We thank the staffs of flow cytometry facility, ABSL2 facility and SPF animal facility of Institut Pasteur of Shanghai for their technical help and advice. This work was supported by the following grants: Strategic Priority Research Program of the Chinese Academy of Sciences (XDB29030103), National Key R&#38;D Program of China (2016YFA0502202), the National Natural Science Foundation of China (31570886).

Animal experiments were approved by the Institutional Animal Care and Use Committee of Institut Pasteur of Shanghai, China. All the experiments were performed based on the Institutional Animal Care and Use Committee-approved animal protocols.
NOTE: Virus infection of mice and isolation of organs should be performed under ABSL2 condition.
1. Inoculation of PR8 influenza virus and recording of mice weight
<ol>
	<li>Prepare 8-week-old male C57BL/6 mi...

<ol>
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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. 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(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. 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Due to specialized roles in providing B-cell help for generating high-affinity antibodies, Tfh cells have been extensively studied in the mechanisms of differentiation and manipulation to provide new strategies for vaccine design. Influenza virus infection induces vigorous Tfh and GC B cells responses, thus being an appropriate model for this field of research. In this paper, we described protocols of influenza virus infection by intranasal inoculation, evaluation of Tfh-associated response by flow cytometry, immunofluor...

In the recent decade, numerous studies have been focused on the newly identified CD4+ T cell subset, Tfh cell subset, for its essential roles in germinal center (GC) B development. B cell lymphoma 6 (Bcl6), which is mainly considered as a gene repressor, is the lineage-defining factor of Tfh cells for the evidence that ectopic expression of Bcl6 is sufficient to drive Tfh differentiation while deficiency of Bcl6 results in vanished Tfh differentiation1,2,3. Unlike other CD4+ T helper subsets performing their effector function by migration to the sites of inflammation, Tfh cells provide the B cell help mainly in the B cell follicular zone of spleen and lymph node. Co-stimulatory molecules ICOS and CD40L, play significant roles in the interaction between Tfh and GC B cells. During Tfh differentiation, ICOS transmits necessary signals from cognate B cells and also acts as a receptor receiving migration signals from bystander B cells for B cell zone localization4,5. CD40L is a mediator of signals from Tfh cells for B cells proliferation and survival6. Another factor playing the similar role as CD40L is the cytokine IL21, which is mainly secreted by Tfh cells. IL21 directly regulates GC B cells development and production of high-affinity antibodies, but its role in Tfh differentiation is still controversial7,8. PD-1 and CXCR5, which are now most frequently used in identifying Tfh cells in flow cytometry analysis, also play significant roles in the differentiation and function of this subset. CXCR5 is the receptor of B cell follicular chemokine and mediates the localization of Tfh cells in B cell follicles9. PD-1 is now identified to not only have the follicular guidance function but also transmit critical signals in the process of GC B cells affinity maturation10. Based on these findings, evaluating the expression of these molecules could basically reflect the maturation and function of Tfh cells.
GC is an induced transient microanatomical structure in secondary lymphoid organs and highly dependent on Tfh cells, thus being a perfect readout to evaluate Tfh response. In GC, after receiving signals mediated by cytokines and co-stimulatory molecules, B cells are subject to class switching and somatic hypermutation to generate high-affinity antibodies11. Differential antibody class switching occurs in differential cytokine niche, in which IL4 and IL21 induce IgG1 class switching while IFN&#947; induces IgG2 class switching12. Plasma cells are the producers of secreted antibodies and are terminally differentiated cells. Like Tfh cells, development of B cells in GC is associated with dynamic expression of many significant molecules. Based on the current study, GC B cells can be identified as B220+PNA+Fas+ or B220+GL7+Fas+ cells and plasma cells, compared to their precursors, downregulate expression of B220 and upregulate CD138 expression13. What is more, both of these characteristics can be detected in flow cytometry and immunofluorescence analysis, thus being appropriate evaluation of GC response.
Robust cellular and humoral responses are induced in influenza virus infection, with Tfh and Th1 cells dominating CD4+ T cell response14, which makes it a perfect model for Tfh cells differentiation study. Influenza A/Puerto Rico/8/34 H1N1(PR8), which is a commonly used mouse-adapted strain, is frequently used in this study14,15,16. Here, we describe some basic protocols of Tfh study-relevant assay in influenza virus infection: 1) intranasal inoculation of PR8 virus; 2) antigen-specific Tfh cells, GC B and plasma cells and IL21 detection with flow cytometry; 3) histological visualization of GC; 4) detection of antigen-specific antibody titer in serum with ELISA. These protocols provide the necessary techniques for new researchers in Tfh-associated study.

<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

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

Characterization of mouse morbidity in influenza virus infection 
After influenza virus infection, mice are less active and anorexic due to illness, which is reflected by severe weight loss, a commonly used symptom to monitor the mouse morbidity19. As shown in Figure 1a, PR8 virus-infected mice started to lose weight on day 6, reached the highest loss level on day 8 and returned to the initial level on day 10. As expected, weight...

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

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

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

T follicular helper cells are newly identified CD4 T cells subset that provide help to B cells in high affinity animal production. Demonstrating the identification of Tfh cells in an influenza virus infection will help new researchers understand how to perform the necessary techniques for Tfh-associated studies. Because an inappropriate virus titer can cause mortality or a too weak immune response, we advise the titer to be determined before performing an experiment.For PR8 virus inoculation of eight-week-old male C57 black 6 mice. Under biosafety level 2 conditions, thaw a minus 80-degree Celsius stored PR8 virus stock on ice. Vortex the virus to obtain a homogenous solution.Dilute the virus to two platforming units per microliter of PBS in a pre-chilled 1.5-milliliter tube and confirm a lack of response toe pinch in the anesthetized animals. Vortex the virus again. Load a 20-microliter pipette tip with 10 microliters of virus.Carefully add drops of virus into one naris of up to three sedated animals before repeating the inoculation on the other side. Then, place all of the mice in sternal recumbency in warm cages with monitoring until full recovery, and weigh each mouse daily for 10 days. At the end of the experiment, place the mice into a biosafety level 2 laminar flow hood, and secure the first animal to an absorbent, paper-covered foam dissection board.Use dissection scissors to cut the skin along the abdominal midline, stretching the skin with tweezers and pinning the skin to the dissection board. Open the peritoneum to collect the spleen, and place the spleen into a 70-micrometer filter with a 6-centimeter dish containing 5 milliliters of DMEM supplemented with 1%FBS. Split the diaphragm and and ribs up to the thymus and secure the rib to the side to expose the thoracic cavity.Move the lung and heart to locate the mediastinal lymph node near the ventral side of the trachea. Then, place the lymph node in its own strainer in a second 6-centimeter dish containing 5 milliliters of medium and use 3-milliliter syringe plungers to gently mash both tissues through the mesh of both strainers. Rinse each strainer with 1 milliliter of fresh DMEM plus FBS, and transfer the cell suspensions to individual 15-milliliter centrifuge tubes.Collect the cells by centrifugation and thoroughly resuspend the pellets in 1 milliliter of fresh DMEM plus FBS per tube. Then add four additional milliliters of medium to the spleen cell suspension and place both tubes on ice. To stain the cells for CXCR5 expression, invert the tube several times to mix and transfer 2 times 10 to the 6 cells per sample into individual FACS tubes.Add 2 milliliters of staining buffer to each tube and mix the cells by vortexing. Pellet the cells by centrifugation and discard the supernatants. Gently tap each tube on absorbent paper twice and gently flick the two bottoms to resuspend the cells in the residual supernatant.Block the nonspecific binding with 0.2 microliters of Fc receptor block per tube, and flick the tube gently to mix before placing the tubes on ice for 10 minutes. At the end of the incubation, add 0.3 microliters of biotin anti-mouse CXCR5 to each tube and mix by gentle flicking. Place the tubes on ice for one hour with gentle flicking at 30 minutes.At the end of the incubation, add 2 milliliters of staining buffer to the tubes and vortex to mix before sedimenting the cells by centrifugation. Discard the supernatant and flick to resuspend the cells in the residual buffer. Then, place the tubes on ice and label the cells with antibodies against the surface marker of interest as outlined in the table as just demonstrated.At the end of the incubation, wash the cells with 2 milliliters of staining buffer and resuspend each pellet in 400 microliters of fresh staining buffer for analysis by flow cytometry. PR8 virus-infected mice begin to lose weight on day six, reaching the highest loss on day eight and returning to initial weight levels on day 10. As expected, weight loss is not observed in PBS-treated control mice.Virus infection also leads to a robust lymphocyte expansion within the draining lymph node. As observed by flow cytometric analysis, T follicular helper cell differentiation initializes at day five and peaks at day 10 post infection with robust numbers of T follicular helper cells induced in influenza virus-infected mice compared to control animals. Further, in both mediastinal lymph nodes and spleens, influenza antigen-specific CD4-positive T cell numbers are induced significantly compared to those observed in control mice with increased populations of PD-1 positive CXCR5-positive cells in PR8 animals.In addition, intracellular staining for IL-21 reveals that PR8 infection induces significantly higher production of this cytokine in response to virus infection. Immunofluorescent staining indicates the presence of an induced germinal center reaction in PR8-infected mice, and ELISA demonstrates the generation of influenza virus-specific antibodies in virus-infected animals. It is important to perform the CXCR5 staining on ice for an hour, as the quality of the stain will be affected by the staining temperature and the time.Although we demonstrated CXR5 and IL-21 staining, other surface markers, transcription factors, and cytokines can be assessed by this method to provide multitudes about Tfh differentiation and function.

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Research

JoVE Journal

Immunology and Infection

5.1K visualizações.  University of Chinese Academy of Sciences. T follicular helper cells are newly identified CD4 T cells subset that provide help to B cells in high affinity animal production. Demonstrating the identification of Tfh cells in an influenza virus infection will help new researchers understand how to perform the necessary techniques for Tfh-associated studies. Because an inappropriate virus titer can cause mortality or a too weak immune response, we advise the titer to be determined before performing an experiment.For PR8 virus inocu...