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 mice for infection at ABSL2 room. 
	NOTE: This protocol is also suitable in experiments with female mice.</li>
	<li>Dilution of PR8 virus: take out the virus from the -80 &#176;C freezer and incubate on ice until it melts into liquid. Vortex the stock virus thoroughly and dilute the virus to 2 PFU/&#181;L with sterile phosphate-buffered saline (PBS, 135 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) in a pre-chilled 1.5 mL tube.</li>
	<li>Mice anesthetization: weigh each mouse and calculate the volume (4-fold (&#181;L) the mouse weight (g)) of sodium pentobarbital (2 mg/mL) to be used. Inject the calculated volume of sodium pentobarbital intraperitoneally. 
	NOTE: This step is to make mice breathe steadily and peacefully, so that accurate titer of virus could be inoculated intranasally. Too fast or slow heartbeats indicate inappropriate anesthetization. In addition, the use of vet ointment is recommended to avoid eye dryness.</li>
	<li>Intranasal Inoculation: vortex the diluted PR8 virus thoroughly. Pipet 10 &#181;L and carefully perform intranasal inoculation on one side drop by drop. After finishing inoculation of all the mice in one cage (maximum 5 mice) on this side, repeat inoculation on the other side (keep the breathing of mouse peaceful and steady all through the inoculation). Each mouse is infected with 40 PFU of PR8 virus in total.</li>
	<li>Place the mice in sternal recumbency in warm cages for better revival.</li>
	<li>Monitor the mouse weight daily for 10 days. (The infection day is recorded as Day 0).</li>
</ol>
2. Isolation of lymphocytes from spleen and mediastinal lymph node (mLN)
<ol>
	<li>Mouse euthanization: put the mice in a small chamber and euthanize the mice by pumping into CO2 peacefully from the bottom of the chamber. Take mice out when they do not move and perform cervical dislocation to ensure mice die completely. Dip the mice with 75% ethanol and transfer to the biosafety hood.</li>
	<li>Immobilize the mice with dissection needles onto the absorbent paper-covered dissection foam plate. Cut the skin along the abdominal midline and the hind legs with dissection scissors and stretch the skin with tweezers. Immobilize the stretched skin with dissection needles.</li>
	<li>Prepare two 6 cm dishes for each mouse and keep them on the ice. Put a 70-&#956;m cell strainer in each dish and add 5 mL of DMEM supplemented with 1% fetal bovine serum (DMEM (1% FBS))</li>
	<li>Spleen isolation: cut the peritoneum to expose the abdominal cavity with dissection scissors. Take the spleen and put it in the prepared dish.</li>
	<li>mLN isolation: cut the diaphragma and the bottom of the cage rib to the vicinity of thymus. Pull the rib aside and pin it with dissection needles to expose the thoracic cavity. Pull the lung aside to the right side and use tweezers to take mLN, underneath the heart and near the ventral side of the trachea.</li>
	<li>Put the mLN in the prepared dish.</li>
	<li>Obtain the single cell suspensions: mesh the spleen or mLN gently with a plunger of 3 mL syringe through the 70-&#956;m cell strainer. Rinse the cell strainer with 1 mL of fresh DMEM (1% FBS). Resuspend the cell suspension and transfer to a 15 mL centrifuge tube.</li>
	<li>Centrifuge the cell suspension at 350 x g for 6 min at 4 &#176;C. Remove supernatant and add 1 mL of DMEM (1% FBS).</li>
	<li>Resuspend the cell pellet with 1 mL-pipette thoroughly. Add 4 mL of DMEM (1% FBS) into spleen cell suspensions and keep them on ice for the following operations. 
	NOTE: It is necessary to resuspend the cell pellet with 1 mL of medium firstly, not 5 mL, for completely isolating single cells from pellet.&#160; 
	From this step onward, all the operations can be performed in the regular lab.</li>
	<li>Spleen cell counting
	<ol>
		<li>Resuspend cells by turning the tubes up and down for several times. Take 10 &#956;L into 90 &#956;L of red blood cell (RBC) lysis buffer (10 mM Tris-HCl pH 7.5, 155mM NH4Cl). Incubate at room temperature (RT) for 3 min and add 900 &#181;L of cold PBS to terminate the reaction.</li>
		<li>Centrifuge at 400 x g for 6 min at 4 &#176;C and remove supernatant. Resuspend with 100 &#956;L of cold PBS. Take 10 &#956;L of cells into 10 &#956;L of 0.4% w/v trypan blue and take 10 &#181;L out of the mixture for cell counting with the hemocytometer.</li>
		<li>Calculation: calculate cells as regular method. In brief, count cell numbers in two diagonal corner squares on the hemocytometer and get N1, N2 for each corner square. The cell concentration of the 5-mL cell suspension should be calculated as (N1+N2)/2 x 104 /mL.</li>
	</ol>
	</li>
</ol>
3. Immunostaining of Polyclonal Tfh cells with PD-1 and CXCR5
<ol>
	<li>Staining with biotin-anti-CXCR5 antibody.
	<ol>
		<li>Resuspend cell suspensions by turning the tube up and down. Take 2 x 106 cells into the FACS tube and add 2 mL of staining buffer (PBS (1% FBS, 1 mM EDTA)). Wash by vortex on the vortex oscillation device.</li>
		<li>Centrifuge at 350 x g for 6 min at 4 &#176;C. Discard the supernatant by pouring out the liquid and dip the tube mouth on the absorbent paper twice.</li>
		<li>Loosen the cell pellet with the residue liquid by tapping the bottom of tube. Put the tube in the tube holder on ice. 
		NOTE: The volume of residue liquid is approximately 25 &#956;L.</li>
		<li>Add 0.2 &#181;L of anti-mouse CD16/CD32 (Fc-receptor blocker) for each tube. Vortex by tapping the tube bottom gently and incubate on ice for 10 min. 
		NOTE: Prepare antibody mixture for multiple samples by dilution with 5&#181;L of staining buffer for each tube. The recipe for mixture should be prepared by dilute (n/10+1) x 0.2 &#181;L Fc-receptor blocker into (n/10+1) x 5 &#181;L staining buffer and add 5.2 &#181;L mixture into each tube.&#160;&#160;</li>
		<li>Add 0.3 &#181;L of biotin-anti mouse CXCR5 into the residue 30 &#181;L of staining buffer for each tube and vortex by tapping the tube bottom. 
		NOTE: Prepare mixture as described in step 3.1.4.</li>
		<li>Incubate on ice for 1 h with gently resuspending cells by tapping the tube at 30 min. 
		NOTE: Vortex at 30 min is to avoid cell aggregates for better staining.</li>
		<li>Add 2 mL of staining buffer and vortex on the vortex oscillation device. Centrifuge at 350 x g for 6 min at 4 &#176;C and discard the supernatant as described in step 3.1.2. Vortex by tapping the tube and incubate on ice for subsequent staining.</li>
	</ol>
	</li>
	<li>Staining with other surface markers.
	<ol>
		<li>Prepare antibody mixture (Table 1) as described in step 3.1.4.</li>
		<li>Add antibody mixture into each tube. Vortex by tapping the tube bottom and incubate on ice for 30 min.</li>
		<li>Wash cells with 2 mL of staining buffer. Centrifuge at 350 x g for 6 min at 4 &#176;C.</li>
		<li>Discard the supernatant and add 400 &#181;L of staining buffer. Vortex the tube on the vortex oscillation device and keep the tube in dark till flow cytometry analysis.&#160;</li>
	</ol>
	</li>
</ol>
4. Immunostaining of PR8 influenza virus NP-specific Tfh cells
NOTE: This protocol of staining NP-specific Tfh cells is from previous studies15,17.
<ol>
	<li>Perform biotin-CXCR5 staining as described in step 3.1 except that the cell number taken for staining is 3 x 106 for enough antigen-specific cells to be recorded in flow cytometry.</li>
	<li>Add 0.3 &#181;L of APC-conjugated-IAbNP311-325 MHC class II (NP311-325) tetramer into the tube from 3.1.7. Prepare mixture for multiple samples as in step 3.1.4 
	NOTE: It is important to stain tetramer before addition of anti-CD4 antibody as the binding between CD4 and anti-CD4 antibody would interfere the optimal tetramer staining.</li>
	<li>Resuspend the cell mixture by gently tapping the tube and incubate in dark at RT for 30 min. 
	NOTE: Cover a wet paper on the mouth of tubes to decrease evaporation</li>
	<li>Add the mixture of other surface markers (Table 1) and continue incubation at RT for 30 min.</li>
	<li>Wash and resuspend cells as described in steps 3.2.3 and 3.2.4.</li>
</ol>
5. Immunostaining of Bcl6 in polyclonal Tfh cells 
<ol>
	<li>Perform surface markers (Table 2) staining as described in section 3 except that the last wash with 2 mL of PBS, instead of staining buffer.</li>
	<li>Centrifuge at 350 x g for 6 min at 4 &#176;C. Discard the supernatant and resuspend cell pellets by gently tapping the tube bottom.</li>
	<li>Add 300 &#181;L of 3.7% formaldehyde solution (diluted from 37% formaldehyde with PBS) into the tube for cell fixation. Vortex on the vortex oscillation device and incubate at RT for 20 min.</li>
	<li>Add 2 mL of staining buffer for wash and centrifuge at 500 x g for 6 min at 4 &#176;C. Discard the supernatant and resuspend cells by gently tapping the tube.</li>
	<li>Add 300 &#181;L of 0.2% Triton-X 100 and resuspend cells by vortex on the vortex oscillation device. Incubate at RT for 15 min.</li>
	<li>Add 2 mL of staining buffer for wash. Centrifuge at 500 x g for 6 min at 4 &#176;C. Discard supernatant and resuspend cells by gently tapping the tube bottom.</li>
	<li>Add 1.5 &#181;L of PE-anti-Bcl6 antibody for each tube. Gently tapping the tube bottom to resuspend the mixture and incubate at RT for 2 h with gently tapping the tube every 30 min. 
	NOTE: Cover a wet paper on the mouth of tubes to decrease mixture evaporation.</li>
	<li>Add 2 mL of PBS supplemented with 0.01% Triton-X 100 into the tube. Vortex and centrifuge at 500 x g for 6 min at 4 &#176;C.</li>
	<li>Repeat wash as step 5.8. Resuspend cells with 400 &#181;L of staining buffer. Keep cells in dark on ice till the flow cytometry analysis.</li>
</ol>
6. Intracellular staining of IL21
<ol>
	<li>Stimulate cells with PMA (phorbol 12-myristate 13-acetate) and ionomycin.
	<ol>
		<li>Take 2 x 106 cells from splenic cell suspension and centrifuge at 350 x g for 6 min at 4 &#176;C. Discard the supernatant and resuspend cell pellet with 500 &#181;L of complete T cell medium. Transfer the cells into the 24-well plate.</li>
		<li>Add 20 nmol PMA and 2 &#181;mol ionomycin into 500 &#181;L of complete medium18 and mix thoroughly by pipetting up and down.</li>
		<li>Add solution prepared in step 6.2 into cell suspension in the 24-well plate and mix by shaking the plate. Set up the unstimulated control by adding 500 &#181;L complete T cell medium without addition of PMA and ionomycin into the cells. Incubate in a&#160;CO2 incubator&#160;at&#160;37&#176;C for 4 h.</li>
		<li>Add 10 &#956;mol BFA (Brefeldin A, dissolved with methanol) into each well to block the Golgi apparatus mediated protein transport. Put the plate back to the cell incubator and incubate for 2 h.</li>
	</ol>
	</li>
	<li>Perform cell surface marker staining.
	<ol>
		<li>Resuspend cells by gently pipetting up and down and transfer the cells into a FACS tube. Add 1 mL of staining buffer into the tube and centrifuge at 350 x g for 6 min at 4 &#176;C.</li>
		<li>Perform Fc-receptor blocker staining as step 3.1.4.</li>
		<li>Perform cell surface markers staining (Table 3) as described in steps 3.2.1 to 3.2.3 except washing cells with 2 mL of PBS.</li>
		<li>Centrifuge at 350 x g for 6 min at 4 &#176;C. Discard the supernatant and resuspend cells by tapping the tube bottom.</li>
	</ol>
	</li>
	<li>Add 0.2 &#181;L of reagent from the Live/Dead Fixable Aqua Dead Cell staining kit and incubate the tube in dark at RT for 10 min to perform the staining of dead cells.</li>
	<li>Add 2 mL of PBS into the tube and vortex on the vortex oscillation device. Centrifuge at 350 x g for 6 min at 4 &#176;C and discard the supernatant.</li>
	<li>Perform the cell fixation as described in steps 5.3 and 5.4.</li>
	<li>Add 300 &#181;L of staining buffer to resuspend cells and store the tubes in the 4 &#176;C refrigerator overnight. Centrifuge at 500 x g for 6 min at 4 &#176;C to remove the supernatant. 
	NOTE: This step could be omitted and continue to step 6.7 directly following step 6.5.</li>
	<li>Add 1 mL of saponin buffer (staining buffer supplemented with 0.2%(w/v) saponin) into the tube and vortex on the vortex oscillation device. Incubate on ice for 20 min to perform cell permeabilization.</li>
	<li>Centrifuge at 500 x g for 6 min at 4 &#176;C and discard supernatant.</li>
	<li>Add 0.5 &#181;L of human Fc-IL21 receptor into each tube. Prepare antibody mixture for multiple as step 3.1.4 except that dilute antibody with saponin buffer instead of staining buffer.</li>
	<li>Incubate at RT for 1 h with gently tapping the tube bottom to resuspend cells at 30 min.</li>
	<li>Add 2 mL of saponin buffer to wash cells and centrifuge at 500 x g for 6 min. Discard supernatant and repeat wash once.</li>
	<li>Add 0.1 &#181;L of APC-anti-human Ig(H+L) into each tube. Prepare mixture for multiple samples as step 3.1.4 except that dilute antibody with saponin buffer instead of staining buffer.</li>
	<li>Incubate the samples on ice for 30 min. and wash as step 6.11.</li>
	<li>Resuspend cells with 400 &#181;L of staining buffer. Keep the sample in dark on ice till the flow cytometry analysis.</li>
</ol>
7. GC B and plasma cells staining
<ol>
	<li>Take cells and perform anti-Fc-receptor antibody staining as steps from 3.1.1 to 3.1.4.</li>
	<li>Perform surface markers staining (Table 4) as steps from 3.1.5 to 3.1.7 except that the incubation time is 30 min instead of 1 h.</li>
	<li>Resuspend cells with 400 &#181;L of staining buffer. Keep the samples in dark on ice till the flow cytometry analysis.</li>
</ol>
8. Isolation of serum from blood
<ol>
	<li>On day 14 post-infection (d.p.i 14), collect the blood from facial vein and keep blood samples in a 4 &#176;C refrigerator overnight. 
	NOTE: Perform blood collection at ABSL2 condition and from this step onward all the procedures could be performed in the regular lab.</li>
	<li>Centrifuge the blood at 400 x g for 10 min at 4 &#176;C. Isolate the serum with the 200 &#181;L pipette carefully to avoid pollution of red cells. Aliquot into 3 tubes for each sample and store them at -80&#176;C.</li>
</ol>
9. Assay of HA-specific antibody titer with ELISA
<ol>
	<li>Coat ELISA plates with 50 &#181;L of 2 &#181;g/mL HA protein solution per well and incubate them in the 4 &#176;C refrigerator overnight.</li>
	<li>Wash three times with 200 &#181;L of PBS-diluted 0.05% tween (PBST). Add 100 &#181;L of PBST-diluted 5% skimmed milk into each well and incubate at RT for 2 h to block the nonspecific binding.</li>
	<li>Serum dilution and incubation: prepare 3% BSA in PBS as the dilution buffer. Dilute the serum in dilution buffer as 1:50, 1:150, 1:450, &#8230;&#8230; to 1:36450 (3-fold serial dilution is recommended). Add 50 &#181;L of diluted serum to each well and incubate in the 4&#176;C refrigerator overnight.</li>
	<li>Discard the serum and quickly wash the wells once by adding 200 &#181;L of PBST into each well (shake it softly, then discard). Then slowly wash the plates on shaker with 200 &#181;L of PBST three times for 5 min each.</li>
	<li>Add 100 &#181;L of HRP-labeled secondary antibody specific for total IgG, IgM, IgG1, IgG2b, IgG2c (1:5000, diluted with PBST) and incubate at RT for 1 h. Wash the plates by PBST as described in 9.4.</li>
	<li>Take out equal volume of Buffer A and Buffer B (TMB) from 4&#160;&#176;C storage and warm up for at least 30 minutes at RT before use. Mix A and B and add 100 &#181;L of TMB into each well and incubate them for 10-30 minutes at RT by shaking softly. 
	NOTE: This is a brief description of the TMB Substrate Reagent Set (BD,555214) manual.</li>
	<li>Pipette 100 &#181;L of 2M H2SO4 into each well to terminate the reaction. Read the OD450 value through instrument.</li>
	<li>Data Analysis: get the final OD450 value by subtracting the background signal (OD450 value of empty well). Draw the curve corresponding to an antibody isotype of each sample with the dilution factor on the X axis and the OD450 value on the Y axis.</li>
</ol>
10. Histology
<ol>
	<li>Isolate the spleens at d.p.i 10. Fix them in 3.7% formaldehyde solution for 1 h at RT. Discard the fixation buffer and wash with PBS for 5 min on the shaker for three times.</li>
	<li>Dehydrate the spleens in PBS (10% sucrose) at 4&#176;C for 1 h and then dehydrate them in PBS (30% sucrose) at 4&#176;C with shaking softly until the spleens sink to the bottom of the 15 mL tube.</li>
	<li>Take out the dehydrated spleens, embed them in optimum cutting temperature compound and cryosectioned.</li>
	<li>Pre-chill the acetone at -20&#176;C. Incubate the tissue sections with pre-chilled acetone for 10 min. Wash the tissue with PBS for three times.</li>
	<li>Permeabilize the tissue sections with PBS containing 0.2% Triton X-100 for 20 min and wash them for three times with PBS.</li>
	<li>Block the non-specific binding with PBS containing 10% normal goat serum (blocking buffer) for 1 h at RT and wash the tissue sections with PBS once.</li>
	<li>Block the non-specific binding with STREPTAVIDIN/BIOTIN blocking kit. 
	NOTE: Do not let the samples dry from this step onward.</li>
	<li>Staining with primary antibody: add blocking buffer-diluted biotin-PNA (25 &#181;g/mL) and rat anti-mouse IgD (2.5 &#181;g/mL) onto the tissue sections carefully. Incubate the tissue sections in the wet chamber in the 4 &#176;C freezer overnight.</li>
	<li>Quickly wash the tissue sections with PBST once. Quickly wash the tissue sections in the PBST with shaking slowly for 5 min. Repeat wash for three times.</li>
	<li>Dilute Alexa Fluor 488-streptavidin (1:500) and Alexa Fluor 555-Goat-anti rat IgG (1:500) antibodies with blocking buffer and add them onto the tissue sections carefully</li>
	<li>Incubate at RT for 1 h.</li>
	<li>Wash the tissue sections as step 10.8 and carefully mount with the prolong solution. Cover the tissue with coverslips carefully and keep them in dark at 4&#176;C until confocal analysis.</li>
	<li>Analyze the magnitude of GC reaction by counting the GC numbers per area size.</li>
</ol>

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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>

The authors have nothing to disclose.

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...

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

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 ...

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Immunology and Infection

5.1K Views.  University of Chinese Academy of Sciences. This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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