Este estudio fue apoyado por becas de la Fundación Nacional de Ciencias Naturales de China (No. 32300785 a X.C.), el Programa Nacional Postdoctoral de China para Talentos Innovadores (No. BX20230449 a X.C.), y el Proyecto Nacional Mayor de Ciencia y Tecnología (No. 2021YFC2300602 a L.Y.).

Descifrando el compromiso temprano de las células T auxiliares foliculares en la infección aguda por LCMV

<ol>
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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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Los autores declaran no tener intereses contrapuestos.

La investigación en el campo de las células TFH se ha destacado desde el descubrimiento de la función especializada de las células TFH para ayudar a las células B. Los estudios acumulados indicaron que la diferenciación de las células TFH es un proceso multietapa y multifactorial30, en el que el compromiso con el destino de las células TFH se determina en la etapa temprana5. Por lo tanto, una mejor comprensión de los meca...

Al encontrarse con diferentes patógenos o amenazas, las células T CD4+ ingenuas adaptan sus respuestas inmunitarias diferenciándose en varios subconjuntos de células T (TH) auxiliarescon funciones especializadas. En el escenario de infección viral aguda, una gran parte de las células T CD4+ vírgenes se diferencian en células T auxiliares foliculares (TFH) que proporcionan ayuda a las células B 2,3. A diferencia de otros subconjuntos de células TH CD4+ (por ejemplo, células TH1, TH2, TH9 y TH17), las células TFH expresan un nivel sustancial de CXCR5, que es el receptor de quimiocinas para la quimiocina CXCL13 de las células B, lo que permite que las células TFH migren a los folículos de las células B. En los folículos de las células B, las células TFH ayudan a las células B afines a iniciar y mantener las reacciones del centro germinal, lo que permite una rápida producción de anticuerpos de alta afinidad y una memoria humoral a largo plazo 2,3.
Tras la infección viral aguda, el compromiso temprano del destino de las células TFH específicas del virus ocurre dentro de las 72 h 4,5 y es controlado por el linfoma represor transcripcional de células B-6 (Bcl-6)5,6,7,8, que actúa como el "regulador maestro" que gobierna las decisiones de destino de las células TFH. La deficiencia de Bcl-6 reduce gravemente la diferenciación de las células TFH, mientras que la expresión ectópica de Bcl-6 promueve sustancialmente el compromiso con el destino de las células TFH. Además de Bcl-6, múltiples moléculas están involucradas en la instrucción del compromiso temprano del destino de las células TFH. Los factores de transcripción TCF-1 y LEF-1 inician la diferenciación de las células TFH a través de la inducción de Bcl-6 9,10,11. La inhibición de Blimp1, tanto por Bcl-6 como por TCF-1, es necesaria para el compromiso temprano del destino de las células TFH 11,12. STAT1 y STAT3 también son necesarios para la diferenciación temprana de las células TFH 13. Además, las modificaciones epigenéticas por la histona metiltransferasa EZH214,15 y la m6A metiltransferasa METTL316 ayudan a estabilizar los programas transcripcionales de las células TFH (especialmente Bcl6 y Tcf7) y, por lo tanto, a preparar el compromiso temprano del destino de las célulasT FH. Si bien se han logrado avances, incluidas las moléculas antes mencionadas y otras, resumidas en otra parte3, en la comprensión de las regulaciones transcripcionales y epigenéticas del compromiso temprano del destino de las células TFH, las moléculas previamente desconocidas aún no se han aprendido.
En el modelo murino de infección por el virus de la coriomeningitis linfocítica aguda (LCMV), las células T CD4+ transgénicas TCR-transgénicas TCR transferidas adoptivamente, que reconocen específicamente el epítopo de la glicoproteína LCMV I-ABGP66-77, experimentan una diferenciación de células TFH o TH1 durante la infección viral. Este patrón de diferenciación de bifurcación TFH/TH1 apoya el avance del modelo de infección por SM/LCMV aguda en el estudio de la biología de las células TFH específicas del virus. De hecho, el modelo de infección por SM/LCMV aguda se ha utilizado ampliamente en el campo de la investigación de células TFH y ha desempeñado un papel crucial en los descubrimientos de hitos en la biología de las células TFH. Esto incluye la identificación del mencionado Bcl-6 como el factor de transcripción que define el linaje de las células TFH 5,6, así como otros factores transcripcionales importantes (por ejemplo, Blimp-16, TCF-1/LEF 9,10,11, STAT1/STAT313, STAT517, KLF218 y Itch19) que guían la diferenciación de las células TFH, las regulaciones postranscripcionales ( por ejemplo, METTL316 y miR-17~9220) de la diferenciación de los linfocitos TFH, la memoria y plasticidad de los linfocitos TFH 21,22 y las estrategias racionales de vacunación dirigidas a los linfocitos TFH (p. ej., selenio23).
El estudio actual describe métodos reproducibles para acceder al compromiso de destino temprano de las células TFH específicas del virus, que incluyen (1) el establecimiento de un modelo de ratón de quimera SM infectado con LCMV agudo adecuado para acceder a las células TFH diferenciadas tempranamente, (2) la realización de tinciones por citometría de flujo de moléculas relacionadas con las células TFH diferenciadas tempranamente y (3) la realización de manipulación génica basada en vectores retrovirales en SM CD4+ Células T. Estos métodos serán útiles para los estudios que investigan el compromiso del destino temprano de las células TFH específicas del virus.

<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

Las células T auxiliares foliculares (TFH) se perciben como un linaje independiente de células T CD4+ que ayuda a las células B afines a producir anticuerpos de alta afinidad, estableciendo así una inmunidad humoral a largo plazo. Durante la infección viral aguda, el compromiso del destino de las células TFH específicas del virus se determina en la fase temprana de infección, y las investigaciones de las células TFH diferenciadas tempranamente son cruciales para comprender la inmunidad humoral dependiente de las células T y optimizar el diseño de la vacuna. En el estudio, utilizando un modelo de ratón de infección por el virus de la coriomeningitis linfocítica aguda (LCMV) y el ratón transgénico TCR SMARTA (SM) con linfocitos T CD4+ que reconocen específicamente el epítopo de glicoproteína LCMVI-A bGP66-77, describimos procedimientos para acceder al compromiso del destino temprano de los linfocitos TFH específicos del virus basados en tinciones por citometría de flujo. Además, mediante la explotación de la transducción retroviral de células T SM CD4+ , también se proporcionan métodos para manipular la expresión génica en células TFH específicas del virus diferenciadas tempranamente. Por lo tanto, estos métodos ayudarán en los estudios que exploran los mecanismos que subyacen al compromiso temprano de las células TFH específicas del virus.

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 destacado: Elucidación de las vías de diferenciación celular de TFH en desafíos agudos de LCMV

Acceso a la diferenciación temprana de células T CD4+ auxiliares foliculares específicas del virus en ratones infectados con LCMV agudo

Características de las células TFH específicas del virus diferenciadas tempranamente durante la infección aguda por LCMV Para probar el compromiso del destino temprano de las células TFH específicas del virus, las células T SM CD4+ congénitas naïve que reconocen específicamente el epítopo I-ABGP GP66-77 de LCMV se transfirieron adoptivamente a receptores de CD45.2+ C57BL/6. Al día siguiente, estos receptores fueron infectado...

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

El estudio actual muestra protocolos para evaluar el compromiso del destino temprano de las células TFH específicas del virus y manipular la expresión génica en estas células.

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

477 vistas.  Shenzhen University. Nuestra investigación describe métodos reproducibles para evaluar el compromiso del destino temprano de las células TFH específicas del virus, incluido el establecimiento de un modelo de ratón monoclonal agudo infectado por LCMV, la realización de tinción por citometría de flujo y la manipulación génica basada en vectores retrovirales. En comparación con la estimulación in vitro de células T, la estimulación in vivo podría activar las células ...

Video: Autor destacado: Elucidación de las vías de diferenciación celular de TFH en desafíos agudos de LCMV

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.