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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Opposing Development of Cytotoxic and Follicular Helper CD4 T Cells Controlled by the TCF-1-Bcl6 Nexus</title><author><name sortKey="Donnarumma, Tiziano" sort="Donnarumma, Tiziano" uniqKey="Donnarumma T" first="Tiziano" last="Donnarumma">Tiziano Donnarumma</name><affiliation><nlm:aff id="aff1">Retroviral Immunology, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK</nlm:aff></affiliation></author><author><name sortKey="Young, George R" sort="Young, George R" uniqKey="Young G" first="George R." last="Young">George R. Young</name><affiliation><nlm:aff id="aff1">Retroviral Immunology, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Retrovirus-Host Interactions, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK</nlm:aff></affiliation></author><author><name sortKey="Merkenschlager, Julia" sort="Merkenschlager, Julia" uniqKey="Merkenschlager J" first="Julia" last="Merkenschlager">Julia Merkenschlager</name><affiliation><nlm:aff id="aff1">Retroviral Immunology, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK</nlm:aff></affiliation></author><author><name sortKey="Eksmond, Urszula" sort="Eksmond, Urszula" uniqKey="Eksmond U" first="Urszula" last="Eksmond">Urszula Eksmond</name><affiliation><nlm:aff id="aff1">Retroviral Immunology, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK</nlm:aff></affiliation></author><author><name sortKey="Bongard, Nadine" sort="Bongard, Nadine" uniqKey="Bongard N" first="Nadine" last="Bongard">Nadine Bongard</name><affiliation><nlm:aff id="aff4">Institute for Virology, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany</nlm:aff></affiliation></author><author><name sortKey="Nutt, Stephen L" sort="Nutt, Stephen L" uniqKey="Nutt S" first="Stephen L." last="Nutt">Stephen L. Nutt</name><affiliation><nlm:aff id="aff5">The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia</nlm:aff></affiliation></author><author><name sortKey="Boyer, Claude" sort="Boyer, Claude" uniqKey="Boyer C" first="Claude" last="Boyer">Claude Boyer</name><affiliation><nlm:aff id="aff6">Centre d’Immunologie de Marseille-Luminy (CIML), Aix-Marseille University, UM2, Marseille 13288, France</nlm:aff></affiliation></author><author><name sortKey="Dittmer, Ulf" sort="Dittmer, Ulf" uniqKey="Dittmer U" first="Ulf" last="Dittmer">Ulf Dittmer</name><affiliation><nlm:aff id="aff4">Institute for Virology, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany</nlm:aff></affiliation></author><author><name sortKey="Le Trilling, Vu Thuy Khanh" sort="Le Trilling, Vu Thuy Khanh" uniqKey="Le Trilling V" first="Vu Thuy Khanh" last="Le-Trilling">Vu Thuy Khanh Le-Trilling</name><affiliation><nlm:aff id="aff4">Institute for Virology, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany</nlm:aff></affiliation></author><author><name sortKey="Trilling, Mirko" sort="Trilling, Mirko" uniqKey="Trilling M" first="Mirko" last="Trilling">Mirko Trilling</name><affiliation><nlm:aff id="aff4">Institute for Virology, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany</nlm:aff></affiliation></author><author><name sortKey="Bayer, Wibke" sort="Bayer, Wibke" uniqKey="Bayer W" first="Wibke" last="Bayer">Wibke Bayer</name><affiliation><nlm:aff id="aff4">Institute for Virology, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany</nlm:aff></affiliation></author><author><name sortKey="Kassiotis, George" sort="Kassiotis, George" uniqKey="Kassiotis G" first="George" last="Kassiotis">George Kassiotis</name><affiliation><nlm:aff id="aff1">Retroviral Immunology, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Department of Medicine, Faculty of Medicine, Imperial College London, London W2 1PG, UK</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">27806296</idno><idno type="pmc">5149578</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5149578</idno><idno type="RBID">PMC:5149578</idno><idno type="doi">10.1016/j.celrep.2016.10.013</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">002372</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">002372</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Opposing Development of Cytotoxic and Follicular Helper CD4 T Cells Controlled by the TCF-1-Bcl6 Nexus</title><author><name sortKey="Donnarumma, Tiziano" sort="Donnarumma, Tiziano" uniqKey="Donnarumma T" first="Tiziano" last="Donnarumma">Tiziano Donnarumma</name><affiliation><nlm:aff id="aff1">Retroviral Immunology, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK</nlm:aff></affiliation></author><author><name sortKey="Young, George R" sort="Young, George R" uniqKey="Young G" first="George R." last="Young">George R. Young</name><affiliation><nlm:aff id="aff1">Retroviral Immunology, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Retrovirus-Host Interactions, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK</nlm:aff></affiliation></author><author><name sortKey="Merkenschlager, Julia" sort="Merkenschlager, Julia" uniqKey="Merkenschlager J" first="Julia" last="Merkenschlager">Julia Merkenschlager</name><affiliation><nlm:aff id="aff1">Retroviral Immunology, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK</nlm:aff></affiliation></author><author><name sortKey="Eksmond, Urszula" sort="Eksmond, Urszula" uniqKey="Eksmond U" first="Urszula" last="Eksmond">Urszula Eksmond</name><affiliation><nlm:aff id="aff1">Retroviral Immunology, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK</nlm:aff></affiliation></author><author><name sortKey="Bongard, Nadine" sort="Bongard, Nadine" uniqKey="Bongard N" first="Nadine" last="Bongard">Nadine Bongard</name><affiliation><nlm:aff id="aff4">Institute for Virology, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany</nlm:aff></affiliation></author><author><name sortKey="Nutt, Stephen L" sort="Nutt, Stephen L" uniqKey="Nutt S" first="Stephen L." last="Nutt">Stephen L. Nutt</name><affiliation><nlm:aff id="aff5">The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia</nlm:aff></affiliation></author><author><name sortKey="Boyer, Claude" sort="Boyer, Claude" uniqKey="Boyer C" first="Claude" last="Boyer">Claude Boyer</name><affiliation><nlm:aff id="aff6">Centre d’Immunologie de Marseille-Luminy (CIML), Aix-Marseille University, UM2, Marseille 13288, France</nlm:aff></affiliation></author><author><name sortKey="Dittmer, Ulf" sort="Dittmer, Ulf" uniqKey="Dittmer U" first="Ulf" last="Dittmer">Ulf Dittmer</name><affiliation><nlm:aff id="aff4">Institute for Virology, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany</nlm:aff></affiliation></author><author><name sortKey="Le Trilling, Vu Thuy Khanh" sort="Le Trilling, Vu Thuy Khanh" uniqKey="Le Trilling V" first="Vu Thuy Khanh" last="Le-Trilling">Vu Thuy Khanh Le-Trilling</name><affiliation><nlm:aff id="aff4">Institute for Virology, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany</nlm:aff></affiliation></author><author><name sortKey="Trilling, Mirko" sort="Trilling, Mirko" uniqKey="Trilling M" first="Mirko" last="Trilling">Mirko Trilling</name><affiliation><nlm:aff id="aff4">Institute for Virology, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany</nlm:aff></affiliation></author><author><name sortKey="Bayer, Wibke" sort="Bayer, Wibke" uniqKey="Bayer W" first="Wibke" last="Bayer">Wibke Bayer</name><affiliation><nlm:aff id="aff4">Institute for Virology, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany</nlm:aff></affiliation></author><author><name sortKey="Kassiotis, George" sort="Kassiotis, George" uniqKey="Kassiotis G" first="George" last="Kassiotis">George Kassiotis</name><affiliation><nlm:aff id="aff1">Retroviral Immunology, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK</nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Department of Medicine, Faculty of Medicine, Imperial College London, London W2 1PG, UK</nlm:aff></affiliation></author></analytic><series><title level="j">Cell Reports</title><idno type="eISSN">2211-1247</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Summary</title><p>CD4<sup>+</sup> T cells develop distinct and often contrasting helper, regulatory, or cytotoxic activities. Typically a property of CD8<sup>+</sup> T cells, granzyme-mediated cytotoxic T cell (CTL) potential is also exerted by CD4<sup>+</sup> T cells. However, the conditions that induce CD4<sup>+</sup> CTLs are not entirely understood. Using single-cell transcriptional profiling, we uncover a unique signature of Granzyme B (GzmB)<sup>+</sup> CD4<sup>+</sup> CTLs, which distinguishes them from other CD4<sup>+</sup> T helper (Th) cells, including Th1 cells, and strongly contrasts with the follicular helper T (Tfh) cell signature. The balance between CD4<sup>+</sup> CTL and Tfh differentiation heavily depends on the class of infecting virus and is jointly regulated by the Tfh-related transcription factors <italic>Bcl6</italic> and <italic>Tcf7</italic> (encoding TCF-1) and by the expression of the inhibitory receptors PD-1 and LAG3. This unique profile of CD4<sup>+</sup> CTLs offers targets for their study, and its antagonism by the Tfh program separates CD4<sup>+</sup> T cells with either helper or killer functions.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Akhmetzyanova, I" uniqKey="Akhmetzyanova I">I. Akhmetzyanova</name></author><author><name sortKey="Zelinskyy, G" uniqKey="Zelinskyy G">G. Zelinskyy</name></author><author><name sortKey="Schimmer, S" uniqKey="Schimmer S">S. Schimmer</name></author><author><name sortKey="Brandau, S" uniqKey="Brandau S">S. Brandau</name></author><author><name sortKey="Altenhoff, P" uniqKey="Altenhoff P">P. Altenhoff</name></author><author><name sortKey="Sparwasser, T" uniqKey="Sparwasser T">T. Sparwasser</name></author><author><name sortKey="Dittmer, U" uniqKey="Dittmer U">U. Dittmer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Antunes, I" uniqKey="Antunes I">I. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Cell Rep</journal-id><journal-id journal-id-type="iso-abbrev">Cell Rep</journal-id><journal-title-group><journal-title>Cell Reports</journal-title></journal-title-group><issn pub-type="epub">2211-1247</issn><publisher><publisher-name>Cell Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">27806296</article-id><article-id pub-id-type="pmc">5149578</article-id><article-id pub-id-type="publisher-id">S2211-1247(16)31399-7</article-id><article-id pub-id-type="doi">10.1016/j.celrep.2016.10.013</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Opposing Development of Cytotoxic and Follicular Helper CD4 T Cells Controlled by the TCF-1-Bcl6 Nexus</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Donnarumma</surname><given-names>Tiziano</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Young</surname><given-names>George R.</given-names></name><xref rid="aff1" ref-type="aff">1</xref><xref rid="aff2" ref-type="aff">2</xref></contrib><contrib contrib-type="author"><name><surname>Merkenschlager</surname><given-names>Julia</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Eksmond</surname><given-names>Urszula</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author"><name><surname>Bongard</surname><given-names>Nadine</given-names></name><xref rid="aff4" ref-type="aff">4</xref></contrib><contrib contrib-type="author"><name><surname>Nutt</surname><given-names>Stephen L.</given-names></name><xref rid="aff5" ref-type="aff">5</xref></contrib><contrib contrib-type="author"><name><surname>Boyer</surname><given-names>Claude</given-names></name><xref rid="aff6" ref-type="aff">6</xref></contrib><contrib contrib-type="author"><name><surname>Dittmer</surname><given-names>Ulf</given-names></name><xref rid="aff4" ref-type="aff">4</xref></contrib><contrib contrib-type="author"><name><surname>Le-Trilling</surname><given-names>Vu Thuy Khanh</given-names></name><xref rid="aff4" ref-type="aff">4</xref></contrib><contrib contrib-type="author"><name><surname>Trilling</surname><given-names>Mirko</given-names></name><xref rid="aff4" ref-type="aff">4</xref></contrib><contrib contrib-type="author"><name><surname>Bayer</surname><given-names>Wibke</given-names></name><xref rid="aff4" ref-type="aff">4</xref></contrib><contrib contrib-type="author"><name><surname>Kassiotis</surname><given-names>George</given-names></name><email>george.kassiotis@crick.ac.uk</email><xref rid="aff1" ref-type="aff">1</xref><xref rid="aff3" ref-type="aff">3</xref><xref rid="fn1" ref-type="fn">7</xref><xref rid="cor1" ref-type="corresp">∗</xref></contrib></contrib-group><aff id="aff1"><label>1</label>Retroviral Immunology, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK</aff><aff id="aff2"><label>2</label>Retrovirus-Host Interactions, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK</aff><aff id="aff3"><label>3</label>Department of Medicine, Faculty of Medicine, Imperial College London, London W2 1PG, UK</aff><aff id="aff4"><label>4</label>Institute for Virology, University Hospital Essen, University Duisburg-Essen, 45122 Essen, Germany</aff><aff id="aff5"><label>5</label>The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia</aff><aff id="aff6"><label>6</label>Centre d’Immunologie de Marseille-Luminy (CIML), Aix-Marseille University, UM2, Marseille 13288, France</aff><author-notes><corresp id="cor1"><label>∗</label>Corresponding author <email>george.kassiotis@crick.ac.uk</email></corresp><fn id="fn1"><label>7</label><p id="ntpara0010">Lead Contact</p></fn></author-notes><pub-date pub-type="pmc-release"><day>01</day><month>11</month><year>2016</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="collection"><day>01</day><month>11</month><year>2016</year></pub-date><pub-date pub-type="epub"><day>01</day><month>11</month><year>2016</year></pub-date><volume>17</volume><issue>6</issue><fpage>1571</fpage><lpage>1583</lpage><history><date date-type="received"><day>7</day><month>6</month><year>2016</year></date><date date-type="rev-recd"><day>31</day><month>8</month><year>2016</year></date><date date-type="accepted"><day>4</day><month>10</month><year>2016</year></date></history><permissions><copyright-statement>© 2016 The Author(s)</copyright-statement><copyright-year>2016</copyright-year><license license-type="CC BY" xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</license-p></license></permissions><abstract id="abs0010"><title>Summary</title><p>CD4<sup>+</sup> T cells develop distinct and often contrasting helper, regulatory, or cytotoxic activities. Typically a property of CD8<sup>+</sup> T cells, granzyme-mediated cytotoxic T cell (CTL) potential is also exerted by CD4<sup>+</sup> T cells. However, the conditions that induce CD4<sup>+</sup> CTLs are not entirely understood. Using single-cell transcriptional profiling, we uncover a unique signature of Granzyme B (GzmB)<sup>+</sup> CD4<sup>+</sup> CTLs, which distinguishes them from other CD4<sup>+</sup> T helper (Th) cells, including Th1 cells, and strongly contrasts with the follicular helper T (Tfh) cell signature. The balance between CD4<sup>+</sup> CTL and Tfh differentiation heavily depends on the class of infecting virus and is jointly regulated by the Tfh-related transcription factors <italic>Bcl6</italic> and <italic>Tcf7</italic> (encoding TCF-1) and by the expression of the inhibitory receptors PD-1 and LAG3. This unique profile of CD4<sup>+</sup> CTLs offers targets for their study, and its antagonism by the Tfh program separates CD4<sup>+</sup> T cells with either helper or killer functions.</p></abstract><abstract abstract-type="graphical" id="abs0015"><title>Graphical Abstract</title><fig id="undfig1" position="anchor"><graphic xlink:href="fx1"></graphic></fig></abstract><abstract abstract-type="author-highlights" id="abs0020"><title>Highlights</title><p><list list-type="simple"><list-item id="u0010"><label>•</label><p>Adenoviruses prime CD4 T cells with CTL potential, but retroviruses do not</p></list-item><list-item id="u0015"><label>•</label><p>CD4 CTLs are transcriptionally distinguishable from other Th cells</p></list-item><list-item id="u0020"><label>•</label><p>The CD4 CTL program is the direct opposite of the Tfh program</p></list-item><list-item id="u0025"><label>•</label><p>CD4 CTLs are restrained by the TCF-1-Bcl6 nexus and by PD-1 and LAG3</p></list-item></list></p></abstract><abstract abstract-type="teaser" id="abs0025"><p>“Helper” CD4 T cells can also exhibit granzyme-mediated cytotoxicity. Donnarumma et al. investigate the conditions that induce CD4 CTLs and describe the dominant effect of the class of infecting virus. They uncover a unique transcriptional signature of CD4 CTLs and its multi-layered control.</p></abstract><kwd-group id="kwrds0010"><title>Keywords</title><kwd>cytotoxic CD4 T cells</kwd><kwd>retroviral infection</kwd><kwd>adenovirus-based vaccination</kwd><kwd>single-cell RNA sequencing</kwd><kwd>inhibitory receptors</kwd><kwd>CD4 T cell differentiation</kwd><kwd>antiviral immunity</kwd></kwd-group></article-meta><notes><p id="misc0010">Published: November 1, 2016</p></notes></front><body><sec id="sec1"><title>Introduction</title><p>CD4<sup>+</sup> TCRαβ T cells centrally orchestrate multiple arms of innate and adaptive immunity using distinct and, often, opposing effector and regulatory functions through the differentiation of distinguishable functional CD4<sup>+</sup> T cell subsets (<xref rid="bib28" ref-type="bibr">O’Shea and Paul, 2010</xref>, <xref rid="bib36" ref-type="bibr">Swain et al., 2012</xref>, <xref rid="bib45" ref-type="bibr">Zhu et al., 2010</xref>). Several such functional subsets are now recognized, including the prototypic Th1 and Th2 subsets but also the Th17, follicular helper (Tfh), and regulatory T (Treg) subsets, each characterized by a well-defined transcriptional program (<xref rid="bib9" ref-type="bibr">Crotty, 2014</xref>, <xref rid="bib28" ref-type="bibr">O’Shea and Paul, 2010</xref>, <xref rid="bib36" ref-type="bibr">Swain et al., 2012</xref>, <xref rid="bib41" ref-type="bibr">Vinuesa et al., 2016</xref>, <xref rid="bib45" ref-type="bibr">Zhu et al., 2010</xref>). Based on the increasingly appreciated diversity of CD4<sup>+</sup> T cell activities, additional functional subsets have been proposed. These include CD4<sup>+</sup> with cytotoxic T cell (CTL) potential, able to kill target cells through the release of granzyme-containing granules (<xref rid="bib5" ref-type="bibr">Brown et al., 2016</xref>, <xref rid="bib6" ref-type="bibr">Cheroutre and Husain, 2013</xref>, <xref rid="bib36" ref-type="bibr">Swain et al., 2012</xref>).</p><p>Cytotoxicity is typically associated with CD8<sup>+</sup> T cells and natural killer (NK) cells and has not been conventionally considered a CD4<sup>+</sup> T cell function (<xref rid="bib10" ref-type="bibr">Cullen et al., 2010</xref>). Indeed, “helper” and “cytotoxic” terms are often used to describe the major histocompatibility complex (MHC) class-II-restricted CD4<sup>+</sup> and MHC class-I-restricted CD8<sup>+</sup> TCRαβ T cell lineages, respectively. Commitment of developing thymocytes to the CD4<sup>+</sup> or CD8<sup>+</sup> lineage and acquisition of either helper or cytotoxic activity is controlled by the antagonistic transcription factors ThPOK and Runx3. ThPOK suppresses the cytotoxic program in CD4<sup>+</sup> thymocytes and mature T cells, whereas Runx3 promotes this program in CD8<sup>+</sup> T cells (<xref rid="bib6" ref-type="bibr">Cheroutre and Husain, 2013</xref>).</p><p>Despite transcriptional repression of the cytotoxic program during CD4<sup>+</sup> T cell development, MHC class-II-restricted, cell-contact-dependent cytotoxicity has long been observed in a variety of conditions, both in humans and experimental animals (<xref rid="bib4" ref-type="bibr">Brown, 2010</xref>, <xref rid="bib5" ref-type="bibr">Brown et al., 2016</xref>, <xref rid="bib6" ref-type="bibr">Cheroutre and Husain, 2013</xref>, <xref rid="bib34" ref-type="bibr">Soghoian and Streeck, 2010</xref>, <xref rid="bib36" ref-type="bibr">Swain et al., 2012</xref>, <xref rid="bib40" ref-type="bibr">van de Berg et al., 2008</xref>). Although CD4<sup>+</sup> T cells can kill target cells also through surface expression of tumor necrosis factor (TNF) family members, including FasL and TRAIL, accumulated evidence has established their ability to develop and use granzyme-mediated cytotoxic activity (<xref rid="bib5" ref-type="bibr">Brown et al., 2016</xref>, <xref rid="bib6" ref-type="bibr">Cheroutre and Husain, 2013</xref>). Pivotal recent studies with a ThPOK-reporter mouse strain uncovered considerable plasticity of the “helper” program in CD4<sup>+</sup> T cells, with loss of ThPOK expression and transcriptional switch to the “cytotoxic” program. Indeed, a sizable fraction of CD4<sup>+</sup> T cells residing in the intestine as intraepithelial lymphocytes (IELs) downregulate the expression of <italic>Zbtb7b</italic> (the gene encoding ThPOK) and acquire the expression of <italic>Runx3</italic> (<xref rid="bib27" ref-type="bibr">Mucida et al., 2013</xref>, <xref rid="bib33" ref-type="bibr">Reis et al., 2013</xref>). This transcriptional reprogramming is accompanied by the expression of genes more characteristic of the CD8<sup>+</sup> lineage, such as <italic>Cd8a</italic>, <italic>Crtam</italic>, and <italic>Eomes</italic>, and, importantly, also by the development of GzmB-mediated cytotoxic potential (<xref rid="bib27" ref-type="bibr">Mucida et al., 2013</xref>, <xref rid="bib33" ref-type="bibr">Reis et al., 2013</xref>).</p><p>These studies emphasize the similarities between CD8<sup>+</sup> T cells and reprogrammed CD4<sup>+</sup> CTLs and suggest that the latter subset should be viewed as distinct from other CD4<sup>+</sup> Th cell subsets (<xref rid="bib6" ref-type="bibr">Cheroutre and Husain, 2013</xref>). Despite these significant advances, the factors that dictate CD4<sup>+</sup> CTL differentiation are still incompletely understood. Also unclear are the precise place of CD4<sup>+</sup> CTLs in the transcriptional spectrum of all Th cell subsets and whether the CD4<sup>+</sup> CTL program is compatible with other Th cell differentiation pathways.</p><p>Here, we investigated the priming requirements for CD4<sup>+</sup> CTLs and the signals that either promote or inhibit CD4<sup>+</sup> CTL differentiation. To overcome the lack of a reliable marker for the unambiguous identification of CD4<sup>+</sup> CTLs, we applied global transcriptional analysis of single CD4<sup>+</sup> T cells. We report that CD4<sup>+</sup> CTL differentiation strictly depends on the infecting or immunizing virus, with retroviral infection and adenovirus-based vaccination at the low and high ends of the spectrum, respectively. Moreover, our results uncovered regulation of the CD4<sup>+</sup> CTL program by expression of inhibitory receptors and direct antagonism by the Tfh program.</p></sec><sec id="sec2"><title>Results</title><sec id="sec2.1"><title>CD4<sup>+</sup> CTL Development Depends on Infecting Virus</title><p>We have previously described an adoptive transfer system that allows the study of the CD4<sup>+</sup> T cell response to the dominant H2-A<sup>b</sup>-restricted env<sub>122–141</sub> epitope within the Friend murine leukemia virus (F-MLV) gp70 glycoprotein (<xref rid="bib24" ref-type="bibr">Merkenschlager et al., 2016</xref>, <xref rid="bib38" ref-type="bibr">Thorborn et al., 2014</xref>). Small numbers of allotypically marked EF4.1 TCRβ-transgenic CD4<sup>+</sup> T cells were transferred into wild-type (WT) C57BL/6 (B6) recipients and primed either by infection with Friend virus (FV) or by immunization with a human Adenovirus 5 (Ad5)-based vector expressing F-MLV gp70 (Ad5.pIX-gp70) (<xref rid="bib3" ref-type="bibr">Bayer et al., 2010</xref>). FV is a retroviral complex of F-MLV and spleen focus-forming virus (SFFV) that causes chronic infection in B6 mice (<xref rid="bib14" ref-type="bibr">Hasenkrug and Chesebro, 1997</xref>, <xref rid="bib39" ref-type="bibr">Tsuji-Kawahara et al., 2013</xref>), whereas the Ad5.pIX-gp70 vaccine vector is replication defective (<xref rid="bib3" ref-type="bibr">Bayer et al., 2010</xref>). Microarray-based comparison of EF4.1 env-reactive CD4<sup>+</sup> T cells primed by Ad5.pIX-gp70 indicated elevated transcription of CTL-related genes, in comparison with T cells primed by FV (<xref rid="bib38" ref-type="bibr">Thorborn et al., 2014</xref>). Indeed, on day 7 of the response, env-reactive effector CD4<sup>+</sup> T cells expressed significantly higher amounts of <italic>Gzmb</italic> mRNA when primed by Ad5.pIX-gp70 than when primed by FV (<xref rid="fig1" ref-type="fig">Figure 1</xref>A). Moreover, the hosts exhibited significantly higher levels of MHC class-II-restricted in vivo cytotoxicity against env<sub>122–141</sub>-pulsed B cell targets when primed by Ad5.pIX-gp70 than when primed by FV (<xref rid="fig1" ref-type="fig">Figure 1</xref>B). More efficient in vivo killing also correlated with enhanced GzmB-mediated in vitro killing, by purified env-reactive CD4<sup>+</sup> T cells, of B cells loaded with a fluorogenic GzmB substrate (<xref rid="fig1" ref-type="fig">Figure 1</xref>C).</p><p>Consistent with higher <italic>Gzmb</italic> expression and GzmB-mediated killing at the population level, env-reactive effector CD4<sup>+</sup> T cells contained a significantly higher proportion of GzmB<sup>+</sup> cells if primed by Ad5.pIX-gp70 than if primed by FV (<xref rid="fig1" ref-type="fig">Figure 1</xref>D). Notably, GzmB protein expression was detected in env-reactive effector CD4<sup>+</sup> T cells even without in vitro restimulation (<xref rid="mmc1" ref-type="supplementary-material">Figure S1</xref>A), suggesting that it reflected in-vivo-induced production. Moreover, EF4.1 env-reactive CD4<sup>+</sup> T cells, additionally carrying an allele encoding a fusion of GzmB and tdTomato fluorescent protein (<xref rid="bib26" ref-type="bibr">Mouchacca et al., 2013</xref>), contained a significantly higher frequency of GzmB-tdTomato<sup>+</sup> cells when primed by Ad5.pIX-gp70 than when primed by FV (<xref rid="mmc1" ref-type="supplementary-material">Figure S1</xref>B). Together, these data support the idea that GzmB production was induced in vivo in splenic CD4<sup>+</sup> T cells during Ad5.pIX-gp70 immunization. Furthermore, Ad5.pIX-gp70 vaccination induced a significantly higher frequency of GzmB<sup>+</sup> cells in splenic host effector CD44<sup>+</sup>IFN-γ<sup>+</sup>CD8<sup>+</sup> T cells than FV infection did (<xref rid="mmc1" ref-type="supplementary-material">Figure S2</xref>), arguing that the difference between the two immunogens was not restricted to CD4<sup>+</sup> T cells or to TCR (T cell-receptor)-transgenic T cells.</p><p>One notable difference between FV infection and Ad5.pIX-gp70 immunization is their ability to prime different TCR clonotypes (<xref rid="bib38" ref-type="bibr">Thorborn et al., 2014</xref>). EF4.1 env-reactive CD4<sup>+</sup> T cells induced by FV are primarily TCR Vα2<sup>+</sup>, whereas those induced by Ad5.pIX-gp70 express a member of the TCR Vα3 family (<xref rid="bib38" ref-type="bibr">Thorborn et al., 2014</xref>). Differences in TCR usage could underlie the distinct ability of FV and Ad5.pIX-gp70 to induce CD4<sup>+</sup> CTLs. Indeed, differentiation of GzmB<sup>+</sup> CD4<sup>+</sup> T cells was moderately higher in Vα3<sup>+</sup> than the Vα2<sup>+</sup> fraction of FV-primed env-reactive CD4<sup>+</sup> T cells (<xref rid="mmc1" ref-type="supplementary-material">Figures S3</xref>A and S3B). Nevertheless, the two fractions differentiated into GzmB<sup>+</sup> CD4<sup>+</sup> T cells with comparable efficiency upon Ad5.pIX-gp70 immunization (<xref rid="mmc1" ref-type="supplementary-material">Figures S3</xref>A and S3B). Moreover, Ad5.pIX-gp70 induced significantly stronger <italic>Gzmb</italic> expression in monoclonal TCR-transgenic EVα2 CD4<sup>+</sup> T cells than FV infection did (<xref rid="mmc1" ref-type="supplementary-material">Figure S3</xref>C). These results indicated a small effect of TCR usage on CD4<sup>+</sup> CTL differentiation, which was, however, overshadowed by other properties of the two viruses.</p><p>Lastly, different immunization regimens elicited distinct frequencies of GzmB<sup>+</sup> cells within env-reactive effector CD4<sup>+</sup> T cells (<xref rid="fig1" ref-type="fig">Figure 1</xref>E). These included non-persisting infection with attenuated N-tropic F-MLV (F-MLV-N) (<xref rid="bib11" ref-type="bibr">Dittmer et al., 1998</xref>) or transient env<sub>124–138</sub> peptide immunization, which failed to induce GzmB<sup>+</sup> cells, and transplantation of the FV-induced FBL-3 tumor cell line (<xref rid="bib20" ref-type="bibr">Klarnet et al., 1989</xref>), which induced moderate levels of GzmB<sup>+</sup> cells (<xref rid="fig1" ref-type="fig">Figure 1</xref>E). They also included infection with a replication-competent and persisting mouse-cytomegalovirus (mCMV)-based vector encoding F-MLV <italic>env</italic>, which also induced readily detectable GzmB<sup>+</sup> cells (<xref rid="fig1" ref-type="fig">Figure 1</xref>E). Thus, properties of the infecting or immunizing virus, independently of its ability to persist in the host, largely determine the efficiency of antigen-specific CD4<sup>+</sup> T cell differentiation into CTLs, with Ad5.pIX-gp70 outperforming FV.</p></sec><sec id="sec2.2"><title>Antagonistic CD4<sup>+</sup> CTL and Tfh Development</title><p>Both TCR usage and the nature of infecting virus can heavily influence Th subset differentiation, which is reflected in the corresponding transcriptional profiles. The necessary cell processing for intracellular GzmB staining precluded further transcriptional analysis between GzmB<sup>+</sup> and GzmB<sup>−</sup> CD4<sup>+</sup> T cells. To overcome this limitation, we performed single-cell RNA sequencing of env-specific CD4<sup>+</sup> T cells primed either by FV or Ad5.pIX-gp70. Consistent with flow-cytometric detection of GzmB production, FV induced <italic>Gzmb</italic> expression in 3/57 and 1/65 cells (an average of 3.2%), whereas Ad5.pIX-gp70 induced <italic>Gzmb</italic> expression in 6/42 and 4/45 cells (an average of 11.5%) analyzed in two independent runs (p = 0.022, Fisher’s exact test) (<xref rid="fig2" ref-type="fig">Figure 2</xref>A). In contrast, expression of other cytotoxic mediators, such as <italic>Tnfa</italic>, <italic>Fasl</italic>, and <italic>Tnfsf10</italic>, was comparable between FV and Ad5.pIX-gp70 priming (<xref rid="mmc1" ref-type="supplementary-material">Figure S4</xref>). It should be noted that gene expression assessment by single-cell RNA sequencing represents the lower limit, as it captures only a fraction of the genes expressed in a given cell. This is evident in the transcription of the <italic>Cd4</italic> gene, which is not detected in all of the CD4<sup>+</sup> T cells analyzed (<xref rid="fig2" ref-type="fig">Figure 2</xref>A).</p><p>Single-cell transcriptional analysis revealed another notable difference between FV-primed and Ad5.pIX-gp70-primed CD4<sup>+</sup> T cells: a significantly higher proportion of the former transcribed <italic>Bcl6</italic> (p = 0.025, Fisher’s exact test) (<xref rid="fig2" ref-type="fig">Figure 2</xref>B), which is essential for Tfh development (<xref rid="bib9" ref-type="bibr">Crotty, 2014</xref>, <xref rid="bib41" ref-type="bibr">Vinuesa et al., 2016</xref>). In contrast, the two types of CD4<sup>+</sup> T cells displayed comparable transcription of <italic>Tcf7</italic> (<xref rid="fig2" ref-type="fig">Figure 2</xref>B), encoding the transcription factor TCF-1, which has been recently found to promote Tfh development at multiple levels, including through induction of <italic>Bcl6</italic> transcription (<xref rid="bib7" ref-type="bibr">Choi et al., 2015</xref>, <xref rid="bib42" ref-type="bibr">Wu et al., 2015</xref>, <xref rid="bib43" ref-type="bibr">Xu et al., 2015</xref>). Independently assessed in CD4<sup>+</sup> T cell populations, levels of <italic>Tcf7</italic> were not significantly lower in Ad5.pIX-gp70-primed than in FV-primed CD4<sup>+</sup> T cells, whereas levels of <italic>Bcl6</italic> were (<xref rid="fig2" ref-type="fig">Figure 2</xref>C). Together, these results suggested that the degree of CTL and Tfh differentiation in env-specific CD4<sup>+</sup> T cells are inversely correlated and dictated by the priming virus.</p><p>To examine whether CTL differentiation was inhibited by competing Th programs, we compared the gene transcripts that distinguished <italic>Gzmb</italic><sup>+</sup> cells (<xref rid="mmc2" ref-type="supplementary-material">Table S1</xref>). Interestingly, <italic>Gzmb</italic><sup>+</sup> cells primed by either virus were characterized by specific loss of <italic>Tcf7</italic> expression, among a selected set of genes (<xref rid="fig2" ref-type="fig">Figure 2</xref>D). Conversely, <italic>Gzmb</italic><sup>+</sup> cells were characterized by elevated expression of several other genes, including <italic>Cxcr6</italic>, <italic>Entpd1</italic> (encoding CD39), <italic>Slamf1</italic> (encoding CD150), and <italic>Cd226</italic> (<xref rid="fig2" ref-type="fig">Figure 2</xref>D), which were further validated by flow cytometry (<xref rid="fig2" ref-type="fig">Figure 2</xref>E). These transcriptional differences were also significant when <italic>Gzmb</italic><sup>−</sup> and <italic>Gzmb</italic><sup>+</sup> cells primed by Ad5.pIX-gp70 only were analyzed (<xref rid="mmc1" ref-type="supplementary-material">Figure S5</xref>). Accordingly, none of the Ad5.pIX-gp70-primed <italic>Gzmb</italic><sup>+</sup> cells expressed <italic>Bcl6</italic>, and half of them expressed the antagonistic transcription factor Blimp-1, encoded by <italic>Prdm1</italic>, in sharp contrast to <italic>Gzmb</italic><sup>−</sup> cells (<xref rid="fig2" ref-type="fig">Figure 2</xref>F). Also in contrast to <italic>Gzmb</italic><sup>−</sup> cells, which were nearly all <italic>Tcf7</italic><sup>+</sup> and most also expressed <italic>Lef1</italic>, encoding the TCF-1 homolog LEF-1, <italic>Gzmb</italic><sup>+</sup> cells only sporadically expressed <italic>Tcf7</italic> and <italic>Lef1</italic> (<xref rid="fig2" ref-type="fig">Figure 2</xref>F; <xref rid="mmc1" ref-type="supplementary-material">Figure S6</xref>A).</p><p>The balance of <italic>Zbtb7b</italic> (encoding ThPOK) and <italic>Runx3</italic> transcription, associated with the CD4<sup>+</sup> and CD8<sup>+</sup> lineages, respectively (<xref rid="bib6" ref-type="bibr">Cheroutre and Husain, 2013</xref>), was also altered in Ad5.pIX-gp70-primed <italic>Gzmb</italic><sup>+</sup> T cells (<xref rid="fig2" ref-type="fig">Figure 2</xref>F; <xref rid="mmc1" ref-type="supplementary-material">Figure S6</xref>A). This observation is consistent with previous reports on intestinal CD4<sup>+</sup> CTLs, in which Runx3 expression is associated with CD8α expression (<xref rid="bib27" ref-type="bibr">Mucida et al., 2013</xref>, <xref rid="bib33" ref-type="bibr">Reis et al., 2013</xref>). In contrast to intestinal CD4<sup>+</sup> CTLs, however, the altered balance of <italic>Zbtb7b</italic> and <italic>Runx3</italic> transcription in splenic <italic>Gzmb</italic><sup>+</sup> CD4<sup>+</sup> T cells induced by Ad5.pIX-gp70 did not lead to transcription of either the <italic>Cd8a</italic> or <italic>Cd8b1</italic> genes or the acquisition of <italic>Crtam</italic> or <italic>Eomes</italic> expression (<xref rid="mmc1" ref-type="supplementary-material">Figure S6</xref>B). Lastly, transcription of <italic>Cd5</italic> and <italic>Nr4a1</italic> (encoding Nur77), which could be indicative of the strength of TCR signaling experienced by env-specific CD4<sup>+</sup> T cells, did not significantly differ between <italic>Gzmb</italic><sup>−</sup> and <italic>Gzmb</italic><sup>+</sup> cells (<xref rid="mmc1" ref-type="supplementary-material">Figure S6</xref>C), suggesting that TCR signal strength is not the primary determinant of CD4<sup>+</sup> CTL differentiation. Collectively, these findings point to a CD4<sup>+</sup> CTL-specific transcriptional signature, characterized by acquisition of <italic>Runx3</italic> transcription and, importantly, downregulation of Tfh-related transcription, particularly of <italic>Tcf7</italic>.</p></sec><sec id="sec2.3"><title>Bcl6 Suppresses CD4<sup>+</sup> CTL Development</title><p>Loss of Tfh-specific transcription in <italic>Gzmb</italic><sup>+</sup> CD4<sup>+</sup> T cells suggested that the TCF-1–Bcl6 nexus was incompatible with, or actively inhibiting, CD4<sup>+</sup> CTL differentiation. To test this possibility, we used conditional ablation of <italic>Bcl6</italic> in env-specific effector CD4<sup>+</sup> T cells, which were transferred into WT hosts (<xref rid="mmc1" ref-type="supplementary-material">Figure S7</xref>). This was achieved by expression of Cre in donor CD4<sup>+</sup> T cells under the control of the <italic>Tnfrsf4</italic> promoter (<italic>Tnfrsf4</italic><sup>Cre</sup>) (<xref rid="bib21" ref-type="bibr">Klinger et al., 2009</xref>). This promoter activates in the majority of CD4<sup>+</sup> T cells, only following antigen recognition, thus avoiding any effects of gene deletion during T cell development and prior to activation (<xref rid="bib23" ref-type="bibr">Marques et al., 2009</xref>). Effector CD4<sup>+</sup> T cells that activated the <italic>Tnfrsf4</italic> promoter were identified using a Cre-conditional yellow fluorescent protein (YFP) reporter (<italic>Gt(ROSA)26Sor</italic><sup>YFP</sup>) allele. A Cre-conditional <italic>Bcl6</italic> (<italic>Bcl6</italic><sup>fl</sup>) allele (<xref rid="bib17" ref-type="bibr">Kaji et al., 2012</xref>) was also introduced in separate donor EF4.1 mice. These combinations created four separate populations of env-specific donor CD4<sup>+</sup> T cells (<xref rid="fig3" ref-type="fig">Figure 3</xref>A; <xref rid="mmc1" ref-type="supplementary-material">Figure S7</xref>). Particularly, YFP<sup>−</sup> CD4<sup>+</sup> T cells with (<italic>Bcl6</italic><sup>fl</sup>) or without the conditional <italic>Bcl6</italic> allele (<italic>Bcl6</italic><sup>wt</sup>) would be comparable, as they retained the capacity to express Bcl6, whereas YFP<sup>+</sup><italic>Bcl6</italic><sup>fl</sup>, but not <italic>Bcl6</italic><sup>wt</sup>, CD4<sup>+</sup> T cells would lose this capacity (<xref rid="bib24" ref-type="bibr">Merkenschlager et al., 2016</xref>).</p><p>Transcriptional analysis of env-specific donor CD4<sup>+</sup> T cell populations primed by either virus confirmed significantly higher <italic>Bcl6</italic> expression in FV-primed than in Ad5.pIX-gp70-primed T cells, regardless of <italic>Tnfrsf4</italic> promoter activity (<xref rid="fig3" ref-type="fig">Figure 3</xref>B). Moreover, <italic>Bcl6</italic> expression was lost in the YFP<sup>+</sup>, but not the YFP<sup>−</sup>, fraction of <italic>Bcl6</italic><sup>fl</sup> T cells (<xref rid="fig3" ref-type="fig">Figure 3</xref>B), validating the approach. Loss of <italic>Bcl6</italic> expression in the YFP<sup>+</sup> fraction was accompanied by significant gain in expression of <italic>Prdm1</italic>, as well as of <italic>Gata3</italic>, <italic>Tbx21</italic>, and <italic>Ifng</italic> (<xref rid="fig3" ref-type="fig">Figure 3</xref>B), but not of <italic>Foxp3</italic> or <italic>Rorc</italic> (<xref rid="mmc1" ref-type="supplementary-material">Figure S8</xref>), suggesting that Bcl6 was suppressing the Th1 and Th2 programs. This effect of <italic>Bcl6</italic> deletion on CD4<sup>+</sup> T cell differentiation was further confirmed by intracellular staining for T-bet (encoded by <italic>Tbx21</italic>) and interferon (IFN)-γ (<xref rid="mmc1" ref-type="supplementary-material">Figure S9</xref>). Higher levels of T-bet and IFN-γ were induced by FV infection than Ad5.pIX-gp70 immunization in <italic>Bcl6</italic><sup>wt</sup> CD4<sup>+</sup> T cells, and these were not further elevated in <italic>Bcl6</italic><sup>fl</sup> CD4<sup>+</sup> T cells (<xref rid="mmc1" ref-type="supplementary-material">Figure S9</xref>). In contrast, the low levels of T-bet and IFN-γ induced by Ad5.pIX-gp70 immunization in <italic>Bcl6</italic><sup>wt</sup> CD4<sup>+</sup> T cells were significantly elevated in <italic>Bcl6</italic><sup>fl</sup> CD4<sup>+</sup> T cells (<xref rid="mmc1" ref-type="supplementary-material">Figure S9</xref>).</p><p>In addition to enhancing differentiation of other Th cell subsets, loss of <italic>Bcl6</italic> expression led to a striking upregulation of <italic>Gzmb</italic> transcription, specifically in Ad5.pIX-gp70-primed CD4<sup>+</sup> T cells (<xref rid="fig3" ref-type="fig">Figure 3</xref>B). Importantly, the gain in <italic>Gzmb</italic> expression in the latter population (>13-fold) was considerably more pronounced than the gain in the transcription of the other genes examined (2.2- to 3.8-fold) (<xref rid="fig3" ref-type="fig">Figure 3</xref>B). The significantly heightened transcription of <italic>Gzmb</italic>, specifically in Ad5.pIX-gp70 immunization, was additionally confirmed by intracellular staining for GzmB in the total env-specific <italic>Bcl6</italic><sup>fl</sup> CD4<sup>+</sup> T cell population, containing both Bcl6-deleted and non-deleted cells (<xref rid="fig4" ref-type="fig">Figure 4</xref>A).</p><p>To gain better insight into the transcriptional profile of only the Bcl6-deleted env-specific CD4<sup>+</sup> T cells, without the need for in vitro restimulation, we next subjected the purified YFP<sup>+</sup> fraction of <italic>Bcl6</italic><sup>fl</sup> CD4<sup>+</sup> T cells to single-cell RNA sequencing. This analysis demonstrated a significant increase in the frequency of <italic>Gzmb</italic><sup>+</sup> cells in Bcl6-deleted env-specific CD4<sup>+</sup> T cells following priming by Ad5.pIX-gp70 (<xref rid="fig4" ref-type="fig">Figure 4</xref>B). Indeed, nearly 40% of these cells were positive for <italic>Gzmb</italic> transcripts (<xref rid="fig4" ref-type="fig">Figure 4</xref>B). In contrast, the frequency of <italic>Gzmb</italic><sup>+</sup> cells in Bcl6-deleted env-specific CD4<sup>+</sup> T cells primed by FV did not change significantly (<xref rid="fig2" ref-type="fig">Figures 2</xref>A and <xref rid="fig4" ref-type="fig">4</xref>B). These data with Bcl6-deleted populations and purified single cells suggested that Bcl6 was restraining the CTL program in env-specific CD4<sup>+</sup> T cells, at least during Ad5.pIX-gp70 priming.</p><p>To relate the effect of Bcl6 deletion on <italic>Gzmb</italic> expression, we examined the transcription of additional effector molecules in single WT or Bcl6-deleted env-specific CD4<sup>+</sup> T cells. A high proportion (68%) of WT CD4<sup>+</sup> T cells primed by FV expressed <italic>Il21</italic> (which, in these settings, was characteristic of the Tfh response), and a smaller proportion (14%) expressed <italic>Il10</italic>, with only partial overlap with <italic>Ifng</italic> expression (<xref rid="fig4" ref-type="fig">Figure 4</xref>C). Although loss of <italic>Bcl6</italic> during FV infection did not markedly reduce <italic>Il21</italic> expression, it did significantly enhance expression of both <italic>Ifng</italic> (2.3-fold) and <italic>Il10</italic> (4.7-fold), which were now co-expressed in the majority of <italic>Bcl6</italic>-deleted CD4<sup>+</sup> T cells (<xref rid="fig4" ref-type="fig">Figure 4</xref>C). In stark contrast to FV infection, Ad5.pIX-gp70 immunization induced very little <italic>Il21</italic> or <italic>Il10</italic> expression in either WT or <italic>Bcl6</italic>-deleted env-specific CD4<sup>+</sup> T cells (<xref rid="fig4" ref-type="fig">Figure 4</xref>C). Moreover, <italic>Bcl6</italic> deletion induced a more modest gain in <italic>Ifng</italic>-expressing cells (1.5-fold) after Ad5.pIX-gp70 priming than after FV priming (<xref rid="fig4" ref-type="fig">Figure 4</xref>C). Collectively, these data argued that Bcl6 suppressed specifically CTL differentiation of env-specific CD4<sup>+</sup> T cells during Ad5.pIX-gp70 priming.</p></sec><sec id="sec2.4"><title>CD4<sup>+</sup> CTL and Th1 Cells Are Transcriptionally Distinct</title><p>GzmB expression is often considered a part of the Th1 program of CD4<sup>+</sup> T cell differentiation. Indeed, some of the genes, such as <italic>Slamf1</italic> (encoding CD150), whose expression characterized <italic>Gzmb</italic><sup>+</sup> cells (<xref rid="fig2" ref-type="fig">Figures 2</xref>D and 2E), are also used to distinguish Th1 from Tfh cells (<xref rid="bib9" ref-type="bibr">Crotty, 2014</xref>, <xref rid="bib41" ref-type="bibr">Vinuesa et al., 2016</xref>). Single-cell transcriptional analysis revealed that, independently of priming virus or Bcl6 sufficiency, over half (57%) of <italic>Gzmb</italic><sup>+</sup> cells co-expressed <italic>Ifng</italic> (<xref rid="fig5" ref-type="fig">Figure 5</xref>A). Similar results were obtained at the protein level (<xref rid="fig5" ref-type="fig">Figure 5</xref>B), indicating a close relationship between GzmB and IFN-γ production. However, the strong <italic>Ifng</italic> expression in FV infection without concomitant <italic>Gzmb</italic> expression, and the inverse during Ad5.pIX-gp70 immunization, particularly after loss of Bcl6 (<xref rid="fig3" ref-type="fig">Figures 3</xref>B, <xref rid="fig4" ref-type="fig">4</xref>A, and 4C), suggested that the profiles of Th1 cells and those that additionally display CTL potential may be separable, both qualitatively and quantitatively. For example, although both Th1 cells and Th1 cells with CTL potential expressed CD150 (encoded by <italic>Slamf1</italic>), a direct comparison of IFN-γ<sup>+</sup> env-specific CD4<sup>+</sup> T cells that co-produced GzmB (IFN-γ<sup>+</sup>GzmB<sup>+</sup>) with those that did not (IFN-γ<sup>+</sup>GzmB<sup>−</sup>) revealed significantly higher CD150 expression in the former (<xref rid="fig5" ref-type="fig">Figure 5</xref>C).</p><p>To comprehensively explore potential transcriptional differences between Th1 and CTL CD4<sup>+</sup> T cells, we compared the transcriptional profiles of env-specific CD4<sup>+</sup> T cells expressing <italic>Ifng</italic>, but not <italic>Gzmb</italic> (<italic>Ifng</italic><sup>+</sup><italic>Gzmb</italic><sup>−</sup>), and those co-expressing <italic>Ifng</italic> and <italic>Gzmb</italic> (<italic>Ifng</italic><sup>+</sup><italic>Gzmb</italic><sup>+</sup>). We reasoned that restricting this comparison only to cells expressing <italic>Ifng</italic> would minimize the effect of comparing mixed Th functional subsets and emphasize differences correlating with Gzmb production. Despite common <italic>Ifng</italic> expression, the transcriptional profile of <italic>Ifng</italic><sup>+</sup><italic>Gzmb</italic><sup>+</sup> cells was readily distinguishable from that of <italic>Ifng</italic><sup>+</sup><italic>Gzmb</italic><sup>−</sup> cells (<xref rid="fig5" ref-type="fig">Figure 5</xref>D). The transcriptional difference between <italic>Ifng</italic><sup>+</sup><italic>Gzmb</italic><sup>+</sup> and <italic>Ifng</italic><sup>+</sup><italic>Gzmb</italic><sup>−</sup> cells remained significant when Ad5.pIX-gp70 priming was analyzed separately, whereas it narrowly lost significance when FV priming was analyzed in isolation due to the low number of <italic>Gzmb</italic><sup>+</sup> cells induced by FV (<xref rid="mmc1" ref-type="supplementary-material">Figure S10</xref>). Moreover, <italic>Ifng</italic><sup>+</sup><italic>Gzmb</italic><sup>+</sup> and <italic>Ifng</italic><sup>+</sup><italic>Gzmb</italic><sup>−</sup> cells remained transcriptionally significantly distinct when Tfh cells were excluded from the analysis, either by virtue of <italic>Cxcr5</italic> expression or by limiting the analysis to Bcl6-deleted CD4<sup>+</sup> T cells, which cannot differentiate into Tfh cells (<xref rid="mmc1" ref-type="supplementary-material">Figure S11</xref>).</p><p>Importantly, the transcriptional differences between <italic>Ifng</italic><sup>+</sup><italic>Gzmb</italic><sup>+</sup> and <italic>Ifng</italic><sup>+</sup><italic>Gzmb</italic><sup>-</sup> cells (<xref rid="fig5" ref-type="fig">Figure 5</xref>D) largely overlapped with those characterizing the <italic>Gzmb</italic><sup>+</sup> subset as a whole (<xref rid="fig2" ref-type="fig">Figure 2</xref>D). This was particularly evident in the opposing expression of <italic>Prdm1</italic> and <italic>Tcf7</italic>. Indeed, whereas most <italic>Ifng</italic><sup>+</sup><italic>Gzmb</italic><sup>−</sup> cells expressed <italic>Tcf7</italic> but not <italic>Prdm1</italic>, most <italic>Ifng</italic><sup>+</sup><italic>Gzmb</italic><sup>+</sup> cells expressed <italic>Prdm1</italic> but not <italic>Tcf7</italic> (<xref rid="fig5" ref-type="fig">Figure 5</xref>E), suggesting that the combination of these two markers was sufficient to distinguish between the Th1 and CD4<sup>+</sup> CTLs.</p><p>To confirm the distinguishing pattern of <italic>Prdm1</italic> and <italic>Tcf7</italic> expression of <italic>Gzmb</italic><sup>+</sup> CD4<sup>+</sup> T cells, we used EF4.1 TCRβ-transgenic CD4<sup>+</sup> T cells additionally carrying a GFP reporter into the <italic>Prdm1</italic> locus (<xref rid="bib18" ref-type="bibr">Kallies et al., 2009</xref>). As GFP insertion disrupts the <italic>Prdm1</italic> gene in these mice (<xref rid="bib18" ref-type="bibr">Kallies et al., 2009</xref>), we used donors heterozygous for the <italic>Prdm1</italic><sup>Gfp</sup> allele to prevent loss of function of the encoded Blimp1 in the adoptively transferred CD4<sup>+</sup> T cells. Following Ad5.pIX-gp70 immunization, a small proportion (∼8%) of donor env-specific effector CD4<sup>+</sup> T cells displayed Blimp1-GFP expression (<xref rid="fig5" ref-type="fig">Figure 5</xref>F) and contained <italic>Prdm1</italic> transcripts (<xref rid="fig5" ref-type="fig">Figure 5</xref>G). Notably, this fraction was also characterized by paucity of <italic>Tcf7</italic> and overabundance of <italic>Gzmb</italic> transcripts, relative to the Blimp1-GFP<sup>−</sup> fraction (<xref rid="fig5" ref-type="fig">Figure 5</xref>G). Thus, loss of <italic>Tcf7</italic> expression and induction of <italic>Prdm1</italic> expression could differentiate <italic>Gzmb</italic><sup>+</sup> CD4<sup>+</sup> T cells from other Th subsets, including Th1 cells.</p></sec><sec id="sec2.5"><title>Layered Checkpoints in CD4<sup>+</sup> CTL Development</title><p>Our findings supported a role for Bcl6 in restraining CD4<sup>+</sup> CTL development, particularly of CD4<sup>+</sup> T cells responding to Ad5.pIX-gp70 immunization (<xref rid="fig3" ref-type="fig">Figures 3</xref> and <xref rid="fig4" ref-type="fig">4</xref>). However, in response to FV infection, <italic>Bcl6</italic> ablation in env-specific CD4<sup>+</sup> T cells failed to promote CD4<sup>+</sup> CTL differentiation, despite elevated <italic>Prdm1</italic> expression (<xref rid="fig3" ref-type="fig">Figure 3</xref>). Therefore, we hypothesized that additional layers of regulation were limiting CD4<sup>+</sup> CTL differentiation specifically in FV infection. To test this idea, we compared the expression of inhibitory receptors in env-specific CD4<sup>+</sup> T cells primed by either FV or Ad5.pIX-gp70. Following FV infection, the majority (>65%) of <italic>Gzmb</italic><sup>−</sup> cells co-expressed <italic>Pcdc1</italic> (encoding PD-1), <italic>Lag3</italic>, and <italic>Ctla4</italic> but completely lacked expression of <italic>Havcr2</italic> (encoding Tim-3) (<xref rid="fig6" ref-type="fig">Figure 6</xref>A). By comparison, following Ad5.pIX-gp70 immunization, <italic>Ctla4</italic> expression was comparable in <italic>Gzmb</italic><sup>−</sup> cells, but expression of <italic>Pcdc1</italic> was reduced to 36%, and expression of <italic>Lag3</italic> was now largely absent (8%) (<xref rid="fig6" ref-type="fig">Figure 6</xref>A). Notably, the difference in expression of inhibitory receptors between the two viruses was more pronounced in <italic>Gzmb</italic><sup>+</sup> cells, which, in the case of FV priming, co-expressed all four inhibitory receptors (<xref rid="fig6" ref-type="fig">Figure 6</xref>A). In contrast, <italic>Gzmb</italic><sup>+</sup> cells primed by Ad5.pIX-gp70 lacked expression of <italic>Pcdc1</italic> and <italic>Lag3</italic> (<xref rid="fig6" ref-type="fig">Figure 6</xref>A). This difference between the two viruses in the induction of <italic>Pcdc1</italic> and <italic>Lag3</italic> expression was also confirmed at the PD-1 and LAG3 protein level. Consistent with the RNA expression data, PD-1 was expressed by nearly all env-specific CD4<sup>+</sup> T cells, but at significantly higher levels in FV infection than in Ad5.pIX-gp70 immunization (<xref rid="fig6" ref-type="fig">Figures 6</xref>B and 6C). Similarly, a much larger fraction of env-specific CD4<sup>+</sup> T cells exhibited LAG3 surface expression when primed by FV than by Ad5.pIX-gp70 (<xref rid="fig6" ref-type="fig">Figure 6</xref>B), a difference that was also reflected in the intensity of LAG3 staining of the entire population (<xref rid="fig6" ref-type="fig">Figure 6</xref>C). Thus, <italic>Gzmb</italic><sup>+</sup> cells displayed considerable expression of <italic>Ctla4</italic> and exclusive expression of <italic>Havcr2</italic>, regardless of the priming virus. Indeed, <italic>Havcr2</italic> expression also distinguished <italic>Ifng</italic><sup>+</sup><italic>Gzmb</italic><sup>+</sup> cells from <italic>Ifng</italic><sup>+</sup><italic>Gzmb</italic><sup>−</sup> cells (<xref rid="fig5" ref-type="fig">Figure 5</xref>C). In contrast, significant expression of both <italic>Pcdc1</italic> and <italic>Lag3</italic> was induced in <italic>Gzmb</italic><sup>+</sup> env-specific CD4<sup>+</sup> T cells uniquely by FV infection but not Ad5.pIX-gp70 immunization.</p><p>The pattern of inhibitory receptors expressed by FV-primed env-specific CD4<sup>+</sup> T cells was suggestive of an exhausted phenotype (<xref rid="bib8" ref-type="bibr">Crawford et al., 2014</xref>), which was investigated further. PD-1 expression was more consistent with antigen-induced activation of effector CD4<sup>+</sup> T cells than with cellular exhaustion, as it was also induced by Ad5.pIX-gp70, albeit to a lower intensity per cell (<xref rid="fig6" ref-type="fig">Figure 6</xref>C), and was also substantially reduced quickly after the peak of the effector response to FV infection (<xref rid="mmc1" ref-type="supplementary-material">Figure S12</xref>). Moreover, effector CD4<sup>+</sup> T cells isolated from acute FV infection were transcriptionally distinct from typical exhausted CD4<sup>+</sup> T cells isolated from chronic lymphocytic choriomeningitis virus (LCMV) infection (<xref rid="bib8" ref-type="bibr">Crawford et al., 2014</xref>; <xref rid="mmc1" ref-type="supplementary-material">Figure S13</xref>A), suggesting that expression of inhibitory receptors by FV-primed effector CD4<sup>+</sup> T cells was part of acute effector differentiation rather than of the exhaustion that characterizes chronic viral infections.</p><p>Nevertheless, expression of inhibitory receptors, particularly of PD-1 and LAG3, by env-specific CD4<sup>+</sup> T cells during acute FV infection could still influence cellular activation or CTL differentiation. To this end, we treated WT recipients of env-specific CD4<sup>+</sup> T cells with PD-1- and LAG3-blocking antibodies during the course of FV infection. Although blockade of either PD-1 or LAG3 separately had only a modest effect, the combined PD-1 and LAG3 blockade significantly increased in the frequency of <italic>Gzmb</italic><sup>+</sup> cells in donor CD4<sup>+</sup> T cells (<xref rid="fig6" ref-type="fig">Figure 6</xref>D; <xref rid="mmc1" ref-type="supplementary-material">Figure S14</xref>), supporting their role in restraining CD4<sup>+</sup> CTL differentiation. In contrast, the PD-1 and LAG3 blockade did not appreciably alter clonal expansion of donor CD4<sup>+</sup> T cells or their production of IFN-γ and TNF-α (<xref rid="mmc1" ref-type="supplementary-material">Figure S15</xref>).</p><p>The high levels of PD-1 expression in env-specific CD4<sup>+</sup> T cells responding to FV infection were previously shown to require cognate interaction between T cells and B cells (<xref rid="bib30" ref-type="bibr">Ploquin et al., 2011</xref>). Therefore, we used B cell deficiency as an alternative to PD-1 blockade. Indeed, a significantly higher proportion of env-specific CD4<sup>+</sup> T cells expressed intracellular GzmB when transferred into B cell-deficient <italic>Ighm</italic><sup>−/−</sup> hosts than into WT hosts (<xref rid="fig6" ref-type="fig">Figure 6</xref>D). These findings supported the premise that PD-1 and LAG3 posed a further block in CD4<sup>+</sup> CTL differentiation, in addition to Bcl6 expression. To test this premise directly, we combined Bcl6 deficiency in env-specific CD4<sup>+</sup> T cells with PD-1 and LAG3 blockade. As before (<xref rid="fig4" ref-type="fig">Figure 4</xref>A), Bcl6 deficiency alone did not significantly enhance CD4<sup>+</sup> CTL differentiation during FV infection (<xref rid="fig6" ref-type="fig">Figure 6</xref>D). In contrast, the combination of Bcl6 deficiency and PD-1 and LAG3 blockade markedly increased the proportion of <italic>Gzmb</italic><sup>+</sup> cells (∼3-fold) (<xref rid="fig6" ref-type="fig">Figure 6</xref>D) to levels comparable with those induced by Bcl6 deficiency in Ad5.pIX-gp70 immunization (<xref rid="fig4" ref-type="fig">Figure 4</xref>B). Thus, PD-1 and LAG3 were preventing Bcl6-deficient env-specific CD4<sup>+</sup> T cells from acquiring GzmB expression during FV infection, representing an additional level of CD4<sup>+</sup> CTL differentiation control.</p></sec></sec><sec id="sec3"><title>Discussion</title><p>Since the earliest descriptions of MHC class-II-restricted cytotoxic activity in CD4<sup>+</sup> T cells nearly 4 decades ago, a number of studies have implicated CD4<sup>+</sup> CTLs in antiviral and antitumor immunity, as well as in autoimmune and inflammatory conditions (<xref rid="bib4" ref-type="bibr">Brown, 2010</xref>, <xref rid="bib5" ref-type="bibr">Brown et al., 2016</xref>, <xref rid="bib6" ref-type="bibr">Cheroutre and Husain, 2013</xref>, <xref rid="bib34" ref-type="bibr">Soghoian and Streeck, 2010</xref>, <xref rid="bib40" ref-type="bibr">van de Berg et al., 2008</xref>). Nevertheless, the priming requirements for CD4<sup>+</sup> CTLs or their phenotypic overlap with other CD4<sup>+</sup> Th subsets have only recently begun to emerge. Here, we described the transcriptional profile of CD4<sup>+</sup> CTLs as the antipode of the Tfh profile. We provided evidence to suggest multilayered control of CD4<sup>+</sup> CTL differentiation: first, by the TCF-1–Bcl6 nexus driving Tfh polarization, and second, inhibition by PD-1 and LAG3.</p><p>Study of CD4<sup>+</sup> CTLs has been hampered by the lack of distinctive markers that are compatible with further characterization of these cells. Although MHC class-II-restricted cytotoxic activity has been amply documented, it has not been consistently attributed to granzyme-mediated killing, as opposed to killing mediated by secreted or membrane-bound cytokines, including IFN-γ, expressed by Th1 cells, or members of the TNF family, expressed by multiple CD4<sup>+</sup> Th cell subsets (<xref rid="bib5" ref-type="bibr">Brown et al., 2016</xref>, <xref rid="bib6" ref-type="bibr">Cheroutre and Husain, 2013</xref>). Even when production of GzmB was used for the identification of CD4<sup>+</sup> CTLs, a certain degree of phenotypic overlap with Th1 cells was noted (<xref rid="bib16" ref-type="bibr">Hua et al., 2013</xref>), and indeed, GzmB-producing CD4<sup>+</sup> T cells are still regarded in the literature as a variant of the Th1 subset. This view is further supported by a potential developmental connection between CD4<sup>+</sup> CTLs and Th1 cells (<xref rid="bib6" ref-type="bibr">Cheroutre and Husain, 2013</xref>). Indeed, GzmB-producing cells often also express typical Th1 products, including IFN-γ. CD4<sup>+</sup> CTL differentiation, particularly in response to interleukin-2 (IL-2) and IFN-α stimulation, has also been suggested to rely on the Th1-related transcription factor T-bet (encoded by the <italic>Tbx21</italic> gene), which can bind directly to the <italic>Gzmb</italic> promoter (<xref rid="bib16" ref-type="bibr">Hua et al., 2013</xref>). However, CD4<sup>+</sup> CTL differentiation has been reported in other studies to depend not on T-bet but on its homolog Eomesodermin (encoded by the <italic>Eomes</italic> gene) (<xref rid="bib15" ref-type="bibr">Hirschhorn-Cymerman et al., 2012</xref>, <xref rid="bib32" ref-type="bibr">Qui et al., 2011</xref>). The differential dependency on either T-bet or Eomesodermin may indicate that, depending on the priming conditions, CD4<sup>+</sup> CTLs can develop through separate developmental pathways, which may or may not overlap with the Th1 differentiation pathway. Using a single-cell RNA-sequencing approach, we were able to contrast the entire transcriptome of <italic>Gzmb</italic><sup>+</sup> CD4<sup>+</sup> T cells with that of other CD4<sup>+</sup> T cells, without prior assumptions of their transcriptional overlap. This approach did not support a correlation between expression of <italic>Gzmb</italic> and expression of either <italic>Tbx21</italic> or <italic>Eomes</italic>, suggesting a certain degree of redundancy. It did, however, reveal a clear distinction between CD4<sup>+</sup> T cells with CTL potential and Th1 cells, exemplified in the reciprocal expression of <italic>Tcf7</italic> and <italic>Prdm1</italic>. Although CD4<sup>+</sup> CTLs are more similar to the Th1 than other Th subsets, loss of <italic>Tcf7</italic> expression and concomitant gain of <italic>Prdm1</italic> expression in CD4<sup>+</sup> CTLs set them apart from all other Th subsets, including Th1 cells. Equally unique was the expression of <italic>Havcr2</italic> (encoding Tim-3), which was found exclusively in CD4<sup>+</sup> CTLs.</p><p>Although our results uncover prominent transcriptional differences between CD4<sup>+</sup> CTLs and Th1 cells, they do not currently inform on any lineage or precursor-product relationship between the two. CD4<sup>+</sup> CTLs may represent an advanced or divergent state of Th1 differentiation, characterized by loss of <italic>Tcf7</italic> and acquisition of <italic>Runx3</italic> and <italic>Prdm1</italic> expression. The markers reported here that distinguish CD4<sup>+</sup> T cells with CTL potential from Th1 cells will be valuable in determining the transcriptional and phenotypic stability of CD4<sup>+</sup> CTLs, or interconversion to a Th1 phenotype, in longitudinal cell-fate studies.</p><p>Another defining characteristic of CD4<sup>+</sup> CTLs that distinguishes them from other Th subsets is the acquisition of CD8<sup>+</sup> lineage-related features. Our analysis confirmed the relative loss of <italic>Zbtb7b</italic> expression (encoding ThPOK) and acquisition of <italic>Runx3</italic> expression, considered responsible for the reprogramming of CD4<sup>+</sup> CTLs (<xref rid="bib27" ref-type="bibr">Mucida et al., 2013</xref>, <xref rid="bib33" ref-type="bibr">Reis et al., 2013</xref>). However, despite acquiring <italic>Runx3</italic> expression under the conditions we have examined, CD4<sup>+</sup> CTLs did not express other CD8<sup>+</sup> lineage-related genes, such as <italic>Cd8a</italic> or <italic>Crtam</italic>, previously observed in CD4<sup>+</sup> CTLs in other conditions (<xref rid="bib27" ref-type="bibr">Mucida et al., 2013</xref>, <xref rid="bib33" ref-type="bibr">Reis et al., 2013</xref>, <xref rid="bib37" ref-type="bibr">Takeuchi et al., 2016</xref>). It is possible that acquisition of CD8<sup>+</sup> lineage-related characteristics is not as extensive in splenic CD4<sup>+</sup> CTLs as in intestine intraepithelial CD4<sup>+</sup> CTLs. It is also possible that further reprogramming of CD4<sup>+</sup> CTLs requires longer antigenic stimulation than the 7 days we have examined here.</p><p>Our ability to contrast the global transcriptional profile of <italic>Gzmb</italic><sup>+</sup> CD4<sup>+</sup> T cells against that of other CD4<sup>+</sup> T cells revealed a principal feature of CD4<sup>+</sup> CTL differentiation; namely, antagonism by the Tfh program. Indeed, loss of <italic>Tcf7</italic> and <italic>Lef1</italic> expression distinguished CD4<sup>+</sup> CTL from other CD4<sup>+</sup> T cell subsets, including <italic>Ifng</italic><sup>+</sup> Th1 cells. TCF-1 (encoded by <italic>Tcf7</italic>) and its homolog, LEF-1, have been recently demonstrated to coordinate Tfh differentiation partly by enhancing <italic>Bcl6</italic> expression during LCMV infection (<xref rid="bib7" ref-type="bibr">Choi et al., 2015</xref>, <xref rid="bib42" ref-type="bibr">Wu et al., 2015</xref>, <xref rid="bib43" ref-type="bibr">Xu et al., 2015</xref>). Interestingly, <italic>Tcf7</italic> and <italic>Lef1</italic> gene deletion in CD4<sup>+</sup> T cells in two of these studies promoted transcriptional features of Th1 cells, as well as of CD4<sup>+</sup> CTLs, including <italic>Gzmb</italic> expression (<xref rid="bib7" ref-type="bibr">Choi et al., 2015</xref>, <xref rid="bib43" ref-type="bibr">Xu et al., 2015</xref>). The acquisition of CTL-related characteristics by <italic>Tcf7</italic>-deficient CD4<sup>+</sup> T cells in these population studies was interpreted as part of enhanced Th1 responses (<xref rid="bib7" ref-type="bibr">Choi et al., 2015</xref>, <xref rid="bib43" ref-type="bibr">Xu et al., 2015</xref>). Our single-cell analysis clearly demonstrated that the blocking of Tfh differentiation can promote Th1 and CTL differentiation of distinct cells. Not only were Th1 cells and CD4<sup>+</sup> CTLs transcriptionally separable, but they were also induced to different degrees in response to different infections. This was exemplified by retroviral infection, where <italic>Bcl6</italic> deficiency promoted Th1, but not CTL, differentiation of CD4<sup>+</sup> T cells and adenoviral vaccination, where <italic>Bcl6</italic> deficiency unleashed specifically CTL differentiation.</p><p>Antagonism between the Tfh and CD4<sup>+</sup> CTL programs is also supported by the contrasting expression of Blimp1 (encoded by <italic>Prdm1</italic>). Blimp1 suppresses both <italic>Bcl6</italic> and <italic>Tcf7</italic> expression and is, in turn, negatively regulated by Bcl6 and TCF-1 (<xref rid="bib7" ref-type="bibr">Choi et al., 2015</xref>, <xref rid="bib42" ref-type="bibr">Wu et al., 2015</xref>, <xref rid="bib43" ref-type="bibr">Xu et al., 2015</xref>). Previous studies demonstrated defective GzmB expression by Blimp1-deficient CD4<sup>+</sup> T cells, whereas Blimp1 overexpression enhances CD4<sup>+</sup> CTL differentiation (<xref rid="bib12" ref-type="bibr">Gong and Malek, 2007</xref>, <xref rid="bib16" ref-type="bibr">Hua et al., 2013</xref>). These studies suggest that CD4<sup>+</sup> CTL differentiation requires Blimp1, which is thought to enhance T-bet binding to the promoters of CTL-related genes, including <italic>Gzmb</italic> (<xref rid="bib12" ref-type="bibr">Gong and Malek, 2007</xref>, <xref rid="bib16" ref-type="bibr">Hua et al., 2013</xref>). Although Blimp1 expression, in combination with loss of TCF-1, was shown here to be a unique characteristic of CD4<sup>+</sup> CTLs, Blimp1 can also be expressed highly by other Th subsets, and its expression alone is not sufficient to drive CD4<sup>+</sup> CTL development in our experimental systems. For example, <italic>Bcl6</italic> deficiency induced significantly elevated expression of both <italic>Prdm1</italic> and <italic>Tbx21</italic> in CD4<sup>+</sup> T cells responding to FV infection, but it did not enhance CTL differentiation, arguing that Blimp1 may be necessary, but not sufficient, to drive the CTL program in CD4<sup>+</sup> T cells.</p><p>Retroviral infection was particularly ineffective at inducing CTL differentiation in responding CD4<sup>+</sup> T cells. This apparent defect was also observed when Tfh differentiation was precluded by deletion of <italic>Bcl6</italic>, suggesting additional blocks in CD4<sup>+</sup> CTL differentiation. Our present findings add the dimension of intrinsic regulation of CD4<sup>+</sup> CTL differentiation by the inhibitory receptors PD-1 and LAG3, induced in response, specifically, to retroviral infection. These receptors represented an additional layer of regulation, which prevented <italic>Bcl6</italic>-deficient CD4<sup>+</sup> T cells acquiring CTL characteristics. Indeed, CD4<sup>+</sup> CTL differentiation in FV infection required the combination of <italic>Bcl6</italic> deletion and PD-1 and LAG3 blockade. In addition to PD-1 and LAG3, CD4<sup>+</sup> CTLs were also characterized by expression of the inhibitory receptor Tim-3 (encoded by <italic>Havcr2</italic>), which was, in fact, entirely restricted to <italic>Gzmb</italic><sup>+</sup> cells. The precise cause of the highly elevated expression of inhibitory receptors in virus-specific effector CD4<sup>+</sup> T cells, particularly, in CD4<sup>+</sup> CTLs, specifically in response to retroviral infection, remains poorly understood. Notably, GzmB production has been previously detected in CD4<sup>+</sup> T cells responding to FV or FV-induced FBL-3 cells, when Treg cells and CD8<sup>+</sup> T cells were depleted (<xref rid="bib1" ref-type="bibr">Akhmetzyanova et al., 2013</xref>, <xref rid="bib22" ref-type="bibr">Manzke et al., 2013</xref>), suggesting extrinsic regulation. These data imply that both extrinsic regulation, in the form of Treg cells, and intrinsic regulation, in the form of inhibitory receptors in effector T cells, are effectively exploited by retroviruses.</p><p>Although incompletely understood, the induction of inhibitory receptors in virus-specific CD4<sup>+</sup> T cells during FV infection requires cognate interaction with B cells (<xref rid="bib30" ref-type="bibr">Ploquin et al., 2011</xref>). Expression of PD-1 in virus-specific CD4<sup>+</sup> T cells, for example, is significantly reduced in the absence of B cell antigen presentation during FV infection (<xref rid="bib30" ref-type="bibr">Ploquin et al., 2011</xref>) or endogenous antigen expression (<xref rid="bib13" ref-type="bibr">Han et al., 2010</xref>). In keeping with these observations, B cell deficiency also enhanced CD4<sup>+</sup> CTL differentiation in this study. These findings are entirely consistent with a critical role for B cells in deciding the balance between Tfh and CTL differentiation of interacting CD4<sup>+</sup> T cells. B cells play a well-described role in stabilizing the Tfh program (<xref rid="bib9" ref-type="bibr">Crotty, 2014</xref>, <xref rid="bib41" ref-type="bibr">Vinuesa et al., 2016</xref>), and, together with inducing PD-1 expression in CD4<sup>+</sup> T cells, they inhibit CD4<sup>+</sup> CTL differentiation. Such a role for B cells would ensure efficient antibody production at the expense of CD4<sup>+</sup> T cell-mediated immunity. Indeed, B cells inhibit antibody-independent CD4<sup>+</sup> T cell-mediated protection against tumors (<xref rid="bib31" ref-type="bibr">Qin et al., 1998</xref>) or FV-induced erythroblastosis (<xref rid="bib29" ref-type="bibr">Pike et al., 2009</xref>).</p><p>A critical influence of the type of antigen-presenting cell on Tfh and CTL differentiation may also underlie the difference in the efficiency with which distinct viruses or viral vaccines elicit either Tfh or CD4<sup>+</sup> CTLs. Elucidation of the role of distinct antigen-presenting cell types in this process may hold the key to both understanding and controlling the balance between Tfh and CD4<sup>+</sup> CTLs.</p><p>Overall, using single-cell analysis, our study revealed the transcriptional signature of <italic>Gzmb</italic>-expressing CD4<sup>+</sup> T cells. Their unique transcriptional features not only support the notion of a distinguishable CD4<sup>+</sup> CTL subset but also provide markers for future identification and further longitudinal study of CD4<sup>+</sup> CTLs. Additionally, regulation of the CD4<sup>+</sup> CTL program by the TCF-1-Bcl6 axis, B cells, and inhibitory receptors, offers a means for manipulating the cytotoxic activity of CD4<sup>+</sup> T cells in health and disease.</p></sec><sec id="sec4"><title>Experimental Procedures</title><sec id="sec4.1"><title>Mice</title><p>Inbred B6 and CD45.1<sup>+</sup> congenic B6 (B6.SJL-<italic>Ptprc</italic><sup><italic>a</italic></sup><italic>Pep3</italic><sup><italic>b</italic></sup>/BoyJ) mice, TCRβ-transgenic EF4.1 mice (<xref rid="bib2" ref-type="bibr">Antunes et al., 2008</xref>), TCRαβ doubly transgenic EVα2 mice (<xref rid="bib24" ref-type="bibr">Merkenschlager et al., 2016</xref>), Rag1-deficient (<italic>Rag1</italic><sup>−/−</sup>) mice (<xref rid="bib25" ref-type="bibr">Mombaerts et al., 1992</xref>), B cell-receptor-deficient (<italic>Ighm</italic><sup>−/−</sup>) mice (<xref rid="bib19" ref-type="bibr">Kitamura et al., 1991</xref>), mice with an activatable YFP gene targeted into the <italic>Gt(ROSA)26Sor</italic> (<italic>R26</italic>) locus (<xref rid="bib35" ref-type="bibr">Srinivas et al., 2001</xref>), mice with a targeted insertion of Cre recombinase into the <italic>Tnfrsf4</italic> locus (<xref rid="bib21" ref-type="bibr">Klinger et al., 2009</xref>) (<italic>Tnfrsf4</italic><sup><italic>Cre</italic></sup>), mice with a conditional <italic>Bcl6</italic> allele (<xref rid="bib17" ref-type="bibr">Kaji et al., 2012</xref>) (<italic>Bcl6</italic><sup>fl</sup>), endogenous ecotropic murine-leukemia-virus-deficient (<italic>Emv2</italic><sup>−/−</sup>) mice (<xref rid="bib44" ref-type="bibr">Young et al., 2012</xref>), mice with a targeted insertion of GFP into the <italic>Prdm1</italic> locus (<xref rid="bib18" ref-type="bibr">Kallies et al., 2009</xref>) (Blimp1-GPF), and mice with a targeted insertion of tdTomato fluorescent protein into the <italic>Gzmb</italic> locus (<xref rid="bib26" ref-type="bibr">Mouchacca et al., 2013</xref>) (GzmB-tdTomato) were all on the B6 genetic background and were maintained at the Francis Crick Institute’s animal facilities. All animal experiments were approved by the ethical committee of the Francis Crick Institute and were conducted according to local guidelines and UK Home Office regulations under the Animals (Scientific Procedures Act) 1986 (ASPA).</p></sec><sec id="sec4.2"><title>Retroviral Infection and Immunization</title><p>Details of infections, immunizations, and other in vivo treatments can be found in the <xref rid="app3" ref-type="sec">Supplemental Information</xref>.</p></sec><sec id="sec4.3"><title>T Cell Purification, Adoptive Transfer, and Recovery</title><p>Single-cell suspensions were prepared from the spleens and lymph nodes of donor CD45.1<sup>+</sup> or CD45.2<sup>+</sup> EF4.1 mice or CD45.2<sup>+</sup> EVα2 mice, and CD4<sup>+</sup> T cells were enriched using immunomagnetic positive selection (StemCell Technologies) at >90% purity. A total of 1 × 10<sup>6</sup> EF4.1 CD4<sup>+</sup> T cells or 1 × 10<sup>5</sup> EVα2 CD4<sup>+</sup> T cells was injected into CD45.1<sup>+</sup>CD45.2<sup>+</sup> recipients via the tail vein. Env-reactive donor CD4<sup>+</sup> T cells were purified (>98% purity) from the spleens of recipient mice by cell sorting, performed on MoFlo cell sorters (Dako-Cytomation).</p></sec><sec id="sec4.4"><title>Flow Cytometry</title><p>Single-cell suspensions were stained with directly conjugated antibodies to surface markers obtained from eBiosciences, CALTAG/Invitrogen, BD Biosciences, or BioLegend.</p><p>For intracellular detection of GzmB, spleen cell suspensions were stimulated for 4 hr with phorbol 14,13 dybutirate (PdBu) and ionomycin (both at 500 ng/ml), in the presence of monensin (2 μg/ml). Cells were then stained for surface antigen and washed; after this step, they were fixed and permeabilized using an anti-mouse/rat FoxP3 staining kit (eBioscience), according to the manufacturer’s instructions. After an additional wash step, cells were stained for intracellular GzmB with Alexa Fluor 647- or FITC (fluorescein isothiocyanate)-conjugated anti-human/mouse GzmB antibodies (clone GB11, Biolegend) and phycoerythrin (PE)-conjugated anti-mouse IFN-γ antibodies (clone XMG1.2, eBioscience). Multi-color cytometry was performed on Canto II or LSRFortessa X-20 flow cytometers (both from BD Biosciences) and analyzed with FlowJo v10 (Tree Star).</p></sec><sec id="sec4.5"><title>Cytotoxicity Assays</title><p>Details of in vitro and in vivo cytotoxicity assays can be found in the <xref rid="app3" ref-type="sec">Supplemental Information</xref>.</p></sec><sec id="sec4.6"><title>PCR-Based Expression Profiling</title><p>Expression of selected genes was quantified in env-reactive CD4<sup>+</sup> T cells by real-time qRT-PCR. The indicated CD4<sup>+</sup> T cell populations were purified by cell sorting, and RNA was isolated using the QIAcube (QIAGEN). Synthesis of cDNA was carried out with the High Capacity Reverse Transcription Kit (Applied Biosystems) with an added RNase inhibitor (Promega Biosciences). A final clean-up was performed with the QIAquick PCR Purification Kit (QIAGEN). Purified cDNA was then used as template for the quantitation of the indicated genes using gene-specific primers (Eurofins MWG Operon) (<xref rid="mmc1" ref-type="supplementary-material">Table S2</xref>). Values were normalized and plotted according to the expression of <italic>Hprt</italic> in the same samples, using a ΔC<sub>T</sub> method.</p></sec><sec id="sec4.7"><title>Single-Cell RNA Sequencing</title><p>Env-reactive CD4<sup>+</sup> T cells from the indicated recipient mice were purified by cell sorting. A detailed description of subsequent single-cell RNA sequencing can be found in the <xref rid="app3" ref-type="sec">Supplemental Information</xref>.</p></sec><sec id="sec4.8"><title>Statistical Analyses</title><p>Statistical comparisons were made using SigmaPlot 12.0 (Systat Software). Parametric comparisons of normally distributed values that satisfied the variance criteria were made by unpaired Student’s t tests or one-way ANOVAs. Data that did not pass the variance test were compared with non-parametric two-tailed Mann-Whitney rank sum tests or ANOVA on ranks tests. Hierarchical clustering and heatmap production was performed with Qlucore Omics Explorer (Qlucore).</p></sec></sec><sec id="sec5"><title>Author Contributions</title><p>Conceptualization, G.K.; Methodology and Formal Analysis, G.R.Y.; Investigation, T.D., J.M., U.E., and N.B.; Resources, W.B., U.D., N.B., V.T.K.L-T., M.T., S.L.N., and C.B.; Funding Acquisition, G.K.; Supervision, G.K.; Writing – Original Draft, T.D., G.R.Y., and G.K.; Writing – Review & Editing, G.K.</p></sec></body><back><ref-list id="cebib0010"><title>References</title><ref id="bib1"><element-citation publication-type="journal" id="sref1"><person-group person-group-type="author"><name><surname>Akhmetzyanova</surname><given-names>I.</given-names></name><name><surname>Zelinskyy</surname><given-names>G.</given-names></name><name><surname>Schimmer</surname><given-names>S.</given-names></name><name><surname>Brandau</surname><given-names>S.</given-names></name><name><surname>Altenhoff</surname><given-names>P.</given-names></name><name><surname>Sparwasser</surname><given-names>T.</given-names></name><name><surname>Dittmer</surname><given-names>U.</given-names></name></person-group><article-title>Tumor-specific CD4+ T cells develop cytotoxic activity and eliminate virus-induced tumor cells in the absence of regulatory T cells</article-title><source>Cancer Immunol. 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Rev. Immunol.</source><volume>28</volume><year>2010</year><fpage>445</fpage><lpage>489</lpage><pub-id pub-id-type="pmid">20192806</pub-id></element-citation></ref></ref-list><sec id="app1"><title>Accession Numbers</title><p>The accession number for the single-cell RNA sequences reported in this paper is ENA: PRJEB14043.</p></sec><sec id="app3" sec-type="supplementary-material"><title>Supplemental Information</title><p><supplementary-material content-type="local-data" id="mmc1"><caption><title>Document S1. Supplemental Experimental Procedures, Figures S1–S15, and Table S2</title></caption><media xlink:href="mmc1.pdf"></media></supplementary-material><supplementary-material content-type="local-data" id="mmc2"><caption><title>Table S1. Differences in Gene Expression between Gzmb+ and Gzmb− Cells, Related to Figure 2</title></caption><media xlink:href="mmc2.xlsx"></media></supplementary-material><supplementary-material content-type="local-data" id="mmc3"><caption><title>Document S2. Article plus Supplemental Information</title></caption><media xlink:href="mmc3.pdf"></media></supplementary-material></p></sec><ack id="ack0010"><title>Acknowledgments</title><p>We wish to thank Dr. Toshitada Takemori for the conditional Bcl6 mice. We are grateful for assistance from the Flow Cytometry and Advanced Sequencing Facilities at the Francis Crick Institute. This work was supported by the <funding-source id="gs1">Francis Crick Institute</funding-source>, which receives its core funding from <funding-source id="gs2">Cancer Research UK</funding-source> (FC001099), the <funding-source id="gs3">UK Medical Research Council</funding-source> (FC001099), and the <funding-source id="gs4">Wellcome Trust</funding-source> (FC001099); and by the <funding-source id="gs5">Deutsche Forschungsgemeinschaft</funding-source> (DFG-TRR60 and DFG-GRK 1949).</p></ack><fn-group><fn id="app2" fn-type="supplementary-material"><p>Supplemental Information includes Supplemental Experimental Procedures, 15 figures, and two tables and can be found with this article online at <ext-link ext-link-type="doi" xlink:href="10.1016/j.celrep.2016.10.013" id="intref0010">http://dx.doi.org/10.1016/j.celrep.2016.10.013</ext-link>.</p></fn></fn-group></back><floats-group><fig id="fig1"><label>Figure 1</label><caption><p>CD4<sup>+</sup> CTL Development Depends on Infecting Virus</p><p>(A) Expression of <italic>Gzmb</italic>, relative to <italic>Hprt</italic>, assessed by qRT-PCR in env-reactive donor EF4.1 CD4<sup>+</sup> T cells purified from the spleens of recipient mice, 7 days after adoptive transfer and FV infection or Ad5.pIX-gp70 immunization. Plotted are the mean values (±SEM) of four technical replicates, from two experiments with five mice per group per experiment.</p><p>(B) Flow-cytometric detection (left) and efficiency of in vivo killing (right) of env<sub>122–141</sub>-pulsed CD45.1<sup>+</sup> and non-pulsed CD45.2<sup>+</sup> B cells in host splenocytes, 24 hr after transfer into CD45.1<sup>+</sup>CD45.2<sup>+</sup> hosts (5 × 10<sup>6</sup> of each per host) that had also received EF4.1 CD4<sup>+</sup> T cells and had either been infected with FV or immunized with Ad5.pIX-gp70 7 days earlier.</p><p>(C) Flow-cytometric detection (left) and efficiency of in vitro killing (right) of env<sub>122–141</sub>-pulsed and non-pulsed B cells, 2 hr after culture with env-reactive donor EF4.1 CD4<sup>+</sup> T cells purified from the spleens of recipient mice, 7 days after adoptive transfer and FV infection or Ad5.pIX-gp70 immunization.</p><p>(D) Flow-cytometric detection of intracellular GzmB host naive (CD44<sup>−</sup>) or env-reactive (CD44<sup>+</sup>) donor EF4.1 CD4<sup>+</sup> T cells (left) and frequency of GzmB<sup>+</sup> cells in env-reactive donor EF4.1 CD4<sup>+</sup> T cells (right) in the spleens of recipient mice, 7 days after adoptive transfer and FV infection or Ad5.pIX-gp70 immunization.</p><p>(E) Frequency of intracellular GzmB<sup>+</sup> cells in env-reactive donor EF4.1 CD4<sup>+</sup> T cells in the spleens of recipient mice, 7 days after adoptive transfer and FV infection, F-MLV-N infection, env<sub>122–141</sub> peptide immunization in the Sigma Adjuvant System, FBL-3 leukemia cell transplantation, mCMV.env infection, or Ad5.pIX-gp70 immunization.</p><p>In (B) to (E), each symbol in the scatterplots represents an individual recipient mouse.</p><p>See also <xref rid="mmc1" ref-type="supplementary-material">Figures S1</xref>, <xref rid="mmc1" ref-type="supplementary-material">S2</xref>, and <xref rid="mmc1" ref-type="supplementary-material">S3</xref>.</p></caption><graphic xlink:href="gr1"></graphic></fig><fig id="fig2"><label>Figure 2</label><caption><p>Antagonistic CD4<sup>+</sup> CTL and Tfh Development</p><p>(A) <italic>Cd4</italic> and <italic>Gzmb</italic> expression, assessed by single-cell RNA sequencing, in env-reactive donor EF4.1 CD4<sup>+</sup> T cells purified from the spleens of recipient mice, 7 days after adoptive transfer and FV infection or Ad5.pIX-gp70 immunization. Each symbol shows the log<sub>2</sub>-transformed normalized reads from an individual cell from one of two experiments. Numbers within the plots denote the number of cells positive for expression of the indicated gene.</p><p>(B) <italic>Tcf7</italic> and <italic>Bcl6</italic> expression in the same cells as in (A).</p><p>(C) Expression of <italic>Tcf7</italic> and <italic>Bcl6</italic>, relative to <italic>Hprt</italic>, assessed by qRT-PCR in bulk env-reactive donor EF4.1 CD4<sup>+</sup> T cells purified from the spleens of recipient mice, 7 days after adoptive transfer and FV infection or Ad5.pIX-gp70 immunization. Plotted are the mean values (±SEM) of four technical replicates from two experiments with four mice per group per experiment.</p><p>(D) Heatmap of gene expression, assessed by single-cell RNA sequencing, comparing <italic>Gzmb</italic><sup><italic>+</italic></sup> and <italic>Gzmb</italic><sup>−</sup> subsets in env-reactive donor EF4.1 CD4<sup>+</sup> T cells purified from the spleens of recipient mice, 7 days after adoptive transfer and priming. CD4<sup>+</sup> T cells from both FV infection and Ad5.pIX-gp70 immunization are included. A select set of genes from the complete list in <xref rid="mmc2" ref-type="supplementary-material">Table S1</xref> is shown.</p><p>(E) Flow-cytometric correlation of intracellular GzmB and surface markers CXCR6, CD39, CD150, and CD226 (left) and frequency of surface marker<sup>+</sup> cells separately in GzmB<sup>−</sup> and GzmB<sup>+</sup> cells within env-reactive (CD44<sup>+</sup>) donor EF4.1 CD4<sup>+</sup> T cells (right) in the spleens of recipient mice, 7 days after adoptive transfer and Ad5.pIX-gp70 immunization. In the scatterplots, each symbol represents an individual recipient.</p><p>(F) Expression of <italic>Prdm1</italic>, <italic>Bcl6</italic>, <italic>Lef1</italic>, <italic>Tcf7</italic>, <italic>Zbtb7b</italic>, and <italic>Runx3</italic> assessed by single-cell RNA sequencing, separately in <italic>Gzmb</italic><sup><italic>+</italic></sup> and <italic>Gzmb</italic><sup><italic>−</italic></sup> env-reactive donor EF4.1 CD4<sup>+</sup> T cells purified from the spleens of recipient mice, 7 days after adoptive transfer and Ad5.pIX-gp70 immunization.</p><p>See also <xref rid="mmc1" ref-type="supplementary-material">Figures S4</xref>, <xref rid="mmc1" ref-type="supplementary-material">S5</xref>, and <xref rid="mmc1" ref-type="supplementary-material">S6</xref> and <xref rid="mmc2" ref-type="supplementary-material">Table S1</xref>.</p></caption><graphic xlink:href="gr2"></graphic></fig><fig id="fig3"><label>Figure 3</label><caption><p>Bcl6 Suppresses CD4<sup>+</sup> CTL Development at the Population Level</p><p>(A) Delineation of env-reactive donor EF4.1 CD4<sup>+</sup> T cells according to YFP expression. Histograms show gated env-reactive EF4.1 CD4<sup>+</sup> T cells from <italic>Bcl6</italic><sup>wt</sup> or <italic>Bcl6</italic><sup>fl</sup> donors, found in the spleens of recipient mice, 7 days after adoptive transfer and FV infection or Ad5.pIX-gp70 immunization. YFP expression reports activation of the <italic>Tnfrsf4</italic> gene and, in the case of <italic>Bcl6</italic><sup>fl</sup> donor CD4<sup>+</sup> T cells, also loss of <italic>Bcl6</italic>. Numbers within the plots denote the proportion of YFP<sup>+</sup> cells.</p><p>(B) Expression of the indicated gene, relative to <italic>Hprt</italic>, assessed by qRT-PCR in the respective bulk subset of env-reactive donor EF4.1 CD4<sup>+</sup> T cells shown immediately above in (A). Plotted are the mean values (±SEM) of two technical replicates, from two experiments with five mice per group per experiment.</p><p>See also <xref rid="mmc1" ref-type="supplementary-material">Figures S7</xref>, <xref rid="mmc1" ref-type="supplementary-material">S8</xref>, and <xref rid="mmc1" ref-type="supplementary-material">S9</xref>.</p></caption><graphic xlink:href="gr3"></graphic></fig><fig id="fig4"><label>Figure 4</label><caption><p>Bcl6 Suppresses CD4<sup>+</sup> CTL Development at the Single-Cell Level</p><p>(A) Frequency of intracellular GzmB<sup>+</sup> cells in bulk <italic>Bcl6</italic><sup>wt</sup> or <italic>Bcl6</italic><sup>fl</sup> env-reactive donor EF4.1 CD4<sup>+</sup> T cells in the spleens of recipient mice, 7 days after adoptive transfer and FV infection or Ad5.pIX-gp70 immunization. Each symbol represents an individual mouse from one representative of two experiments.</p><p>(B) <italic>Cd4</italic> and <italic>Gzmb</italic> expression, assessed by single-cell RNA sequencing, in YFP<sup>+</sup><italic>Bcl6</italic><sup>fl</sup> env-reactive donor EF4.1 CD4<sup>+</sup> T cells purified from the spleens of recipient mice, 7 days after adoptive transfer and FV infection or Ad5.pIX-gp70 immunization.</p><p>(C) <italic>Il21</italic>, <italic>Il10</italic>, and <italic>Ifng</italic> expression, assessed by single-cell RNA sequencing, in YFP<sup>+</sup><italic>Bcl6</italic><sup>wt</sup>, and <italic>Bcl6</italic><sup>fl</sup> env-reactive donor EF4.1 CD4<sup>+</sup> T cells purified from the spleens of recipient mice, 7 days after adoptive transfer and FV infection or Ad5.pIX-gp70 immunization.</p></caption><graphic xlink:href="gr4"></graphic></fig><fig id="fig5"><label>Figure 5</label><caption><p>CD4<sup>+</sup> CTL and Th1 Cells Are Transcriptionally Distinct</p><p>(A) <italic>Gzmb</italic> and <italic>Ifng</italic> expression, assessed by single-cell RNA sequencing, in env-reactive donor EF4.1 CD4<sup>+</sup> T cells purified from the spleens of recipient mice, 7 days after adoptive transfer and priming. Both <italic>Bcl6</italic><sup>wt</sup> and <italic>Bcl6</italic><sup>fl</sup> CD4<sup>+</sup> T cells from both FV infection and Ad5.pIX-gp70 immunization are included.</p><p>(B) Flow-cytometric detection of intracellular GzmB and IFN-γ in host naive (CD44<sup>−</sup>) or env-reactive (CD44<sup>+</sup>) donor EF4.1 CD4<sup>+</sup> T cells in the spleens of recipient mice, 7 days after adoptive transfer and Ad5.pIX-gp70 immunization. The plot is representative of four recipients.</p><p>(C) Flow-cytometric correlation of intracellular GzmB and surface CD150 expression (left) and frequency of CD150<sup>+</sup> cells separately in GzmB<sup>−</sup> and GzmB<sup>+</sup> cells within IFN-γ<sup>+</sup> env-reactive (CD44<sup>+</sup>) donor EF4.1 CD4<sup>+</sup> T cells (right) in the spleens of recipient mice, 7 days after adoptive transfer and Ad5.pIX-gp70 immunization. In the scatterplot, each symbol represents an individual recipient.</p><p>(D) Heatmap of significantly (p = 3.95 × 10<sup>−4</sup>) regulated gene expression, assessed by single-cell RNA sequencing, comparing <italic>Ifng</italic><sup><italic>+</italic></sup><italic>Gzmb</italic><sup>+</sup> and <italic>Ifng</italic><sup><italic>+</italic></sup><italic>Gzmb</italic><sup>−</sup> subsets in the same cells as in (A).</p><p>(E) <italic>Prdm1</italic> and <italic>Tcf7</italic> expression, assessed by single-cell RNA sequencing, in the same cells as in (A).</p><p>(F) Flow-cytometric detection of Blimp1-GFP and CD44 expression in host and env-reactive donor CD4<sup>+</sup> T cells in the spleens of recipient mice, 7 days after adoptive transfer and Ad5.pIX-gp70 immunization.</p><p>(G) Expression of <italic>Prdm1</italic>, <italic>Tcf7</italic>, and <italic>Gzmb</italic>, relative to <italic>Hprt</italic>, assessed by qRT-PCR in bulk Blimp1-GFP<sup>+</sup> and Blimp1-GFP<sup>-</sup> subsets in env-reactive donor EF4.1 CD4<sup>+</sup> T cells purified from the spleens of recipient mice, 7 days after adoptive transfer and Ad5.pIX-gp70 immunization. Plotted are the mean values (±SEM) of two technical replicates from one experiment with five mice per group.</p><p>See also <xref rid="mmc1" ref-type="supplementary-material">Figures S10</xref> and <xref rid="mmc1" ref-type="supplementary-material">S11</xref>.</p></caption><graphic xlink:href="gr5"></graphic></fig><fig id="fig6"><label>Figure 6</label><caption><p>Layered Checkpoints in CD4<sup>+</sup> CTL Development</p><p>(A) <italic>Pcdc1</italic>, <italic>Lag3</italic>, <italic>Ctla4</italic>, and <italic>Havcr2</italic> expression, assessed by single-cell RNA sequencing, in env-reactive donor EF4.1 CD4<sup>+</sup> T cells purified from the spleens of recipient mice, 7 days after adoptive transfer and FV infection or Ad5.pIX-gp70 immunization. Both <italic>Bcl6</italic><sup>wt</sup> and <italic>Bcl6</italic><sup>fl</sup> CD4<sup>+</sup> T cells are included.</p><p>(B) Flow-cytometric detection of PD-1 and LAG3 expression in host naive (CD44<sup>−</sup>) and env-reactive (CD44<sup>+</sup>) donor CD4<sup>+</sup> T cells in the spleens of recipient mice, 7 days after adoptive transfer and FV infection or Ad5.pIX-gp70 immunization. Numbers within the plots denote the proportion of PD-1<sup>+</sup> and LAG3<sup>+</sup> cells in env-reactive donor CD4<sup>+</sup> T cells only.</p><p>(C) Median fluorescence intensity (MFI) of PD-1 and LAG3 staining in the same cells as in (B). Each symbol represents an individual recipient. The dashed line represents the MFI of PD-1 and LAG3 staining in host naive CD4<sup>+</sup> T cells.</p><p>(D) Frequency of intracellular GzmB<sup>+</sup> cells in bulk <italic>Bcl6</italic><sup>wt</sup> and <italic>Bcl6</italic><sup>fl</sup> env-reactive donor EF4.1 CD4<sup>+</sup> T cells in the spleens of either WT or B cell-deficient <italic>Ighm</italic><sup>−/−</sup> recipient mice, 7 days after adoptive transfer and FV infection. The indicated groups additionally received treatment with PD-1- and LAG3-blocking antibodies. Each symbol represents an individual recipient.</p><p>See also <xref rid="mmc1" ref-type="supplementary-material">Figures S12</xref>, <xref rid="mmc1" ref-type="supplementary-material">S13</xref>, <xref rid="mmc1" ref-type="supplementary-material">S14</xref>, and <xref rid="mmc1" ref-type="supplementary-material">S15</xref>.</p></caption><graphic xlink:href="gr6"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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