Agradecemos al personal de las instalaciones de citometría de flujo, las instalaciones de ABSL2 y las instalaciones de animales SPF del Institut Pasteur de Shanghai por su ayuda técnica y asesoramiento. Este trabajo fue apoyado por las siguientes subvenciones: Programa de Investigación Estratégica Prioritaria de la Academia China de Ciencias (XDB29030103), Programa Nacional clave de I+D de China (2016YFA0502202), la Fundación Nacional de Ciencias Naturales de China (31570886).

<ol>
	<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>Pedros, C., 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=A+TRAF-like+motif+of+the+inducible+costimulator+ICOS+controls+development+of+germinal+center+TFH+cells+via+the+kinase+TBK1.">A TRAF-like motif of the inducible costimulator ICOS controls development of germinal center TFH cells via the kinase TBK1.</a> Nature Immunology. 17 (7), 825-833 (2016).</li><li>Xu, H., 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=Follicular+T-helper+cell+recruitment+governed+by+bystander+B+cells+and+ICOS-driven+motility.">Follicular T-helper cell recruitment governed by bystander B cells and ICOS-driven motility.</a> Nature. 496 (7446), 523-527 (2013).</li><li>Lee, S. K., 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=B+cell+priming+for+extrafollicular+antibody+responses+requires+Bcl-6+expression+by+T+cells.">B cell priming for extrafollicular antibody responses requires Bcl-6 expression by T cells.</a> The Journal of Experimental Medicine. 208 (7), 1377-1388 (2011).</li><li>Zotos, 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=IL-21+regulates+germinal+center+B+cell+differentiation+and+proliferation+through+a+B+cell-intrinsic+mechanism.">IL-21 regulates germinal center B cell differentiation and proliferation through a B cell-intrinsic mechanism.</a> The Journal of Experimental Medicine. 207 (2), 365-378 (2010).</li><li>Vogelzang, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fundamental+role+for+interleukin-21+in+the+generation+of+T+follicular+helper+cells.">A fundamental role for interleukin-21 in the generation of T follicular helper cells.</a> Immunity. 29 (1), 127-137 (2008).</li><li>Ansel, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemokine-driven+positive+feedback+loop+organizes+lymphoid+follicles.">A chemokine-driven positive feedback loop organizes lymphoid follicles.</a> Nature. 406 (6793), 309-314 (2000).</li><li>Shi, 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=PD-1+Controls+Follicular+T+Helper+Cell+Positioning+and+Function.">PD-1 Controls Follicular T Helper Cell Positioning and Function.</a> Immunity. 49 (2), 264-274 (2018).</li><li>Methot, S. P., Di Noia, J. M. <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+of+Somatic+Hypermutation+and+Class+Switch+Recombination.">Molecular Mechanisms of Somatic Hypermutation and Class Switch Recombination.</a> Advanced ImmunoChemical. 133, 37-87 (2017).</li><li>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=Follicular+helper+CD4+T+cells+(TFH).">Follicular helper CD4 T cells (TFH).</a> Annual Review of Immunology. 29, 621-663 (2011).</li><li>Calame, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+cells:+finding+new+light+at+the+end+of+B+cell+development.">Plasma cells: finding new light at the end of B cell development.</a> Nature Immunology. 2 (12), 1103-1108 (2001).</li><li>Yoo, J. K., Fish, E. N., Braciale, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LAPCs+promote+follicular+helper+T+cell+differentiation+of+Ag-primed+CD4++T+cells+during+respiratory+virus+infection.">LAPCs promote follicular helper T cell differentiation of Ag-primed CD4+ T cells during respiratory virus infection.</a> The Journal of Experimental Medicine. 209 (10), 1853-1867 (2012).</li><li>Leon, B., Bradley, J. E., Lund, F. E., Randall, T. D., Ballesteros-Tato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxP3++regulatory+T+cells+promote+influenza-specific+Tfh+responses+by+controlling+IL-2+availability.">FoxP3+ regulatory T cells promote influenza-specific Tfh responses by controlling IL-2 availability.</a> Nature Communications. 5, 3495 (2014).</li><li>He, 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=Extracellular+matrix+protein+1+promotes+follicular+helper+T+cell+differentiation+and+antibody+production.">Extracellular matrix protein 1 promotes follicular helper T cell differentiation and antibody production.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (34), 8621-8626 (2018).</li><li>Le&#243;n, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+TH2+development+by+CXCR5++dendritic+cells+and+lymphotoxin-expressing+B+cells.">Regulation of TH2 development by CXCR5+ dendritic cells and lymphotoxin-expressing B cells.</a> Nature Immunology. 13 (7), 681-690 (2012).</li><li>Wang, H., 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+Foxp1+is+a+critical+negative+regulator+of+the+differentiation+of+follicular+helper+T+cells.">The transcription factor Foxp1 is a critical negative regulator of the differentiation of follicular helper T cells.</a> Nature Immunology. 15 (7), 667-675 (2014).</li><li>Bouvier, N. M., Lowen, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+for+Influenza+Virus+Pathogenesis+and+Transmission.">Animal Models for Influenza Virus Pathogenesis and Transmission.</a> Viruses. 2 (8), 1530-1563 (2010).</li><li>Miyauchi, K., 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=Protective+neutralizing+influenza+antibody+response+in+the+absence+of+T+follicular+helper+cells.">Protective neutralizing influenza antibody response in the absence of T follicular helper cells.</a> Nature Immunology. 17 (12), 1447-1458 (2016).</li><li>Rodriguez, L., Nogales, A., Martinez-Sobrido, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+A+Virus+Studies+in+a+Mouse+Model+of+Infection.">Influenza A Virus Studies in a Mouse Model of Infection.</a> Journal of Visualized Experiments. (127), (2017).</li><li>Kitano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl6+protein+expression+shapes+pre-germinal+center+B+cell+dynamics+and+follicular+helper+T+cell+heterogeneity.">Bcl6 protein expression shapes pre-germinal center B cell dynamics and follicular helper T cell heterogeneity.</a> Immunity. 34 (6), 961-972 (2011).</li><li>Yusuf, 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=Germinal+center+T+follicular+helper+cell+IL-4+production+is+dependent+on+signaling+lymphocytic+activation+molecule+receptor+(CD150).">Germinal center T follicular helper cell IL-4 production is dependent on signaling lymphocytic activation molecule receptor (CD150).</a> The Journal of Immunology. 185 (1), 190-202 (2010).</li><li>Sun, J., Dodd, H., Moser, E. K., Sharma, R., Braciale, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++T+cell+help+and+innate-derived+IL-27+induce+Blimp-1-dependent+IL-10+production+by+antiviral+CTLs.">CD4+ T cell help and innate-derived IL-27 induce Blimp-1-dependent IL-10 production by antiviral CTLs.</a> Nature Immunology. 12 (4), 327-334 (2011).</li></ol>

Los autores no tienen nada que revelar.

Debido a los roles especializados en proporcionar ayuda de células B para generar anticuerpos de alta afinidad, las células Tfh han sido ampliamente estudiadas en los mecanismos de diferenciación y manipulación para proporcionar nuevas estrategias para el diseño de vacunas. La infección por el virus de la gripe induce vigorosas respuestas de las células Tfh y GC B, siendo así un modelo adecuado para este campo de investigación. En este documento, describimos protocolos de infección por el virus de la gripe por ...

En la última década, numerosos estudios se han centrado en el subconjunto de células CD4+ T recientemente identificado, subconjunto de células Tfh, por sus funciones esenciales en el desarrollo del centro germinal (GC) B. El linfoma de células B 6 (Bcl6), que se considera principalmente como represor genético, es el factor definitorio del linaje de las células Tfh para la evidencia de que la expresión ectopic de Bcl6 es suficiente para impulsar la diferenciación tfh, mientras que la deficiencia de Bcl6 resulta en la diferenciación tfh desaparecida1,2,3. A diferencia de otros subconjuntos auxiliares CD4+ T que realizan su función de efector mediante la migración a los sitios de inflamación, las células Tfh proporcionan la ayuda celular B principalmente en la zona folicular de células B de bazo y ganglio linfático. Las moléculas co-estimulantes ICOS y CD40L, desempeñan un papel significativo en la interacción entre las células Tfh y GC B. Durante la diferenciación tfh, ICOS transmite las señales necesarias de las células B del cognato y también actúa como un receptor que recibe señales de migración de las células B del transeúnte para la localización de la zona celular B4,5. CD40L es un mediador de señales de células Tfh para la proliferación de células B y supervivencia6. Otro factor que juega el papel similar a CD40L es la citoquina IL21, que es secreta principalmente por las células Tfh. IL21 regula directamente el desarrollo de células GC B y la producción de anticuerpos de alta afinidad, pero su papel en la diferenciación de Tfh sigue siendo controvertido7,8. PD-1 y CXCR5, que ahora se utilizan con mayor frecuencia en la identificación de células Tfh en el análisis de citometría de flujo, también desempeñan un papel significativo en la diferenciación y función de este subconjunto. CXCR5 es el receptor de la quimiocina folicular de células B y media la localización de células Tfh en folículos celulares B9. Pd-1 ahora se identifica no sólo para tener la función de orientación folicular, sino también transmitir señales críticas en el proceso de maduración de afinidad de células GC B10. Sobre la base de estos hallazgos, evaluar la expresión de estas moléculas podría básicamente reflejar la maduración y la función de las células Tfh.
El GC es una estructura microanatomical transitoria inducida en órganos linfoides secundarios y altamente dependiente de las células Tfh, siendo así una lectura perfecta para evaluar la respuesta de Tfh. En GC, después de recibir señales mediadas por citoquinas y moléculas co-estimulantes, las células B están sujetas a conmutación de clases e hipermutación somática para generar anticuerpos de alta afinidad11. La conmutación diferencial de la clase de anticuerpos se produce en el nicho diferencial de citoquinas, en el que IL4 e IL21 inducen la conmutación de la clase IgG1 mientras que el IFNḥ induce el cambio de clase IgG212. Las células plasmáticas son los productores de anticuerpos secretos y son células diferenciadas terminalmente. Al igual que las células Tfh, el desarrollo de células B en GC se asocia con la expresión dinámica de muchas moléculas significativas. Según el estudio actual, las células GC B se pueden identificar como B220+PNA+Fas+ o B220+GL7+Fas+ células y células plasmáticas, en comparación con sus precursores, expresión downregulate de B220 y cd138 upregulate expresión133. Además, ambas características se pueden detectar en el análisis de citometría de flujo e inmunofluorescencia, siendo así una evaluación adecuada de la respuesta de GC.
Respuestas celulares y humorales robustas son inducidas en la infección por el virus de la gripe, con células Tfh y Th1 dominando CD4+ T respuesta celular14,lo que lo convierte en un modelo perfecto para el estudio de diferenciación de células Tfh. Influenza A/Puerto Rico/8/34 H1N1(PR8), que es una cepa adaptada al ratón de uso común, se utiliza con frecuencia en este estudio14,15,16. Aquí, describimos algunos protocolos básicos del ensayo relevante para el estudio de Tfh en la infección por el virus de la gripe: 1) inoculación intranasal del virus PR8; 2) células Tfh específicas de antígenos, células GC B y plasma y detección IL21 con citometría de flujo; 3) visualización histológica de GC; 4) detección de titer de anticuerpos específicos de antígenos en suero con ELISA. Estos protocolos proporcionan las técnicas necesarias para nuevos investigadores en el estudio asociado a Tfh.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Immunostaining of Tfh cells, NP-specific Tfh cells and Bcl-6</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37% formaldehyde</td>
			<td>Sigma</td>
			<td>F1635</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD16/32 mouse</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-0161-86</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-conjugated-IAbNP311-325 MHC class II tetramer</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bcl-6 PE</td>
			<td>Biolegend</td>
			<td>358504</td>
			<td>clone:7D1</td>
		</tr>
		<tr>
			<td>Biotin-CXCR5</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-7185-82</td>
			<td>clone: SPRCL5</td>
		</tr>
		<tr>
			<td>CD4 Percp-eFluor 710</td>
			<td>Thermo Fisher Scientific</td>
			<td>46-0041-82</td>
			<td>clone:GK1.5</td>
		</tr>
		<tr>
			<td>CD44 eVolve 605</td>
			<td>Thermo Fisher Scientifi</td>
			<td>83-0441-42</td>
			<td>clone:IM7</td>
		</tr>
		<tr>
			<td>CD44 FITC</td>
			<td>Thermo Fisher Scientifi</td>
			<td>11-0441-82</td>
			<td>clone:IM7</td>
		</tr>
		<tr>
			<td>CD62L FITC</td>
			<td>BD Pharmingen</td>
			<td>553150</td>
			<td>clone:MEL-14</td>
		</tr>
		<tr>
			<td>ICOS BV421</td>
			<td>Biolegend</td>
			<td>564070</td>
			<td>clone:7E.17G9</td>
		</tr>
		<tr>
			<td>PD1 PE/Cy7</td>
			<td>Biolegend</td>
			<td>135216</td>
			<td>clone:29F.1A12</td>
		</tr>
		<tr>
			<td>Streptavidin BV421</td>
			<td>BD Pharmingen</td>
			<td>563259</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin PE</td>
			<td>BD Pharmingen</td>
			<td>554081</td>
			<td></td>
		</tr>
		<tr>
			<td>Intracelluar staining of IL21</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37% formaldehyde</td>
			<td>Sigma</td>
			<td>F1635</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human IgG</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>109-605-098</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>Sigma</td>
			<td>B6542</td>
			<td></td>
		</tr>
		<tr>
			<td>human FCc IL-21 receptor</td>
			<td>R&#38;D System</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ionomycin</td>
			<td>Sigma</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead Fixable Aqua Dead Cell staining kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>L34966</td>
			<td></td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Sigma</td>
			<td>P1585</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>MP</td>
			<td>102855</td>
			<td></td>
		</tr>
		<tr>
			<td>GC B and plasma cells staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B220 APC</td>
			<td>Thermo Fisher Scientific</td>
			<td>17-0452-81</td>
			<td>clone:RA3-6B2</td>
		</tr>
		<tr>
			<td>CD138 PE</td>
			<td>BD Pharmingen</td>
			<td>561070</td>
			<td>clone:281-2</td>
		</tr>
		<tr>
			<td>CD95 (FAS) PE/Cy7</td>
			<td>BD Pharmingen</td>
			<td>557653</td>
			<td>clone:Jo2</td>
		</tr>
		<tr>
			<td>IgD eFluor 450</td>
			<td>Thermo Fisher Scientific</td>
			<td>48-5993-82</td>
			<td>clone:11-26c</td>
		</tr>
		<tr>
			<td>PNA FITC</td>
			<td>Sigma</td>
			<td>L7381</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay of HA-specific antibody titer with ELISA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PR8-HA</td>
			<td>Sino Biological</td>
			<td>11684-V08H</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>SSBC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti mouse Ig (SBA Clonotyping System-HRP)</td>
			<td>SouthernBiotech</td>
			<td>5300-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti mouse IgM (SBA Clonotyping System-HRP)</td>
			<td>SouthernBiotech</td>
			<td>5300-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti mouse IgG1 (SBA Clonotyping System-HRP)</td>
			<td>SouthernBiotech</td>
			<td>5300-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti mouse IgG2b (SBA Clonotyping System-HRP)</td>
			<td>SouthernBiotech</td>
			<td>5300-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti mouse IgG2c (SBA Clonotyping System-HRP)</td>
			<td>SouthernBiotech</td>
			<td>5300-05</td>
			<td></td>
		</tr>
		<tr>
			<td>TMB Substrate Reagent Set</td>
			<td>BD Pharmingen</td>
			<td>555214</td>
			<td></td>
		</tr>
		<tr>
			<td>Histology</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 555-Goat-anti rat IgG</td>
			<td>Life Technology</td>
			<td>A21434</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse IgD</td>
			<td>Biolegend</td>
			<td>405702</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated PNA</td>
			<td>Vector laboratories</td>
			<td>B-1075</td>
			<td></td>
		</tr>
		<tr>
			<td>dilute Alexa Fluor 488-streptavidin</td>
			<td>Life Technology</td>
			<td>S11223</td>
			<td></td>
		</tr>
		<tr>
			<td>normal goat serum</td>
			<td>SouthernBiotech</td>
			<td>0060-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Pro-long gold antifade reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>P3630</td>
			<td></td>
		</tr>
		<tr>
			<td>STREPTAVIDIN/BIOTIN blocking kit</td>
			<td>Vector laboratories</td>
			<td>SP-2002</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of t follicular helper cells and germinal center response during influenza a virus infection in mice

Las células T Follicular Helper (Tfh) son un subconjunto independiente de células CD4+ T especializado en proporcionar ayuda para el desarrollo del centro germinal (GC) y la generación de anticuerpos de alta afinidad. En la infección por el virus de la gripe, se inducen respuestas sólidas de células Tfh y GC B para facilitar la erradicación eficaz del virus, lo que confiere un modelo de ratón cualificado para el estudio asociado a Tfh. En este artículo, describimos protocolos en la detección de la respuesta inmune básica asociada a Tfh durante la infección por el virus de la gripe en ratones. Estos protocolos incluyen: inoculación intranasal del virus de la gripe; flujo de tinción de citometría y análisis de células Tfh policlonales y específicas de antígenos, células GC B y células plasmáticas; detección de inmunofluorescencia de GCs; ensayo inmunosorbente vinculado a enzimas (ELISA) de anticuerpos específicos del virus de la gripe en suero. Estos ensayos básicamente cuantifican la diferenciación y la función de las células Tfh en la infección por el virus de la gripe, proporcionando así ayuda para estudios sobre el mecanismo de diferenciación y estrategia de manipulación.

Caracterización de la morbilidad del ratón en la infección por el virus de la gripe Después de la infección por el virus de la gripe, los ratones son menos activos y anoréxicos debido a una enfermedad, que se refleja en la pérdida de peso grave, un síntoma comúnmente utilizado para controlar la morbilidad del ratón19. Como se muestra en la Figura 1a , ratones infectados por el virus PR8 comenzaron a perder peso en el dí...

Watch this Scientific Journal Video about Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice at JoVE.com

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

Este documento describe los protocolos de evaluación de la respuesta Tfh y GC B en el modelo de ratón de infección por el virus de la gripe.

Evaluación de las células auxiliares foliculares T y la respuesta del centro germinal durante la infección por el virus de la gripe A en ratones

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

evaluation-t-follicular-helper-cells-germinal-center-response-during

Research

JoVE Journal

Immunology and Infection

5.1K vistas.  University of Chinese Academy of Sciences. Este documento describe los protocolos de evaluación de la respuesta Tfh y GC B en el modelo de ratón de infección por el virus de la gripe.

Video: Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Utilizando imágenes bioluminiscentes encargado de investigar la sinergia entre Streptococcus pneumoniae Y el Virus de la Influenza en ratones lactantes

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Investigación

Inmunología e Infección

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

El análisis in vitro de la respuesta inmune celular Myd88 mediada por el Virus del Nilo Occidental cepa mutante Infección

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Propagación del virus de la influenza en embrionados de pollo Huevos

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

Medición de apego y Internalización del Virus de la Influenza en células A549 por Citometría de Flujo

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Aislamiento de neutrófilos y análisis para determinar su papel en el linfoma de células sensibles a los agentes terapéuticos

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

In Vitro El desmontaje de virus de influenza A cápsides por centrifugación en gradiente

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Estudios de Virus de influenza A en un modelo murino de infección

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Evaluación de respuestas de células T Zika Virus-específica en Immunoprivileged órganos de ratones- / - Ifnar1 infectados

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

Proyección de imagen de interacción de células en Mucosa traqueal durante la infección del Virus de la Influenza usando microscopia Intravital de dos fotones

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

Un virus de la gripe Reportero fluorescente de Luciferase para imágenes en vivo y cuantificación de la infección viral

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...