Diese Studie wurde durch Zuschüsse der National Natural Science Foundation of China (Nr. 32300785 bis X.C.), des China National Postdoctoral Program for Innovative Talents (Nr. BX20230449 bis X.C.) und das National Science and Technology Major Project (Nr. 2021YFC2300602 bis L.Y.).

Entschlüsselung der frühen Bindung follikulärer Helfer-T-Zellen bei akuter LCMV-Infektion

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl6+expressing+follicular+helper+CD4+T+cells+are+fate+committed+early+and+have+the+capacity+to+form+memory.">Bcl6 expressing follicular helper CD4 T cells are fate committed early and have the capacity to form memory.</a> J Immunol. 190 (8), 4014-4026 (2013).</li><li>Johnston, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl6+and+BLIMP-1+are+reciprocal+and+antagonistic+regulators+of+T+follicular+helper+cell+differentiation.">Bcl6 and BLIMP-1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation.</a> Science. 325 (5943), 1006-1010 (2009).</li><li>Nurieva, R. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl6+mediates+the+development+of+T+follicular+helper+cells.">Bcl6 mediates the development of T follicular helper cells.</a> Science. 325 (5943), 1001-1005 (2009).</li><li>Yu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptional+repressor+bcl-6+directs+T+follicular+helper+cell+lineage+commitment.">The transcriptional repressor bcl-6 directs T follicular helper cell lineage commitment.</a> Immunity. 31 (3), 457-468 (2009).</li><li>Choi, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LEF1+and+TCF1+orchestrate+T(FH)+differentiation+by+regulating+differentiation+circuits+upstream+of+the+transcriptional+repressor+bcl6.">LEF1 and TCF1 orchestrate T(FH) differentiation by regulating differentiation circuits upstream of the transcriptional repressor bcl6.</a> Nat Immunol. 16 (9), 980-990 (2015).</li><li>Wu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TCF1+is+required+for+the+t+follicular+helper+cell+response+to+viral+infection.">TCF1 is required for the t follicular helper cell response to viral infection.</a> Cell Rep. 12 (12), 2099-2110 (2015).</li><li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+TCF-1+initiates+the+differentiation+of+T(FH)+cells+during+acute+viral+infection.">The transcription factor TCF-1 initiates the differentiation of T(FH) cells during acute viral infection.</a> Nat Immunol. 16 (9), 991-999 (2015).</li><li>Oestreich, K. J., Mohn, S. E., Weinmann, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+that+control+the+expression+and+activity+of+bcl-6+in+th1+cells+to+regulate+flexibility+with+a+TFH-like+gene+profile.">Molecular mechanisms that control the expression and activity of bcl-6 in th1 cells to regulate flexibility with a TFH-like gene profile.</a> Nat Immunol. 13 (4), 405-411 (2012).</li><li>Choi, Y. S., Eto, D., Yang, J. A., Lao, C., Crotty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+STAT1+is+required+for+IL-6-mediated+bcl6+induction+for+early+follicular+helper+cell+differentiation.">Cutting edge: STAT1 is required for IL-6-mediated bcl6 induction for early follicular helper cell differentiation.</a> J Immunol. 190 (7), 3049-3053 (2013).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histone+methyltransferase+ezh2+primes+the+early+differentiation+of+follicular+helper+T+cells+during+acute+viral+infection.">The histone methyltransferase ezh2 primes the early differentiation of follicular helper T cells during acute viral infection.</a> Cell Mol Immunol. 17 (3), 247-260 (2020).</li><li>Li, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ezh2+programs+T(FH)+differentiation+by+integrating+phosphorylation-dependent+activation+of+bcl6+and+polycomb-dependent+repression+of+p19ARF.">Ezh2 programs T(FH) differentiation by integrating phosphorylation-dependent activation of bcl6 and polycomb-dependent repression of p19ARF.</a> Nat Commun. 9 (1), 5452 (2018).</li><li>Yao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=METTL3-dependent+m(6)A+modification+programs+T+follicular+helper+cell+differentiation.">METTL3-dependent m(6)A modification programs T follicular helper cell differentiation.</a> Nat Commun. 12 (1), 1333 (2021).</li><li>Johnston, R. J., Choi, Y. S., Diamond, J., Yang, J. A., Crotty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT5+is+a+potent+negative+regulator+of+TFH+cell+differentiation.">STAT5 is a potent negative regulator of TFH cell differentiation.</a> J Exp Med. 209 (2), 243-250 (2012).</li><li>Lee, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+KLF2+restrains+CD4++T+follicular+helper+cell+differentiation.">The transcription factor KLF2 restrains CD4+ T follicular helper cell differentiation.</a> Immunity. 42 (2), 252-264 (2015).</li><li>Xiao, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+e3+ubiquitin+ligase+itch+is+required+for+the+differentiation+of+follicular+helper+t+cells.">The e3 ubiquitin ligase itch is required for the differentiation of follicular helper t cells.</a> Nat Immunol. 15 (7), 657-666 (2014).</li><li>Baumjohann, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+cluster+mir-17~92+promotes+TFH+cell+differentiation+and+represses+subset-inappropriate+gene+expression.">The microRNA cluster mir-17~92 promotes TFH cell differentiation and represses subset-inappropriate gene expression.</a> Nat Immunol. 14 (8), 840-848 (2013).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kinase+complex+mTORC2+promotes+the+longevity+of+virus-specific+memory+CD4(+)+T+cells+by+preventing+ferroptosis.">The kinase complex mTORC2 promotes the longevity of virus-specific memory CD4(+) T cells by preventing ferroptosis.</a> Nat Immunol. 23 (2), 303-317 (2022).</li><li>Hale, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+memory+CD4++T+cells+with+commitment+to+T+follicular+helper-+and+T+helper+1-cell+lineages+are+generated+after+acute+viral+infection.">Distinct memory CD4+ T cells with commitment to T follicular helper- and T helper 1-cell lineages are generated after acute viral infection.</a> Immunity. 38 (4), 805-817 (2013).</li><li>Yao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selenium-GPX4+axis+protects+follicular+helper+t+cells+from+ferroptosis.">Selenium-GPX4 axis protects follicular helper t cells from ferroptosis.</a> Nat Immunol. 22 (9), 1127-1139 (2021).</li><li>Oxenius, A., Bachmann, M. F., Zinkernagel, R. 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Die Autoren erklären, dass keine konkurrierenden Interessen bestehen.

Die Forschung auf dem Gebiet derT-FH-Zellen ist seit der Entdeckung der spezialisierten Funktion vonT-FH-Zellen bei der Unterstützung von B-Zellen hervorgehoben worden. Häufende Studien deuteten darauf hin, dass dieT-FH-Zelldifferenzierung ein mehrstufiger und multifaktorieller Prozess30 ist, wobei die Schicksalsbindung derT-FH-Zellen im frühen Stadium bestimmt wird5. Daher ist ein besseres Verständnis der Mechanismen, die fr?...

Wenn naive CD4+ T-Zellen auf unterschiedliche Krankheitserreger oder Bedrohungen treffen, passen sie ihre Immunantworten an, indem sie sich in verschiedene T-Helfer-Zell-Untergruppen (TH) mit spezialisierten Funktionen differenzieren1. Im Szenario einer akuten Virusinfektion differenziert sich ein großer Teil der naiven CD4+ T-Zellen zu follikulären Helfer-T (TFH)-Zellen, die den B-Zellen helfen 2,3. Im Unterschied zu anderen CD4+ TH Zell-Untergruppen (z. B. TH1, TH2, TH9 und TH17 Zellen) exprimieren TFH-Zellen eine beträchtliche Menge an CXCR5, dem Chemokinrezeptor für das B-Zell-Homing-Chemokin CXCL13, das es TFH-Zellen ermöglicht, in B-Zellfollikel zu wandern. In den B-Zell-Follikeln unterstützenT-FH-Zellen verwandte B-Zellen bei der Initiierung und Aufrechterhaltung von Keimzentrumsreaktionen und ermöglichen so eine schnelle Produktion von Antikörpern mit hoher Affinität und ein humorales Langzeitgedächtnis 2,3.
Nach einer akuten Virusinfektion erfolgt die frühe Schicksalsbindung von virusspezifischenT-FH-Zellen innerhalb von 72 h 4,5 und wird durch den transkriptionellen Repressor B-Zell-Lymphom-6 (Bcl-6)5,6,7,8 gesteuert, der als "Master-Regulator" fungiert und über das Schicksal vonT-FH-Zellen entscheidet. Ein Mangel an Bcl-6 stumpft die Differenzierung derT-FH-Zellen stark ab, während die ektopische Bcl-6-Expression die Bindung der T-FH-Zellen an das Schicksal derT-FH-Zellen erheblich fördert. Zusätzlich zu Bcl-6 sind mehrere Moleküle an der Instruktion des frühen Schicksals derT-FH-Zellen beteiligt. Die Transkriptionsfaktoren TCF-1 und LEF-1 initiieren die Differenzierung derT-FH-Zellen über die Induktion von Bcl-6 9,10,11. Die Hemmung von Blimp1 sowohl durch Bcl-6 als auch durch TCF-1 ist für die frühe Bindung vonT-FH-Zellen erforderlich11,12. STAT1 und STAT3 werden auch für die frühe Differenzierung vonT-FH-Zellen benötigt13. Darüber hinaus tragen epigenetische Modifikationen durch die Histon-Methyltransferase EZH214,15 und die m6A-Methyltransferase METTL316 dazu bei, die Transkriptionsprogramme derT-FH-Zellen (insbesondere Bcl6 und Tcf7) zu stabilisieren und damit das frühe Schicksal derT-FH-Zellen zu unterstützen. Während Fortschritte, einschließlich der oben genannten Moleküle und anderer, die an anderer Stellezusammengefasst sind 3, beim Verständnis der transkriptionellen und epigenetischen Regulation des frühen Schicksals vonT-FH-Zellen gemacht wurden, müssen bisher unbekannte Moleküle noch gelernt werden.
Im Mausmodell der akuten lymphozytären Choriomeningitis-Virus (LCMV)-Infektion durchlaufen adoptiv übertragene kongene TCR-transgene SMARTA (SM) CD4+ T-Zellen, die spezifisch das LCMV-Glykoprotein-Epitop I-AbGP66-77 erkennen, während der Virusinfektion entweder eineT-FH- oder eineT-H1-Zelldifferenzierung. Dieses TFH/TH1-Bifurkationsdifferenzierungsmuster unterstützt die Weiterentwicklung des SM/akuten LCMV-Infektionsmodells bei der Untersuchung der Biologie virusspezifischer TFH-Zellen. In der Tat ist das SM/akute LCMV-Infektionsmodell in derT-FH-Zellforschung weit verbreitet und hat eine entscheidende Rolle bei bahnbrechenden Entdeckungen in derT-FH-Zellbiologie gespielt. Dies beinhaltet die Identifizierung des bereits erwähnten Bcl-6 als liniendefinierenden Transkriptionsfaktor derT-FH-Zellen 5,6 sowie anderer wichtiger transkriptioneller Faktoren (z. B. Blimp-16, TCF-1/LEF 9,10,11, STAT1/STAT313, STAT517, KLF218 und Itch19), die die Differenzierungvon T-FH-Zellen steuern, posttranskriptionelle Regulierungen ( z.B. METTL316 und miR-17~9220) der T-FH-Zelldifferenzierung, des T-FH-Zellgedächtnisses und der Plastizität21,22 und rationaler Impfstrategien, die auf T-FH-Zellen abzielen (z. B. Selen23).
Die vorliegende Studie beschreibt reproduzierbare Methoden für den Zugang zur frühen Schicksalsbindung von virusspezifischenT-FH-Zellen , einschließlich (1) der Etablierung eines akuten LCMV-infizierten SM-Chimären-Mausmodells, das für den Zugang zu früh differenziertenT-FH-Zellen geeignet ist, (2) der Durchführung von Durchflusszytometrie-Färbungen von Molekülen, die mit frühdifferenziertenT-FH-Zellen verwandt sind, und (3) der Durchführung retroviraler vektorbasierter Genmanipulation in SM CD4+ T-Zellen. Diese Methoden werden für Studien nützlich sein, die die frühe Schicksalsbindung von virusspezifischenT-FH-Zellen untersuchen.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Corning</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde Fix Solution, 4% PFA</td>
			<td>Beyotime</td>
			<td>P0099-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainer</td>
			<td>Merck</td>
			<td>CLS431751</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 anti-mouse TCR V&#945;2 (clone B20.1)</td>
			<td>Biolegend</td>
			<td>127812</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 700 anti-mouse CD45.1 (clone A20)</td>
			<td>Biolegend</td>
			<td>110724</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>APC anti-mouse CD25 (clone PC61)</td>
			<td>Biolegend</td>
			<td>101910</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>B6 CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ) mouse</td>
			<td>The Jackson Laboratory</td>
			<td>002014</td>
			<td></td>
		</tr>
		<tr>
			<td>BeaverBeads Streptavidin</td>
			<td>Beaver</td>
			<td>22321-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin anti-mouse F4/80 Antibody (clone BM8)</td>
			<td>Biolegend</td>
			<td>123106</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Biotin Rat anti-mouse CD11c (clone N418)</td>
			<td>Biolegend</td>
			<td>117304</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Biotin Rat anti-Mouse CD19 (clone 6D5)</td>
			<td>Biolegend</td>
			<td>115504</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Biotin Rat anti-Mouse CD8a (clone 53-6.7)</td>
			<td>Biolegend</td>
			<td>100704</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Biotin Rat anti-mouse NK-1.1 (clone PK136)</td>
			<td>Biolegend</td>
			<td>108704</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Biotin Rat anti-mouse TER-119/Erythroid Cells (clone TER-119)</td>
			<td>Biolegend</td>
			<td>116204</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>bovine serum albumin, BSA</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 421 anti-T-bet (clone 4B10)</td>
			<td>Biolegend</td>
			<td>644816</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>Brilliant Violet 605 anti-mouse CD279 (PD-1) (clone 29F.1A12)</td>
			<td>Biolegend</td>
			<td>135220</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>C57BL/6J (B6) mouse</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>CXCR5-GFP knock-in reporter mouse</td>
			<td></td>
			<td></td>
			<td>In house; the CXCR5-GFP knock-in mouse line was generated by the insertion of an IRES-GFP construct after the open reading frame of Cxcr5.</td>
		</tr>
		<tr>
			<td>DMEM 10% medium</td>
			<td></td>
			<td></td>
			<td>DMEM medium containing 10% FBS</td>
		</tr>
		<tr>
			<td>DMEM medium</td>
			<td>Gibco</td>
			<td>11885092</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSFortesa</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum, FBS</td>
			<td>Sigma</td>
			<td>F8318</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo (version 10.4.0)</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foxp3/Transcription Factor Staining Buffer Set</td>
			<td>Invitrogen</td>
			<td>00-5523-00</td>
			<td>The kit contains three reagents: a. Fixation/Permeabilization Concentrate (4X); b. Fixation / Permeabilization Diluent; c. Permeabilization Buffer.</td>
		</tr>
		<tr>
			<td>Goat Anti-Rat IgG Antibody (H+L), Biotinylated</td>
			<td>Vector laboratories</td>
			<td>BA-9400-1.5</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Invitrogen EVOS FL Auto Cell Imaging System</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isolation buffer</td>
			<td></td>
			<td></td>
			<td>FACS buffer containing 0.5% BSA and 2mM EDTA</td>
		</tr>
		<tr>
			<td>LCMV GP61-77 peptide (GLKGPDIYKGVYQFKSV)</td>
			<td>Chinese Peptide Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm excitation</td>
			<td>Life Technologies</td>
			<td>L10199</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>MigR1</td>
			<td>addgene</td>
			<td>#27490</td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM medium</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>pCL-Eco</td>
			<td>addgene</td>
			<td>#12371</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD69 (clone H1.2F3)</td>
			<td>Biolegend</td>
			<td>104508</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>PE Mouse anti-Bcl-6 (clone K112-91)</td>
			<td>BD Biosciences</td>
			<td>561522</td>
			<td>1:50 dilution</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, PBS</td>
			<td>Gibco</td>
			<td>10010072</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Solarbio</td>
			<td>H8761</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Rat Anti-Mouse CXCR5 (clone 2G8)</td>
			<td>BD Biosciences</td>
			<td>551961</td>
			<td>1:50 dilution</td>
		</tr>
		<tr>
			<td>Rat monoclonal PerCP anti-mouse CD4 (clone RM4-5)</td>
			<td>Biolegend</td>
			<td>100538</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>recombinant murine IL-2</td>
			<td>Gibco</td>
			<td>212-12-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer</td>
			<td>Beyotime</td>
			<td>C3702-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Sigma</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 2%</td>
			<td></td>
			<td></td>
			<td>RPMI 1640 medium containing 2% FBS</td>
		</tr>
		<tr>
			<td>SMARTA (SM) TCR transgenic mouse</td>
			<td></td>
			<td></td>
			<td>SM TCR transgenic line in our lab is a gift from Dr. Rafi Ahmed (Emory University). Additionally, this mouse line can also be obtained from The Jackson Laboratory (stain#: 030450).</td>
		</tr>
		<tr>
			<td>Staining buffer</td>
			<td></td>
			<td></td>
			<td>PBS containing 2% FBS and 0.01% NaN3</td>
		</tr>
		<tr>
			<td>Streptavidin PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-4317-82</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>TCF1/TCF7 (C63D9) Rabbit mAb (Alexa Fluor 488 Conjugate)&#160;</td>
			<td>Cell signaling technology</td>
			<td>6444S</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>TFH cell staining buffer</td>
			<td></td>
			<td></td>
			<td>FACS buffer containing 1% BSA and 2% mouse serum</td>
		</tr>
		<tr>
			<td>TransIT-293 reagent</td>
			<td>Mirus Bio</td>
			<td>MIRUMIR2700</td>
			<td></td>
		</tr>
	</tbody>
</table>

accessing early differentiation of virus-specific follicular helper cd4+ t cell in acute lcmv-infected mice

Follikuläre T-Helferzellen (TFH) werden als eigenständige CD4+ T-Zelllinie wahrgenommen, die verwandte B-Zellen bei der Produktion von hochaffinen Antikörpern unterstützt und so eine langfristige humorale Immunität aufbaut. Während einer akuten Virusinfektion wird das Schicksal virusspezifischerT-FH-Zellen in der frühen Infektionsphase bestimmt, und Untersuchungen der früh differenziertenT-FH-Zellen sind entscheidend für das Verständnis der T-Zell-abhängigen humoralen Immunität und die Optimierung des Impfstoffdesigns. In der Studie haben wir unter Verwendung eines Mausmodells für eine akute Infektion mit dem lymphozytären Choriomeningitis-Virus (LCMV) und der TCR-transgenen SMARTA (SM)-Maus mit CD4+ T-Zellen, die spezifisch das LCMV-Glykoprotein-Epitop I-AbGP66-77 erkennen, Verfahren beschrieben, um auf der Grundlage von Durchflusszytometrie-Färbungen auf das frühe Schicksal von virusspezifischenT-FH-Zellen zuzugreifen. Darüber hinaus werden durch die Ausnutzung der retroviralen Transduktion von SM CD4+ T-Zellen auch Methoden zur Manipulation der Genexpression in früh differenzierten virusspezifischenT-FH-Zellen bereitgestellt. Daher werden diese Methoden bei Studien helfen, die den Mechanismus untersuchen, der der frühen Bindung von virusspezifischenT-FH-Zellen zugrunde liegt.

Encountering different pathogens or threats, na&#239;ve CD4+ T cells tailor their immune responses by differentiating into various helper T (TH) cell subsets with specialized functions1. In the scenario of acute viral infection, a large portion of na&#239;ve CD4+ T cells differentiate into follicular helper T (TFH) cells that provide help to B cells2,3. Distinct from other CD4+ TH cell subsets (e.g., TH1, TH2, TH9, and TH17 cells), TFH cells express a substantial level of CXCR5, which is the chemokine receptor for the B cell homing chemokine CXCL13, enabling TFH cells to migrate into B cell follicles. In the B cell follicles, TFH cells assist cognate B cells in initiating and maintaining germinal center reactions, thus enabling rapid high-affinity antibody production and long-term humoral memory2,3.
Upon acute viral infection, the early fate commitment of virus-specific TFH cells occurs within 72 h4,5and is controlled by the transcriptional repressor B cell lymphoma-6 (Bcl-6)5,6,7,8, which acts as the &#34;master regulator&#34; governing TFH cell fate decisions. Deficiency of Bcl-6 severely blunts TFH cell differentiation, while ectopic Bcl-6 expression substantially promotes TFH cell fate commitment. In addition to Bcl-6, multiple molecules are involved in instructing early TFH cell fate commitment. Transcription factors TCF-1 and LEF-1 initiate TFH cell differentiation via the induction of Bcl-69,10,11. The inhibition of Blimp1, by both Bcl-6 and TCF-1, is required for early TFH cell fate commitment11,12. STAT1 and STAT3 are also required for early TFH cell differentiation13. Besides, epigenetic modifications by histone methyltransferase EZH214,15and m6A methyltransferase METTL316 help to stabilize TFH cell transcriptional programs (especially Bcl6 and Tcf7) and thus prime early TFH cell fate commitment. While advances, including the aforementioned molecules and others, summarized elsewhere3, have been made in understanding the transcriptional and epigenetic regulations of early TFH&#160;cell fate commitment, previously unknown molecules remain to be learned.
In the mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection, adoptively transferred congenic TCR-transgenic SMARTA (SM) CD4+ T cells, which specifically recognize the LCMV glycoprotein epitope I-AbGP66-77, undergo either TFH or TH1 cell differentiation during viral infection. This TFH/TH1 bifurcation differentiation pattern supports the advancement of the SM/acute LCMV infection model in studying the biology of virus-specific TFH cells. Indeed, the SM/acute LCMV infection model has been widely used in the TFH cell research field and has played a crucial role in milestone discoveries in TFH cell biology. This includes the identification of the aforementioned Bcl-6 as the lineage-defining transcription factor of TFH cells5,6, as well as other important transcriptional factors (e.g., Blimp-16, TCF-1/LEF9,10,11, STAT1/STAT313, STAT517, KLF218, and Itch19) guiding TFH cell differentiation, post-transcriptional regulations (e.g., METTL316 and miR-17~9220) of TFH cell differentiation, TFH cell memory and plasticity21,22, and rational vaccination strategies targeting TFH cells (e.g., selenium23).
The current study describes reproducible methods for accessing the early fate commitment of virus-specific TFH cells, including (1) establishing an acute LCMV-infected SM chimera mouse model suitable for accessing early-differentiated TFH cells, (2) conducting flow cytometry stainings of molecules related to early-differentiated TFH cells, and (3) performing retroviral vector-based gene manipulation in SM CD4+ T cells. These methods will be useful for studies investigating the early fate commitment of virus-specific TFH cells.

Autor im Rampenlicht: Aufklärung der Wege der TFH-Zelldifferenzierung bei akuten LCMV-Herausforderungen

Zugang zur frühen Differenzierung der virusspezifischen follikulären Helferzelle CD4+ T-Zellen in akuten LCMV-infizierten Mäusen

Our research describes reproducible methods for assessing the early fate commitment of virus-specific TFH cells, including establishing acute LCMV-infected monoclonal mouse model, conducting flow cytometry staining, and performing retroviral-vector-based gene manipulation. Compared to in-vitro T-cell stimulation, in-vivo stimulation could activate T cells more quickly, even in 12 hours, and it's fully activated for the next retrovirus transduction. Our findings will help in studies exploring the mechanisms underlying the early commitment of various specific TFH cells.Furthermore, our research will contribute to understanding T-cell-dependent humoral immunity and optimizing vaccine design.

Eigenschaften von früh differenzierten virusspezifischenT-FH-Zellen während einer akuten LCMV-Infektion Um die frühe Schicksalsbindung von virusspezifischenT-FH-Zellen zu untersuchen, wurden naive kongene SM CD4+ T-Zellen, die spezifisch das LCMV-GP-Epitop I-AbGP66-77 erkennen, adoptiv in CD45.2+ C57BL/6-Empfänger übertragen. Am nächsten Tag wurden diese Empfänger intravenös mit einer hohen Dosierung des akut aufgelösten LCMV A...

Read this Scientific Article Protocol about Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice at JoVE.com

Die aktuelle Studie zeigt Protokolle zur Bewertung der frühen Schicksalsbindung von virusspezifischenT-FH-Zellen und zur Manipulation der Genexpression in diesen Zellen.

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, ...

accessing-early-differentiation-virus-specific-follicular-helper-cd4

Research

JoVE Journal

Immunology and Infection

Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice

436 Aufrufe.  Shenzhen University. Unsere Forschung beschreibt reproduzierbare Methoden zur Beurteilung des frühen Verbleibs von virusspezifischen TFH-Zellen, einschließlich der Etablierung eines akuten LCMV-infizierten monoklonalen Mausmodells, der Durchführung von Durchflusszytometrie-Färbungen und der Durchführung retroviraler Vektor-basierter Genmanipulationen. Im Vergleich zur In-vitro-T-Zell-Stimulation kann die In-vivo-Stimulation die T-Zellen schneller aktivieren, sogar innerhalb ...

Serie: Autor im Rampenlicht: Aufklärung der Wege der TFH-Zelldifferenzierung bei akuten LCMV-Herausforderungen

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, thus establishing long-term humoral immunity. During acute viral infection, the fate commitment of virus-specific TFH cells is determined in the early infection phase, and investigations of the early-differentiated TFH cells are crucial in understanding T cell-dependent humoral immunity and optimizing vaccine design. In the study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection and the TCR-transgenic SMARTA (SM) mouse with CD4+ T cells specifically recognizing LCMV glycoprotein epitope I-AbGP66-77, we described procedures to access the early fate commitment of virus-specific TFH cells based on flow cytometry stainings. Furthermore, by exploiting retroviral transduction of SM CD4+ T cells, methods to manipulate gene expression in early-differentiated virus-specific TFH cells are also provided. Hence, these methods will help in studies exploring the mechanism(s) underlying the early commitment of virus-specific TFH cells.

The current study showcases protocols for assessing the early fate commitment of virus-specific TFH cells and manipulating gene expression in these cells.

Forschung

Immunologie und Infektion

Mapping the Early Development of LCMV-Specific Follicular Helper T-Cells in Mice

To begin, obtain a six to eight week old CD 45.1 positive SMARTA mouse and inject it with 200 micrograms of LCMV GP 61-77 peptide in 100 microliters of RPMI medium intravenously. After acquiring the splenocytes from the mouse, suspend them in 2%RPMI to achieve a cell density of one times 10 to the eighT-cells per milliliter and place the tube with cells on ice. Dispense 50 microliters of cell suspension along with 150 microliters of staining buffer to each well of a round-bottom 96-well plate.Centrifuge the 96-well plate at 800 G for one minute at four degrees Celsius and carefully decant the supernatant. Vortex the plate and add 200 microliters of staining buffer to each well. After pelleting the cells, resuspend and incubate them with surface antibody cocktail on ice for 30 minutes in the dark.After washing two times, resuspend the cells in 200 microliters of staining buffer and transfer them into a flow cytometry tube. Perform flow cytometry analysis to check the activation status of SMARTA CD4 positive T-cells. Then seed one times 10 the six SMARTA CD4 positive T-cells per well into a 24 well plate.Spin down the cells at 800 G for three minutes at four degrees Celsius and carefully remove the supernatant medium. Next, add one milliliter of retrovirus medium and eight micrograms of polybrene to each well. Spin transduce the cells at 800 G for two hours at 37 degrees Celsius.After discarding the retrovirus medium, incubate the cells with one milliliter of prewarmed 10%RPMI supplemented with interleukin 2. Transfer the cell suspension into a 15 milliliter conical tube and centrifuge it. After discarding the supernatant, resuspend the cells with 2%RPMI to achieve a cell density of one times 10 to the eighth cells per milliliter.For assessing the transduction efficiency, aliquot 50 microliters of cell suspension into a round bottom 96-well plate. Incubate them on ice with the surface antibody cocktail including CD4, V alpha two, and vector tag-associated antibody. Wash the cells and perform flow cytometry as demonstrated earlier.Then inject one times 10 of the six CD 45.1 positive SMARTA CD4 positive T-cells to a C57BL/6 recipient mouse intravenously followed by one times 10 to the six plaque forming units of LCMV Armstrong the next day. After acquiring the splenocytes from the mouse, stain them to check the desired markers. To detect transcriptional factors, incubate the cells with 200 microliters of 2%paraformaldehyde at room temperature for 20 minutes in the dark.Finally, perform flow cytometry to detect factors at the early acute LCMV infection stage. On day three after infection, a balanced bifurcation of follicular helper T-cells and T-helper 1 cells Among MIGR1 SMARTA CD4 positive T-cells was found. A predominant follicular helper T-cell directed differentiation and enhanced BCL6 expression levels were observed in MIGR1 BCL6 SMARTA CD4 positive T-cells.