of Viral Hepatitis. 17 (1), 16-22 (2010).</li><li>Kurktschiev, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysfunctional+CD8++T+cells+in+hepatitis+B+and+C+are+characterized+by+a+lack+of+antigen-specific+T-bet+induction.">Dysfunctional CD8+ T cells in hepatitis B and C are characterized by a lack of antigen-specific T-bet induction.</a> Journal of Experimental Medicine. 211 (10), 2047-2059 (2014).</li><li>Fisicaro, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiviral+intrahepatic+T-cell+responses+can+be+restored+by+blocking+programmed+death-1+pathway+in+chronic+hepatitis+B.">Antiviral intrahepatic T-cell responses can be restored by blocking programmed death-1 pathway in chronic hepatitis B.</a> Gastroenterology. 138 (2), 682-693 (2010).</li><li>Schurich, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+signal+cytokine+IL-12+rescues+the+anti-viral+function+of+exhausted+HBV-specific+CD8+T+cells.">The third signal cytokine IL-12 rescues the anti-viral function of exhausted HBV-specific CD8 T cells.</a> PLoS Pathogens. 9 (3), 1003208 (2013).</li><li>Gehring, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+virus-specific+T+cells+that+target+HBV+infected+hepatocytes+and+hepatocellular+carcinoma+cell+lines.">Engineering virus-specific T cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines.</a> Journal of Hepatology. 55 (1), 103-110 (2011).</li><li>Xia, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-gamma+and+Tumor+Necrosis+Factor-alpha+Produced+by+T+Cells+Reduce+the+HBV+Persistence+Form,+cccDNA,+Without+Cytolysis.">Interferon-gamma and Tumor Necrosis Factor-alpha Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis.</a> Gastroenterology. 150 (1), 194-205 (2016).</li><li>Huang, L. R., Wu, H. L., Chen, P. J., Chen, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immunocompetent+mouse+model+for+the+tolerance+of+human+chronic+hepatitis+B+virus+infection.">An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection.</a> Proceedings of the National Academy of Sciences U.S.A. 103 (47), 17862-17867 (2006).</li><li>Wong, P., Pamer, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8+T+cell+responses+to+infectious+pathogens.">CD8 T cell responses to infectious pathogens.</a> Annual Review of Immunology. 21, 29-70 (2003).</li><li>Murray, J. M., Wieland, S. F., Purcell, R. H., Chisari, F. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+hepatitis+B+virus+clearance+in+chimpanzees.">Dynamics of hepatitis B virus clearance in chimpanzees.</a> Proceedings of the National Academy of Sciences USA. 102 (49), 17780-17785 (2005).</li><li>Hinrichs, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptively+transferred+effector+cells+derived+from+naive+rather+than+central+memory+CD8++T+cells+mediate+superior+antitumor+immunity.">Adoptively transferred effector cells derived from naive rather than central memory CD8+ T cells mediate superior antitumor immunity.</a> Proceedings of the National Academy of Sciences U.S.A. 106 (41), 17469-17474 (2009).</li><li>Hinrichs, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+effector+CD8++T+cells+derived+from+naive+rather+than+memory+subsets+possess+superior+traits+for+adoptive+immunotherapy.">Human effector CD8+ T cells derived from naive rather than memory subsets possess superior traits for adoptive immunotherapy.</a> Blood. 117 (3), 808-814 (2011).</li><li>Kerkar, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+of+murine+CD8++and+CD4++T+cells+for+preclinical+adoptive+immunotherapy+studies.">Genetic engineering of murine CD8+ and CD4+ T cells for preclinical adoptive immunotherapy studies.</a> Journal of Immunotherapy. 34 (4), 343-352 (2011).</li><li>Kuball, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitating+matched+pairing+and+expression+of+TCR+chains+introduced+into+human+T+cells.">Facilitating matched pairing and expression of TCR chains introduced into human T cells.</a> Blood. 109 (6), 2331-2338 (2007).</li><li>van Loenen, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixed+T+cell+receptor+dimers+harbor+potentially+harmful+neoreactivity.">Mixed T cell receptor dimers harbor potentially harmful neoreactivity.</a> Proceedings of the National Academy of Sciences USA. 107 (24), 10972-10977 (2010).</li><li>Cameron, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+Titin-derived+HLA-A1-presented+peptide+as+a+cross-reactive+target+for+engineered+MAGE+A3-directed+T+cells.">Identification of a Titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells.</a> Science Translational Medicine. 5 (197), (2013).</li><li>Fedorov, V. D., Themeli, M., Sadelain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-1-+and+CTLA-4-based+inhibitory+chimeric+antigen+receptors+(iCARs)+divert+off-target+immunotherapy+responses.">PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses.</a> Science Translational Medicine. 5 (215), (2013).</li><li>Maus, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cells+expressing+chimeric+antigen+receptors+can+cause+anaphylaxis+in+humans.">T cells expressing chimeric antigen receptors can cause anaphylaxis in humans.</a> Cancer Immunolology Research. 1 (1), 26-31 (2013).</li><li>Haque, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programming+of+regulatory+T+cells+from+pluripotent+stem+cells+and+prevention+of+autoimmunity.">Programming of regulatory T cells from pluripotent stem cells and prevention of autoimmunity.</a> Journal of Immunology. 189 (3), 1228-1236 (2012).</li><li>Vizcardo, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+human+tumor+antigen-specific+T+cells+from+iPSCs+derived+from+mature+CD8(+)+T+cells.">Regeneration of human tumor antigen-specific T cells from iPSCs derived from mature CD8(+) T cells.</a> Cell Stem Cell. 12 (1), 31-36 (2013).</li><li>Nishimura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+rejuvenated+antigen-specific+T+cells+by+reprogramming+to+pluripotency+and+redifferentiation.">Generation of rejuvenated antigen-specific T cells by reprogramming to pluripotency and redifferentiation.</a> Cell Stem Cell. 12 (1), 114-126 (2013).</li><li>Lei, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+programming+of+tumor+antigen-specific+T+lymphocytes+from+pluripotent+stem+cells+to+promote+cancer+immunosurveillance.">In vivo programming of tumor antigen-specific T lymphocytes from pluripotent stem cells to promote cancer immunosurveillance.</a> Cancer Research. 71 (14), 4742-4747 (2011).</li><li>Kim, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oct4-induced+pluripotency+in+adult+neural+stem+cells.">Oct4-induced pluripotency in adult neural stem cells.</a> Cell. 136 (3), 411-419 (2009).</li><li>Lei, F., Haque, R., Xiong, X., Song, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+induced+pluripotent+stem+cells+towards+T+lymphocytes.">Directed differentiation of induced pluripotent stem cells towards T lymphocytes.</a> Journal of Visualized Experiments. (63), e3986 (2012).</li><li>Lei, F., Haque, M., Sandhu, P., Ravi, S., Ni, Y., Zheng, S., Fang, D., Jia, H., Yang, J. M., Song, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+naive+single-type+tumor+antigen-specific+CD8++T+lymphocytes+from+murine+pluripotent+stem+cells.">Development and characterization of naive single-type tumor antigen-specific CD8+ T lymphocytes from murine pluripotent stem cells.</a> OncoImmunology. 6, (2017).</li><li>Haque, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanoma+Immunotherapy+in+Mice+Using+Genetically+Engineered+Pluripotent+Stem+Cells.">Melanoma Immunotherapy in Mice Using Genetically Engineered Pluripotent Stem Cells.</a> Cell Transplantation. 25 (5), 811-827 (2016).</li><li>Tan, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Expression+Profiles+of+HBV+DNA+Integrated+Into+Genomes+of+Hepatocellular+Carcinoma+Cells+to+Select+T+Cells+for+Immunotherapy.">Use of Expression Profiles of HBV DNA Integrated Into Genomes of Hepatocellular Carcinoma Cells to Select T Cells for Immunotherapy.</a> Gastroenterology. , (2019).</li><li>Wu, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ly6C(+)+Monocytes+and+Kupffer+Cells+Orchestrate+Liver+Immune+Responses+Against+Hepatitis+B+Virus+in+Mice.">Ly6C(+) Monocytes and Kupffer Cells Orchestrate Liver Immune Responses Against Hepatitis B Virus in Mice.</a> Hepatology. , (2019).</li><li>Haque, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-derived+tissue-associated+regulatory+T+cells+suppress+the+activity+of+pathogenic+cells+in+autoimmune+diabetes.">Stem cell-derived tissue-associated regulatory T cells suppress the activity of pathogenic cells in autoimmune diabetes.</a> Journal of Clinical Investigation Insights. , (2019).</li><li>Chisari, F. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+pathological+effects+of+synthesis+of+hepatitis+B+virus+large+envelope+polypeptide+in+transgenic+mice.">Structural and pathological effects of synthesis of hepatitis B virus large envelope polypeptide in transgenic mice.</a> Proceedings of the National Academy of Sciences USA. 84 (19), 6909-6913 (1987).</li><li>Wirth, S., Guidotti, L. G., Ando, K., Schlicht, H. J., Chisari, F. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+tolerance+leads+to+autoantibody+production+but+not+autoimmune+liver+disease+in+hepatitis+B+virus+envelope+transgenic+mice.">Breaking tolerance leads to autoantibody production but not autoimmune liver disease in hepatitis B virus envelope transgenic mice.</a> Journal of Immunology. 154 (5), 2504-2515 (1995).</li></ol>

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...

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 (&#62;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 development. Standard treatment for HBV infection includes IFN-&#945;, nucleoside, and nucleotide analogues. These agents have direct antiviral activity and immune modulatory capacities. Nevertheless, seroconversion of HBe antigen (Ag)+ carriers with anti-HBe antibody (Ab) and loss of serum HBV deoxyribonucleic acid&#160;(DNA) appear individually in approximately 20% of treated patients, and whole immunological control of the virus verified by the deprivation of the HBsAg is no more than 5%3. Moreover, the response to treatment is often not durable. Prophylactic vaccination with recombinant HBs Ag is highly effective in preventing infection, but therapeutic HBs Ag vaccination is not effective. Clearly, T cell-mediated immune responses play a critical role in controlling HBV infection and liver impairment; however, in chronic hepatitis patients, HBV-reactive T cells are often deleted, dysfunctional, or convert exhausted4,5,6. Consequently, in individuals with persistent HBV infection, no attempts to reinstate HBV-specific immunity (i.e., T cell-based immunity) by means of anti-viral remedy, immuno-modulatory cytokines, or curative immunization have achieved success.
Adoptive cell transfer (ACT) of HBV Ag-specific T cells is an efficient treatment directed to eventually eradicate remaining hepatocytes wih HBV7,8. ACT of HBV-specific CTLs into HBV-infected mice has been shown to cause transient, mild hepatitis, and a dramatic drop in HBV ribonucleic acid (RNA) transcripts in hepatocytes. In these studies, CTLs did not inhibit transcription of HBV genes but enhanced the degradation of HBV transcripts9. HBV-specific CTLs are important to prevent viral infection and mediate the clearance of HBV10,11. For ACT-based remedies, in vitro expansion of HBV-specific T cells with a high reactivity for in vivo resettlement has been suggested to be an ideal method12,13,14; nevertheless, the present approaches are restricted regarding their abilities to generate, separate, and grow appropriate quantities and qualities of HBV-specific T cells from patients for the potential therapies.
Although clinical trials present safety, practicability, and prospective therapeutic activity of cell-based treatments by means of engineered T cells that are specific to HBV virus-infected hepatocytes, there are worries about the unfavorable effects occurring from autoimmune responses because of cross-reactivity from mispairing T cell receptor (TCR)15,16, off-target Ag recognition by non-specific TCR17 and on-target off-toxicity by a chimeric Ag receptor (CAR)18,19 with healthy tissues. Currently, the genetically modified T cells, which only have short-term persistence in vivo, are usually intermediate or later effector T cells. To date, pluripotent stem cells (PSCs) are the only source available to generate high numbers of naive single-type Ag-specific T cells20,21,22,23. Induced PSCs (iPSCs) are simply converted from a patient&#8217;s somatic cells through the use of gene transduction of several transcription factors. As a result, the iPSCs have similar characteristics as those of embryonic stem cells (ESCs)24. Owing to the flexibility and possibility for the infinite ability to self-renew, in addition to tissue replacement, iPSC-based treatments may be widely applied in regenerative medicine. Furthermore, the regiments underlying iPSCs may substantially improve current cell-based therapies.
The overall goal of this method is to generate a large amount of HBV-specific CTLs from iPSCs (i.e., iPSC-CTLs) for ACT-based immunotherapy. The advantages over alternative techniques are that HBV-specific iPSC-CTLs have a single-type TCR and naive phenotype, which results in more memory T cell development after the ACT. It is demonstrated that the ACT of HBV-specific iPSC-CTLs increases the migration of functional CD8+ T cells in the liver and reduces HBV replication in both the livers and blood of administered mice. This method reveals a potential use of viral Ag-specific iPSC-CTLs for HBV immunotherapy and may be adapted to generate other viral Ag-specific iPSC-T cells for viral immunotherapy.

<table>
	<tbody>
		<tr>
			<td>HHD mice</td>
			<td>Institut Pasteur, Paris, France</td>
			<td></td>
			<td>H-2 class I knockout, HLA-A2.1-transgenic (HHD) mice</td>
		</tr>
		<tr>
			<td>iPS-MEF-Ng-20D-17</td>
			<td>RIKEN Cell Bank</td>
			<td>APS0001</td>
			<td></td>
		</tr>
		<tr>
			<td>SNL76/7</td>
			<td>ATCC</td>
			<td>SCRC-1049</td>
			<td></td>
		</tr>
		<tr>
			<td>OP9</td>
			<td>ATCC</td>
			<td>CRL-2749</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV/HBV1.2 plasmid</td>
			<td>Dr. Dr. Pei-Jer Chen (National Taiwan University Hospital, Taiwan)</td>
			<td></td>
			<td>HBV DNA construct</td>
		</tr>
		<tr>
			<td>HBs183-91(s183) (FLLTRILTI)-specific TCR genes</td>
			<td>Dr. Adam J Gehring (Toronto General Hospital Research Institute, Toronto, Canada)</td>
			<td></td>
			<td>FLLTRILTI-specific A2-restricted human-murine hybrid TCR genes (V&#945;34 and V&#946;28)</td>
		</tr>
		<tr>
			<td>OVA257&#8211;264-specific TCR genes</td>
			<td>Dr. Dario A. Vignali (University of Pittsburgh, PA)</td>
			<td></td>
			<td>SIINFEKL-specific H-2Kb-restricted TCR genes</td>
		</tr>
		<tr>
			<td>Anti-CD3 (17A2) antibody</td>
			<td>Biolegend</td>
			<td>100236</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD44 (IM7) antibody</td>
			<td>BD Pharmingen</td>
			<td>103012</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD4 (GK1.5) antibody</td>
			<td>Biolegend</td>
			<td>100408</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD8 (53-6.7) antibody</td>
			<td>Biolegend</td>
			<td>100732</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-IFN-&#947; (XMG1.2) antibody</td>
			<td>Biolegend</td>
			<td>505810</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-TNF-a (MP6-XT22) antibody</td>
			<td>Biolegend</td>
			<td>506306</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Invitrogen</td>
			<td>A10490-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-HBs antibody</td>
			<td>Thermo Fisher</td>
			<td>MA5-13059</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysis buffer</td>
			<td>Lonza</td>
			<td>10-548E</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>Sigma</td>
			<td>B7651</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>ABCD1234</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SH3007.01</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSAria Fusion cell sorter</td>
			<td>BD</td>
			<td>656700</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>MilliporeSigma</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneJammer</td>
			<td>Agilent</td>
			<td>204130</td>
			<td></td>
		</tr>
		<tr>
			<td>HLA-A201-HBs183-91-PE pentamer</td>
			<td>Proimmune</td>
			<td>F027-4A - 27</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP Anti-Mouse Secondary Antibody</td>
			<td>Invitrogen</td>
			<td>A27025</td>
			<td></td>
		</tr>
		<tr>
			<td>mFlt-3L</td>
			<td>Peprotech</td>
			<td>250-31L</td>
			<td></td>
		</tr>
		<tr>
			<td>mIL-7</td>
			<td>Peprotech</td>
			<td>217-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease S7</td>
			<td>Roche</td>
			<td>10107921001</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>MilliporeSigma</td>
			<td>P6148-500G</td>
			<td>Caution: Allergenic, Carcenogenic, Toxic</td>
		</tr>
		<tr>
			<td>Permeabilization buffer</td>
			<td>Biolegend</td>
			<td>421002</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>MilliporeSigma</td>
			<td>107689</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong&#8482; Gold Antifade Mountant with DAPI</td>
			<td>Invitrogen</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp MinElute Virus Spin Kit</td>
			<td>Qiagen</td>
			<td>57704</td>
			<td></td>
		</tr>
	</tbody>
</table>

stem cell-derived viral ag-specific t lymphocytes suppress hbv replication in mice

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.

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 (HBs183-191-specific, s183), then the gene-transduced iPSCs are co-cultured with OP9-DL1/DL4 cells expressing Notch ligands (both DL1 and...

Watch this Scientific Journal Video about Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice at JoVE.com

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

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.

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...

Research

JoVE Journal

Immunology and Infection

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