Building and Optimizing a Virus-specific T Cell Receptor Library for Targeted Immunotherapy in Viral Infections
Identifieur interne : 000005 ( Pmc/Curation ); précédent : 000004; suivant : 000006Building and Optimizing a Virus-specific T Cell Receptor Library for Targeted Immunotherapy in Viral Infections
Auteurs : Nasirah Banu [Singapour] ; Adeline Chia [Singapour] ; Zi Zong Ho [Singapour] ; Alfonso Tan Garcia [Singapour] ; Komathi Paravasivam [Singapour] ; Gijsbert M. Grotenbreg [Singapour] ; Antonio Bertoletti [Singapour] ; Adam J. Gehring [Singapour, États-Unis]Source :
- Scientific Reports [ 2045-2322 ] ; 2014.
Abstract
Restoration of antigen-specific T cell immunity has the potential to clear persistent viral infection. T cell receptor (TCR) gene therapy can reconstitute CD8 T cell immunity in chronic patients. We cloned 10 virus-specific TCRs targeting 5 different viruses, causing chronic and acute infection. All 10 TCR genetic constructs were optimized for expression using a P2A sequence, codon optimization and the addition of a non-native disulfide bond. However, maximum TCR expression was only achieved after establishing the optimal orientation of the alpha and beta chains in the expression cassette; 9/10 TCRs favored the beta-P2A-alpha orientation over alpha-P2A-beta. Optimal TCR expression was associated with a significant increase in the frequency of IFN-gamma+ T cells. In addition, activating cells for transduction in the presence of Toll-like receptor ligands further enhanced IFN-gamma production. Thus, we have built a virus-specific TCR library that has potential for therapeutic intervention in chronic viral infection or virus-related cancers.
Url:
DOI: 10.1038/srep04166
PubMed: 24566718
PubMed Central: 3933865
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Grotenbreg</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Departments of Microbiology and Biological Sciences, Immunology Programme, National University of Singapore</institution>, Singapore</nlm:aff><country xml:lang="fr">Singapour</country><wicri:regionArea></wicri:regionArea></affiliation></author><author><name sortKey="Bertoletti, Antonio" sort="Bertoletti, Antonio" uniqKey="Bertoletti A" first="Antonio" last="Bertoletti">Antonio Bertoletti</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Singapore Institute for Clinical Sciences, Agency for Science Technology and Research (A*Star)</institution>, Singapore</nlm:aff><country xml:lang="fr">Singapour</country><wicri:regionArea></wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a3"><institution>Program of Emerging Viral Diseases, Duke-NUS Graduate Medical School, National University of Singapore</institution>, Singapore</nlm:aff><country xml:lang="fr">Singapour</country><wicri:regionArea></wicri:regionArea></affiliation></author><author><name sortKey="Gehring, Adam J" sort="Gehring, Adam J" uniqKey="Gehring A" first="Adam J." last="Gehring">Adam J. Gehring</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Singapore Institute for Clinical Sciences, Agency for Science Technology and Research (A*Star)</institution>, Singapore</nlm:aff><country xml:lang="fr">Singapour</country><wicri:regionArea></wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a4"><institution>Molecular Microbiology and Immunology, Saint Louis University School of Medicine</institution>, Saint Louis, Missouri,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a5"><institution>Saint Louis University Liver Center, Saint Louis University School of Medicine</institution>, Saint Louis, Missouri,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24566718</idno><idno type="pmc">3933865</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3933865</idno><idno type="RBID">PMC:3933865</idno><idno type="doi">10.1038/srep04166</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000005</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000005</idno><idno type="wicri:Area/Pmc/Curation">000005</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000005</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Building and Optimizing a Virus-specific T Cell Receptor Library for Targeted Immunotherapy in Viral Infections</title><author><name sortKey="Banu, Nasirah" sort="Banu, Nasirah" uniqKey="Banu N" first="Nasirah" last="Banu">Nasirah Banu</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Singapore Institute for Clinical Sciences, Agency for Science Technology and Research (A*Star)</institution>, Singapore</nlm:aff><country xml:lang="fr">Singapour</country><wicri:regionArea></wicri:regionArea></affiliation></author><author><name sortKey="Chia, Adeline" sort="Chia, Adeline" uniqKey="Chia A" first="Adeline" last="Chia">Adeline Chia</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Singapore Institute for Clinical Sciences, Agency for Science Technology and Research (A*Star)</institution>, Singapore</nlm:aff><country xml:lang="fr">Singapour</country><wicri:regionArea></wicri:regionArea></affiliation></author><author><name sortKey="Ho, Zi Zong" sort="Ho, Zi Zong" uniqKey="Ho Z" first="Zi Zong" last="Ho">Zi Zong Ho</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Singapore Institute for Clinical Sciences, Agency for Science Technology and Research (A*Star)</institution>, Singapore</nlm:aff><country xml:lang="fr">Singapour</country><wicri:regionArea></wicri:regionArea></affiliation></author><author><name sortKey="Garcia, Alfonso Tan" sort="Garcia, Alfonso Tan" uniqKey="Garcia A" first="Alfonso Tan" last="Garcia">Alfonso Tan Garcia</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Singapore Institute for Clinical Sciences, Agency for Science Technology and Research (A*Star)</institution>, Singapore</nlm:aff><country xml:lang="fr">Singapour</country><wicri:regionArea></wicri:regionArea></affiliation></author><author><name sortKey="Paravasivam, Komathi" sort="Paravasivam, Komathi" uniqKey="Paravasivam K" first="Komathi" last="Paravasivam">Komathi Paravasivam</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Singapore Institute for Clinical Sciences, Agency for Science Technology and Research (A*Star)</institution>, Singapore</nlm:aff><country xml:lang="fr">Singapour</country><wicri:regionArea></wicri:regionArea></affiliation></author><author><name sortKey="Grotenbreg, Gijsbert M" sort="Grotenbreg, Gijsbert M" uniqKey="Grotenbreg G" first="Gijsbert M." last="Grotenbreg">Gijsbert M. Grotenbreg</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Departments of Microbiology and Biological Sciences, Immunology Programme, National University of Singapore</institution>, Singapore</nlm:aff><country xml:lang="fr">Singapour</country><wicri:regionArea></wicri:regionArea></affiliation></author><author><name sortKey="Bertoletti, Antonio" sort="Bertoletti, Antonio" uniqKey="Bertoletti A" first="Antonio" last="Bertoletti">Antonio Bertoletti</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Singapore Institute for Clinical Sciences, Agency for Science Technology and Research (A*Star)</institution>, Singapore</nlm:aff><country xml:lang="fr">Singapour</country><wicri:regionArea></wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a3"><institution>Program of Emerging Viral Diseases, Duke-NUS Graduate Medical School, National University of Singapore</institution>, Singapore</nlm:aff><country xml:lang="fr">Singapour</country><wicri:regionArea></wicri:regionArea></affiliation></author><author><name sortKey="Gehring, Adam J" sort="Gehring, Adam J" uniqKey="Gehring A" first="Adam J." last="Gehring">Adam J. Gehring</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Singapore Institute for Clinical Sciences, Agency for Science Technology and Research (A*Star)</institution>, Singapore</nlm:aff><country xml:lang="fr">Singapour</country><wicri:regionArea></wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a4"><institution>Molecular Microbiology and Immunology, Saint Louis University School of Medicine</institution>, Saint Louis, Missouri,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation><affiliation wicri:level="1"><nlm:aff id="a5"><institution>Saint Louis University Liver Center, Saint Louis University School of Medicine</institution>, Saint Louis, Missouri,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Scientific Reports</title><idno type="eISSN">2045-2322</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Restoration of antigen-specific T cell immunity has the potential to clear persistent viral infection. T cell receptor (TCR) gene therapy can reconstitute CD8 T cell immunity in chronic patients. We cloned 10 virus-specific TCRs targeting 5 different viruses, causing chronic and acute infection. All 10 TCR genetic constructs were optimized for expression using a P2A sequence, codon optimization and the addition of a non-native disulfide bond. However, maximum TCR expression was only achieved after establishing the optimal orientation of the alpha and beta chains in the expression cassette; 9/10 TCRs favored the beta-P2A-alpha orientation over alpha-P2A-beta. Optimal TCR expression was associated with a significant increase in the frequency of IFN-gamma+ T cells. In addition, activating cells for transduction in the presence of Toll-like receptor ligands further enhanced IFN-gamma production. Thus, we have built a virus-specific TCR library that has potential for therapeutic intervention in chronic viral infection or virus-related cancers.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Bertoletti, A" uniqKey="Bertoletti A">A. Bertoletti</name></author><author><name sortKey="Ferrari, C" uniqKey="Ferrari C">C. Ferrari</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pagliara, D" uniqKey="Pagliara D">D. Pagliara</name></author><author><name sortKey="Savoldo, B" uniqKey="Savoldo B">B. Savoldo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lau, G K" uniqKey="Lau G">G. K. Lau</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lau, G K K" uniqKey="Lau G">G. K. K. Lau</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Loggi, E" uniqKey="Loggi E">E. Loggi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cobbold, M" uniqKey="Cobbold M">M. Cobbold</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rooney, C M" uniqKey="Rooney C">C. M. Rooney</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kreijtz, J H C M" uniqKey="Kreijtz J">J. H. C. M. Kreijtz</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sridhar, S" uniqKey="Sridhar S">S. Sridhar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Oh, H L J" uniqKey="Oh H">H. L. J. Oh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gehring, A J" uniqKey="Gehring A">A. J. Gehring</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sung, W K" uniqKey="Sung W">W.-K. Sung</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Szymczak, A L" uniqKey="Szymczak A">A. L. Szymczak</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yang, S" uniqKey="Yang S">S. Yang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kuball, J" uniqKey="Kuball J">J. Kuball</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cohen, C J" uniqKey="Cohen C">C. J. Cohen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Scholten, K B J" uniqKey="Scholten K">K. B. J. Scholten</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Engels, B" uniqKey="Engels B">B. Engels</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Leisegang, M" uniqKey="Leisegang M">M. Leisegang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Caron, G" uniqKey="Caron G">G. Caron</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhu, Q" uniqKey="Zhu Q">Q. Zhu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Haga Friedman, A" uniqKey="Haga Friedman A">A. Haga-Friedman</name></author><author><name sortKey="Horovitz Fried, M" uniqKey="Horovitz Fried M">M. Horovitz-Fried</name></author><author><name sortKey="Cohen, C J" uniqKey="Cohen C">C. J. Cohen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Okamoto, S" uniqKey="Okamoto S">S. Okamoto</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Aggen, D H" uniqKey="Aggen D">D. H. Aggen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sommermeyer, D" uniqKey="Sommermeyer D">D. Sommermeyer</name></author><author><name sortKey="Uckert, W" uniqKey="Uckert W">W. Uckert</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bialer, G" uniqKey="Bialer G">G. Bialer</name></author><author><name sortKey="Horovitz Fried, M" uniqKey="Horovitz Fried M">M. Horovitz-Fried</name></author><author><name sortKey="Ya Acobi, S" uniqKey="Ya Acobi S">S. Ya'acobi</name></author><author><name sortKey="Morgan, R A" uniqKey="Morgan R">R. A. Morgan</name></author><author><name sortKey="Cohen, C J" uniqKey="Cohen C">C. J. Cohen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Voss, R H" uniqKey="Voss R">R.-H. Voss</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sebestyen, Z" uniqKey="Sebestyen Z">Z. Sebestyén</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cohen, C J" uniqKey="Cohen C">C. J. Cohen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yang, S" uniqKey="Yang S">S. Yang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hinrichs, C S" uniqKey="Hinrichs C">C. S. Hinrichs</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Koh, S" uniqKey="Koh S">S. Koh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kearse, K P" uniqKey="Kearse K">K. P. Kearse</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bonifacino, J S" uniqKey="Bonifacino J">J. S. Bonifacino</name></author><author><name sortKey="Suzuki, C K" uniqKey="Suzuki C">C. K. Suzuki</name></author><author><name sortKey="Klausner, R D" uniqKey="Klausner R">R. D. Klausner</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Shin, J" uniqKey="Shin J">J. Shin</name></author><author><name sortKey="Lee, S" uniqKey="Lee S">S. Lee</name></author><author><name sortKey="Strominger, J L" uniqKey="Strominger J">J. L. Strominger</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Labrecque, N" uniqKey="Labrecque N">N. Labrecque</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Arber, C" uniqKey="Arber C">C. Arber</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Leen, A M" uniqKey="Leen A">A. M. Leen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chang, C X L" uniqKey="Chang C">C. X. L. Chang</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Sci Rep</journal-id><journal-id journal-id-type="iso-abbrev">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type="epub">2045-2322</issn><publisher><publisher-name>Nature Publishing Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24566718</article-id><article-id pub-id-type="pmc">3933865</article-id><article-id pub-id-type="pii">srep04166</article-id><article-id pub-id-type="doi">10.1038/srep04166</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Building and Optimizing a Virus-specific T Cell Receptor Library for Targeted Immunotherapy in Viral Infections</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Banu</surname><given-names>Nasirah</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Chia</surname><given-names>Adeline</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Ho</surname><given-names>Zi Zong</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Garcia</surname><given-names>Alfonso Tan</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Paravasivam</surname><given-names>Komathi</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Grotenbreg</surname><given-names>Gijsbert M.</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Bertoletti</surname><given-names>Antonio</given-names></name><xref ref-type="corresp" rid="c2">b</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Gehring</surname><given-names>Adam J.</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a4">4</xref><xref ref-type="aff" rid="a5">5</xref></contrib><aff id="a1"><label>1</label><institution>Singapore Institute for Clinical Sciences, Agency for Science Technology and Research (A*Star)</institution>, Singapore</aff><aff id="a2"><label>2</label><institution>Departments of Microbiology and Biological Sciences, Immunology Programme, National University of Singapore</institution>, Singapore</aff><aff id="a3"><label>3</label><institution>Program of Emerging Viral Diseases, Duke-NUS Graduate Medical School, National University of Singapore</institution>, Singapore</aff><aff id="a4"><label>4</label><institution>Molecular Microbiology and Immunology, Saint Louis University School of Medicine</institution>, Saint Louis, Missouri,<country>USA</country></aff><aff id="a5"><label>5</label><institution>Saint Louis University Liver Center, Saint Louis University School of Medicine</institution>, Saint Louis, Missouri,<country>USA</country></aff></contrib-group><author-notes><corresp id="c1"><label>a</label><email>gehringa@slu.edu</email></corresp><corresp id="c2"><label>b</label><email>Antonio@sics.a-star.edu.sg</email></corresp></author-notes><pub-date pub-type="epub"><day>25</day><month>02</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>4</volume><elocation-id>4166</elocation-id><history><date date-type="received"><day>05</day><month>09</month><year>2013</year></date><date date-type="accepted"><day>03</day><month>02</month><year>2014</year></date></history><permissions><copyright-statement>Copyright © 2014, Macmillan Publishers Limited. All rights reserved</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Macmillan Publishers Limited. All rights reserved</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by-nc-sa/3.0/"><pmc-comment>author-paid</pmc-comment>
<license-p>This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc-sa/3.0/">http://creativecommons.org/licenses/by-nc-sa/3.0/</ext-link></license-p></license></permissions><abstract><p>Restoration of antigen-specific T cell immunity has the potential to clear persistent viral infection. T cell receptor (TCR) gene therapy can reconstitute CD8 T cell immunity in chronic patients. We cloned 10 virus-specific TCRs targeting 5 different viruses, causing chronic and acute infection. All 10 TCR genetic constructs were optimized for expression using a P2A sequence, codon optimization and the addition of a non-native disulfide bond. However, maximum TCR expression was only achieved after establishing the optimal orientation of the alpha and beta chains in the expression cassette; 9/10 TCRs favored the beta-P2A-alpha orientation over alpha-P2A-beta. Optimal TCR expression was associated with a significant increase in the frequency of IFN-gamma+ T cells. In addition, activating cells for transduction in the presence of Toll-like receptor ligands further enhanced IFN-gamma production. Thus, we have built a virus-specific TCR library that has potential for therapeutic intervention in chronic viral infection or virus-related cancers.</p></abstract></article-meta></front><floats-group><fig id="f1"><label>Figure 1</label><caption><title>Optimization strategies to increase TCR expression.</title><p>(A) Frequency of CD8+, multimer+ T cells following transduction with the different formats of TCR gene cassettes: α + β (separate vectors), α-β-Opt (P2A linked, optimized, single vector), α-βcys-Opt (P2A linked, optimized, non-native disulfide, single vector). (B) Dot plots of 3 TCRs showing expression differences between TCRs without and with the additional disulphid bond. Values in upper right quadrant is mean fluorescence intensity of CD8+ multimer+ T cells. Experiments were performed at least twice for each TCR.</p></caption><graphic xlink:href="srep04166-f1"></graphic></fig><fig id="f2"><label>Figure 2</label><caption><title>Orientation of the alpha and beta genes within the cassettes impacts expression.</title><p>(A) Diagram describing the different orientations of the TCR cassette. (B) Representative dot plots using HBV env 171-80 TCR showing difference in TCR beta staining with each orientation. (C) Summary of Vbeta staining from both orientations for all TCRs (D) Mean frequency of Vbeta+ CD8+ T cells when TCRs were grouped according to their postive (higher) and negative (lower) expression. (E) Representative dot plots using HBV env 171-80 TCR showing difference in HLA tetramer staining with each orientation. (F) Summary of HLA-multimer staining from both orientations for all TCRs (G) Mean frequency of HLA-multimer+ CD8+ T cells when TCRs were grouped according to their postive (higher) and negative (lower) expression. There was a statistically significant difference in expression between the positive and negative orientation using the paired t test.</p></caption><graphic xlink:href="srep04166-f2"></graphic></fig><fig id="f3"><label>Figure 3</label><caption><title>TCR orientation significantly impacts T cell function.</title><p>(A) Representative dot plots showing the difference in IFN-γ+ CD8+ T cell frequency using the HBV env 183-91 TCR in both orientations. (B) Frequency of IFN-γ+ CD8+ T cells for each TCR in both orientations. (C) Mean frequency of IFN-γ+ CD8+ T cells when TCRs were grouped according to their postive (higher) and negative (lower) expression. There was a statistically significant difference in IFN-γ+ T cells between the positive and negative orientation deteremined by paired t test. Positive orientation of the TCR increased the production of (D) TNF-α and (E) IL-2 in the transduced T cell population.</p></caption><graphic xlink:href="srep04166-f3"></graphic></fig><fig id="f4"><label>Figure 4</label><caption><title>TLR ligands increase functionality of transduced T cells.</title><p>(A) Pentamer staining following transduction of T cells activated in the presence of IL-2 alone or IL-2 + TLR ligands. (B) Frequency of pentamer+ CD8+ T cells after activation in different conditions. (C) Frequency of IFN-γ+ CD8+ T cells after activation in different conditions. (D) Percent increase in IFN-γ+ CD8+ T cells compared to T cells activated in IL-2 alone. (E) Frequency of pentamer+ CD4+ T cells after activation in different conditions. (F) Frequency of IFN-γ+ CD4+ T cells after activation in different conditions. (G) Percent increase in IFN-γ+ CD4+ T cells compared to T cells activated in IL-2 alone. Experiment is representative of 3 separate donors.</p></caption><graphic xlink:href="srep04166-f4"></graphic></fig><fig id="f5"><label>Figure 5</label><caption><title>Cytokines in culture supernatant during T cell activation in the presence of TLR ligands.</title><p>(A) Immunoregulatory/Th2 cyotkines in the culture medium 48 h after T cell activation. (B) Inflammatory/Th1 cytokines in the culture medium 48 h after T cell activation.</p></caption><graphic xlink:href="srep04166-f5"></graphic></fig><fig id="f6"><label>Figure 6</label><caption><title>IFN-γ production and cytotoxic function of TCR+ T cells.</title><p>(A) Frequency of HLA-pentamer+ CD8+ T cells following transduction of T cells activated in the presence of IL-2 alone or IL-2 + polyI:C or ssRNA40. Mock cells are untransduced T cells as negative control (B) Frequency of IFN-γ+ T cells in each transduction condition. Bars show total frequency of IFN-γ+ CD8+ T cells. Black portion constitutes the IFN-γ+/pentamer+ T cell fraction. (C) Dot plots showing pentamer vs. IFN-γ staining on CD8+ T cells transduced under different conditions. (D) Frequency of TCR beta+ CD8+ T cells following transduction of T cells activated in the presence of IL-2 alone or IL-2 + polyI:C or ssRNA40. (E) Frequency of IFN-γ+ T cells in each transduction condition. Bars show total frequency of IFN-γ+ CD8+ T cells. (F) Dot plots showing TCR beta vs. IFN-γ staining on CD8+ T cells transduced under different conditions. (G) Specific lysis of hepatocyte cell lines expressing HBV antigens with sorted HBV core18–27 TCR, V beta+ CD8+ transduced T cells cultured under different conditions. HG2sAg expresses irrelevant antigen as negative control and HG2cAg expresses HBV core antigen.</p></caption><graphic xlink:href="srep04166-f6"></graphic></fig><table-wrap position="float" id="t1"><label>Table 1</label><caption><title>Cloned Virus-specific T cell receptors</title></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col><col align="center"></col></colgroup><thead valign="bottom"><tr><th align="justify" valign="top" charoff="50">#</th><th align="justify" valign="top" charoff="50">Virus</th><th align="justify" valign="top" charoff="50">Ag</th><th align="center" valign="top" charoff="50">aa position</th><th align="justify" valign="top" charoff="50">Peptide Sequence</th><th align="justify" valign="top" charoff="50">HLA</th><th align="justify" valign="top" charoff="50">Optimal orientation</th><th align="center" valign="top" charoff="50">Vβ<xref ref-type="fn" rid="t1-fn1">a</xref></th><th align="center" valign="top" charoff="50">Pent<xref ref-type="fn" rid="t1-fn1">a</xref></th><th align="center" valign="top" charoff="50">IFN-<italic>γ</italic><xref ref-type="fn" rid="t1-fn1">a</xref></th></tr></thead><tbody valign="top"><tr><td align="justify" valign="top" charoff="50">1</td><td align="justify" valign="top" charoff="50">CMV</td><td align="justify" valign="top" charoff="50">IE1</td><td align="char" valign="top" char="-" charoff="50">42–50</td><td align="justify" valign="top" charoff="50">KEVNSQLSL</td><td align="justify" valign="top" charoff="50">B4001</td><td align="justify" valign="top" charoff="50">Vβ27-P2A-Vα26</td><td align="char" valign="top" char="." charoff="50">1.2</td><td align="char" valign="top" char="." charoff="50">3.5</td><td align="char" valign="top" char="." charoff="50">1.3</td></tr><tr><td align="justify" valign="top" charoff="50">2</td><td align="justify" valign="top" charoff="50">CMV</td><td align="justify" valign="top" charoff="50">pp65</td><td align="char" valign="top" char="-" charoff="50">501–09</td><td align="justify" valign="top" charoff="50">ATVQGQNLK</td><td align="justify" valign="top" charoff="50">A1101</td><td align="justify" valign="top" charoff="50">Vβ9-P2A-Vα29</td><td align="char" valign="top" char="." charoff="50">4.1</td><td align="char" valign="top" char="." charoff="50">8.1</td><td align="char" valign="top" char="." charoff="50">1.6</td></tr><tr><td align="justify" valign="top" charoff="50">3</td><td align="justify" valign="top" charoff="50">CMV</td><td align="justify" valign="top" charoff="50">pp65</td><td align="char" valign="top" char="-" charoff="50">495–505</td><td align="justify" valign="top" charoff="50">NLVPMVATV</td><td align="justify" valign="top" charoff="50">A0201</td><td align="justify" valign="top" charoff="50">Vβ12-P2A-Vα5</td><td align="char" valign="top" char="." charoff="50">1.1</td><td align="char" valign="top" char="." charoff="50">1</td><td align="char" valign="top" char="." charoff="50">1.2</td></tr><tr><td align="justify" valign="top" charoff="50">4</td><td align="justify" valign="top" charoff="50">EBV</td><td align="justify" valign="top" charoff="50">EBNA-4NP</td><td align="char" valign="top" char="-" charoff="50">399–408</td><td align="justify" valign="top" charoff="50">AVFDRKSDAK</td><td align="justify" valign="top" charoff="50">A11</td><td align="justify" valign="top" charoff="50">Vβ5-P2A-Vα19</td><td align="char" valign="top" char="." charoff="50">2.4</td><td align="left" valign="top" charoff="50"> </td><td align="char" valign="top" char="." charoff="50">3.2</td></tr><tr><td align="justify" valign="top" charoff="50">5</td><td align="justify" valign="top" charoff="50">HBV</td><td align="justify" valign="top" charoff="50">env</td><td align="char" valign="top" char="-" charoff="50">171–80</td><td align="justify" valign="top" charoff="50">FLGPLLVLQA</td><td align="justify" valign="top" charoff="50">Cw0801</td><td align="justify" valign="top" charoff="50">Vβ20.1-P2A-Vα5</td><td align="char" valign="top" char="." charoff="50">2.5</td><td align="char" valign="top" char="." charoff="50">6.9</td><td align="char" valign="top" char="." charoff="50">6.3</td></tr><tr><td align="justify" valign="top" charoff="50">6</td><td align="justify" valign="top" charoff="50">HBV</td><td align="justify" valign="top" charoff="50">core</td><td align="char" valign="top" char="-" charoff="50">18–27 V</td><td align="justify" valign="top" charoff="50">FLPSDFFPSV</td><td align="justify" valign="top" charoff="50">A0201</td><td align="justify" valign="top" charoff="50">Vα17-P2A-Vβ12-4</td><td align="char" valign="top" char="." charoff="50">2.9</td><td align="char" valign="top" char="." charoff="50">2.4</td><td align="char" valign="top" char="." charoff="50">3.7</td></tr><tr><td align="justify" valign="top" charoff="50">7</td><td align="justify" valign="top" charoff="50">HBV</td><td align="justify" valign="top" charoff="50">env</td><td align="char" valign="top" char="-" charoff="50">370–379</td><td align="justify" valign="top" charoff="50">SIVSPFIPLL</td><td align="justify" valign="top" charoff="50">A0201</td><td align="justify" valign="top" charoff="50">Vβ7.8-P2A-Vα12</td><td align="left" valign="top" charoff="50"> </td><td align="char" valign="top" char="." charoff="50">9.8</td><td align="char" valign="top" char="." charoff="50">5.4</td></tr><tr><td align="justify" valign="top" charoff="50">8</td><td align="justify" valign="top" charoff="50">HBV</td><td align="justify" valign="top" charoff="50">env</td><td align="char" valign="top" char="-" charoff="50">183–191</td><td align="justify" valign="top" charoff="50">FLLTRILTI</td><td align="justify" valign="top" charoff="50">A0201</td><td align="justify" valign="top" charoff="50">Vβ28-P2A-Vα34.1</td><td align="char" valign="top" char="." charoff="50">1.2</td><td align="char" valign="top" char="." charoff="50">1.9</td><td align="char" valign="top" char="." charoff="50">1.9</td></tr><tr><td align="justify" valign="top" charoff="50">9</td><td align="justify" valign="top" charoff="50">SARS</td><td align="justify" valign="top" charoff="50">NP</td><td align="char" valign="top" char="-" charoff="50">216–225</td><td align="justify" valign="top" charoff="50">GETALALLLL</td><td align="justify" valign="top" charoff="50">B4001</td><td align="justify" valign="top" charoff="50">Vβ4.3-P2A-Vα4.1</td><td align="char" valign="top" char="." charoff="50">1</td><td align="char" valign="top" char="." charoff="50">2.7</td><td align="char" valign="top" char="." charoff="50">1.4</td></tr><tr><td align="justify" valign="top" charoff="50">10</td><td align="justify" valign="top" charoff="50">Flu</td><td align="justify" valign="top" charoff="50">M1</td><td align="char" valign="top" char="-" charoff="50">58–66</td><td align="justify" valign="top" charoff="50">GILGFVFTL</td><td align="justify" valign="top" charoff="50">A0201</td><td align="justify" valign="top" charoff="50">Vβ19.1-P2A-Vα27</td><td align="char" valign="top" char="." charoff="50">1</td><td align="char" valign="top" char="." charoff="50">1.3</td><td align="char" valign="top" char="." charoff="50">1.1</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="left" valign="top" charoff="50"> </td><td align="left" valign="top" charoff="50"> </td><td align="left" valign="top" charoff="50"> </td><td align="left" valign="top" charoff="50"> </td><td align="left" valign="top" charoff="50"> </td><td align="justify" valign="top" charoff="50">Mean</td><td align="char" valign="top" char="." charoff="50">1.9</td><td align="char" valign="top" char="." charoff="50">4.2</td><td align="char" valign="top" char="." charoff="50">2.7</td></tr></tbody></table><table-wrap-foot><fn id="t1-fn1"><p><sup>a</sup>Fold increase based on positive orientation of TCR 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