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

Summary

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

Abstract

T Follicular Helper (Tfh) cells are an independent CD4⁺ 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.

Introduction

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 differentiation^1,2,3. 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 localization^4,5. 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 controversial^7,8. 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 IFNγ 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 study^14,15,16. 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.

Protocol

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

1. Prepare 8-week-old male C57BL/6 mice for infection at ABSL2 room.
   NOTE: This protocol is also suitable in experiments with female mice.
2. Dilution of PR8 virus: take out the virus from the -80 °C freezer and incubate on ice until it melts into liquid. Vortex the stock virus thoroughly and dilute the virus to 2 PFU/µL 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.
3. Mice anesthetization: weigh each mouse and calculate the volume (4-fold (µL) 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.
4. Intranasal Inoculation: vortex the diluted PR8 virus thoroughly. Pipet 10 µL 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.
5. Place the mice in sternal recumbency in warm cages for better revival.
6. Monitor the mouse weight daily for 10 days. (The infection day is recorded as Day 0).

2. Isolation of lymphocytes from spleen and mediastinal lymph node (mLN)

1. 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.
2. 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.
3. Prepare two 6 cm dishes for each mouse and keep them on the ice. Put a 70-μm cell strainer in each dish and add 5 mL of DMEM supplemented with 1% fetal bovine serum (DMEM (1% FBS))
4. Spleen isolation: cut the peritoneum to expose the abdominal cavity with dissection scissors. Take the spleen and put it in the prepared dish.
5. 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.
6. Put the mLN in the prepared dish.
7. Obtain the single cell suspensions: mesh the spleen or mLN gently with a plunger of 3 mL syringe through the 70-μm 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.
8. Centrifuge the cell suspension at 350 x g for 6 min at 4 °C. Remove supernatant and add 1 mL of DMEM (1% FBS).
9. 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. 
   From this step onward, all the operations can be performed in the regular lab.
10. Spleen cell counting
    1. Resuspend cells by turning the tubes up and down for several times. Take 10 μL into 90 μL 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 µL of cold PBS to terminate the reaction.
    2. Centrifuge at 400 x g for 6 min at 4 °C and remove supernatant. Resuspend with 100 μL of cold PBS. Take 10 μL of cells into 10 μL of 0.4% w/v trypan blue and take 10 µL out of the mixture for cell counting with the hemocytometer.
    3. 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.

3. Immunostaining of Polyclonal Tfh cells with PD-1 and CXCR5

1. Staining with biotin-anti-CXCR5 antibody.
   1. 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.
   2. Centrifuge at 350 x g for 6 min at 4 °C. Discard the supernatant by pouring out the liquid and dip the tube mouth on the absorbent paper twice.
   3. 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 μL.
   4. Add 0.2 µL 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 5µL of staining buffer for each tube. The recipe for mixture should be prepared by dilute (n/10+1) x 0.2 µL Fc-receptor blocker into (n/10+1) x 5 µL staining buffer and add 5.2 µL mixture into each tube.  
   5. Add 0.3 µL of biotin-anti mouse CXCR5 into the residue 30 µL of staining buffer for each tube and vortex by tapping the tube bottom.
      NOTE: Prepare mixture as described in step 3.1.4.
   6. 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.
   7. Add 2 mL of staining buffer and vortex on the vortex oscillation device. Centrifuge at 350 x g for 6 min at 4 °C and discard the supernatant as described in step 3.1.2. Vortex by tapping the tube and incubate on ice for subsequent staining.
2. Staining with other surface markers.
   1. Prepare antibody mixture (Table 1) as described in step 3.1.4.
   2. Add antibody mixture into each tube. Vortex by tapping the tube bottom and incubate on ice for 30 min.
   3. Wash cells with 2 mL of staining buffer. Centrifuge at 350 x g for 6 min at 4 °C.
   4. Discard the supernatant and add 400 µL of staining buffer. Vortex the tube on the vortex oscillation device and keep the tube in dark till flow cytometry analysis. 

4. Immunostaining of PR8 influenza virus NP-specific Tfh cells

NOTE: This protocol of staining NP-specific Tfh cells is from previous studies^15,17.

1. 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.
2. Add 0.3 µL of APC-conjugated-IAbNP311-325 MHC class II (NP_311-325) 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.
3. 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
4. Add the mixture of other surface markers (Table 1) and continue incubation at RT for 30 min.
5. Wash and resuspend cells as described in steps 3.2.3 and 3.2.4.

5. Immunostaining of Bcl6 in polyclonal Tfh cells

1. 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.
2. Centrifuge at 350 x g for 6 min at 4 °C. Discard the supernatant and resuspend cell pellets by gently tapping the tube bottom.
3. Add 300 µL 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.
4. Add 2 mL of staining buffer for wash and centrifuge at 500 x g for 6 min at 4 °C. Discard the supernatant and resuspend cells by gently tapping the tube.
5. Add 300 µL of 0.2% Triton-X 100 and resuspend cells by vortex on the vortex oscillation device. Incubate at RT for 15 min.
6. Add 2 mL of staining buffer for wash. Centrifuge at 500 x g for 6 min at 4 °C. Discard supernatant and resuspend cells by gently tapping the tube bottom.
7. Add 1.5 µL 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.
8. 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 °C.
9. Repeat wash as step 5.8. Resuspend cells with 400 µL of staining buffer. Keep cells in dark on ice till the flow cytometry analysis.

6. Intracellular staining of IL21

1. Stimulate cells with PMA (phorbol 12-myristate 13-acetate) and ionomycin.
   1. Take 2 x 10⁶ cells from splenic cell suspension and centrifuge at 350 x g for 6 min at 4 °C. Discard the supernatant and resuspend cell pellet with 500 µL of complete T cell medium. Transfer the cells into the 24-well plate.
   2. Add 20 nmol PMA and 2 µmol ionomycin into 500 µL of complete medium¹⁸ and mix thoroughly by pipetting up and down.
   3. 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 µL complete T cell medium without addition of PMA and ionomycin into the cells. Incubate in a CO2 incubator at 37°C for 4 h.
   4. Add 10 μmol 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.
2. Perform cell surface marker staining.
   1. 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 °C.
   2. Perform Fc-receptor blocker staining as step 3.1.4.
   3. 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.
   4. Centrifuge at 350 x g for 6 min at 4 °C. Discard the supernatant and resuspend cells by tapping the tube bottom.
3. Add 0.2 µL 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.
4. 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 °C and discard the supernatant.
5. Perform the cell fixation as described in steps 5.3 and 5.4.
6. Add 300 µL of staining buffer to resuspend cells and store the tubes in the 4 °C refrigerator overnight. Centrifuge at 500 x g for 6 min at 4 °C to remove the supernatant.
   NOTE: This step could be omitted and continue to step 6.7 directly following step 6.5.
7. 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.
8. Centrifuge at 500 x g for 6 min at 4 °C and discard supernatant.
9. Add 0.5 µL 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.
10. Incubate at RT for 1 h with gently tapping the tube bottom to resuspend cells at 30 min.
11. Add 2 mL of saponin buffer to wash cells and centrifuge at 500 x g for 6 min. Discard supernatant and repeat wash once.
12. Add 0.1 µL 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.
13. Incubate the samples on ice for 30 min. and wash as step 6.11.
14. Resuspend cells with 400 µL of staining buffer. Keep the sample in dark on ice till the flow cytometry analysis.

7. GC B and plasma cells staining

1. Take cells and perform anti-Fc-receptor antibody staining as steps from 3.1.1 to 3.1.4.
2. 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.
3. Resuspend cells with 400 µL of staining buffer. Keep the samples in dark on ice till the flow cytometry analysis.

8. Isolation of serum from blood

1. On day 14 post-infection (d.p.i 14), collect the blood from facial vein and keep blood samples in a 4 °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.
2. Centrifuge the blood at 400 x g for 10 min at 4 °C. Isolate the serum with the 200 µL pipette carefully to avoid pollution of red cells. Aliquot into 3 tubes for each sample and store them at -80°C.

9. Assay of HA-specific antibody titer with ELISA

1. Coat ELISA plates with 50 µL of 2 µg/mL HA protein solution per well and incubate them in the 4 °C refrigerator overnight.
2. Wash three times with 200 µL of PBS-diluted 0.05% tween (PBST). Add 100 µL of PBST-diluted 5% skimmed milk into each well and incubate at RT for 2 h to block the nonspecific binding.
3. 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, …… to 1:36450 (3-fold serial dilution is recommended). Add 50 µL of diluted serum to each well and incubate in the 4°C refrigerator overnight.
4. Discard the serum and quickly wash the wells once by adding 200 µL of PBST into each well (shake it softly, then discard). Then slowly wash the plates on shaker with 200 µL of PBST three times for 5 min each.
5. Add 100 µL 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.
6. Take out equal volume of Buffer A and Buffer B (TMB) from 4 °C storage and warm up for at least 30 minutes at RT before use. Mix A and B and add 100 µL 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.
7. Pipette 100 µL of 2M H₂SO₄ into each well to terminate the reaction. Read the OD450 value through instrument.
8. 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.

10. Histology

1. 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.
2. Dehydrate the spleens in PBS (10% sucrose) at 4°C for 1 h and then dehydrate them in PBS (30% sucrose) at 4°C with shaking softly until the spleens sink to the bottom of the 15 mL tube.
3. Take out the dehydrated spleens, embed them in optimum cutting temperature compound and cryosectioned.
4. Pre-chill the acetone at -20°C. Incubate the tissue sections with pre-chilled acetone for 10 min. Wash the tissue with PBS for three times.
5. Permeabilize the tissue sections with PBS containing 0.2% Triton X-100 for 20 min and wash them for three times with PBS.
6. 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.
7. Block the non-specific binding with STREPTAVIDIN/BIOTIN blocking kit.
   NOTE: Do not let the samples dry from this step onward.
8. Staining with primary antibody: add blocking buffer-diluted biotin-PNA (25 µg/mL) and rat anti-mouse IgD (2.5 µg/mL) onto the tissue sections carefully. Incubate the tissue sections in the wet chamber in the 4 °C freezer overnight.
9. 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.
10. 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
11. Incubate at RT for 1 h.
12. 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°C until confocal analysis.
13. Analyze the magnitude of GC reaction by counting the GC numbers per area size.

Representative Results

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 loss was not observed all through the period in PBS-treated control mice. For in vivo symptoms, virus infection leads to robust lymphocytes expansion in the draining lymph node, mLN in this case. Therefore, significantly larger size of mLNs were observed in PR8 virus-infected mice than in control mice (Figure 1b). Taken together, these mice all showed expected symptoms and were qualified for the subsequent Tfh-associated immune response study.

Detection of Tfh differentiation and function-associated molecules
To analyze Tfh differentiation, mice were sacrificed on day 5, 7, 10 and 14 after infection and mLNs or spleens were isolated for flow cytometry analysis. Figure 2a and Figure 2b show the Tfh population gating strategy, with Tfh gated as PD-1^hi CXCR5^hi cells and non-Tfh as PD-1^lowCXCR5^low cells. With this gating strategy, the kinetics of Tfh differentiation during influenza virus infection were assayed. As shown in Figure 2c, Tfh differentiation initialized at day 5 and peaked at day 10. So we took samples of day 10 for further analysis. As shown in Figure 3a, robust Tfh cell differentiation was induced in influenza virus-infected mice compared with control mice. To analyze Influenza virus-specific Tfh cells, fluorochrome-labeled IAbNP311–325 MHC class II tetramers (NP_311-325) were added in the polyclonal Tfh cells staining panel (Table 1). Both in mLNs and spleens from influenza virus-infected mice, NP_311-325-specific CD4⁺ T cells were significantly induced and NP_311-325-specific Tfh cells could be analyzed by addition of PD-1 and CXCR5 into analysis(Figure 3e). Because of essential roles of Bcl6 in Tfh differentiation, Bcl6⁺CXCR5⁺ cells can also represent the Tfh population. Consistently, Tfh cells identified with this strategy were also induced robustly (Figure 3b). We further analyzed expression of Bcl6 in Tfh and non-Tfh cells. As shown in Figure 3c, higher expression of Bcl6 in Tfh cells than that in non-Tfh cells indicates successful Bcl6 staining. With similar strategy, ICOS, another Tfh-associated molecule was also analyzed (Figure 3d). Due to the specialized role of Tfh cells in providing help for B cells, assay of the expression of IL21, which is secreted mainly by Tfh cells and demonstrated to directly regulate B cells survival and proliferation, could reveal Tfh cells function to some extent. As shown in Figure 3f, intracellular staining of IL21 revealed that PR8 infection induced significantly higher production of this cytokine, with unstimulated cells as gating control. Taken together, these assays could reflect basic information of Tfh differentiation and provide the insights into the B cell-help ability.

Detection of GC B and plasma cells development and influenza virus-specific antibodies in serum
The main function of Tfh cells is to provide B cell help in GCs, in which antibody class switching and affinity maturation occur. So GC B development could indirectly reflect differentiation and function of Tfh cells. GC B cells could be gated as B220⁺PNA⁺Fas⁺ cells (Figure 2d). Through this gating strategy, we assayed the kinetics of GC B cell response and found that GC B response started at day 10 and continued to increase at day 14 (Figure 2e). Comparison between PR8 virus-infected and control mice showed robust GC B were induced both in mLN and spleen after influenza virus infection(Figure 4a), which is consistent with the induced Tfh differentiation in PRB virus-infected mice. In addition, Immunofluorescence staining with IgD and PNA provides visualized images indicating induced GC reaction (green areas) in PR8 virus-infected mice (Figure 4d). Plasma cells, identified as IgD^lowCD138⁺ cells(Figure 2d), were also generated in PR8 virus-infected mice (Figure 4b). Previous studies have identified that IFNƳ and IL21 could be secreted from both Th1 and Tfh cells in virus infection and induce IgG2 and IgG1 class switching, respectively²⁰. Figure 4c depicts the generation of influenza virus-specific antibody by ELISA assay of HA-specific IgM, total IgG, IgG1, IgG2b and IgG2C. Together, all of these assays reflect the Tfh-associated B cell responses in influenza virus infection.

Figure 1: Characterization of mouse morbidity. 8-week-old male mice were infected with 40 PFU of PR8 influenza virus by intranasal inoculation. Mice were weighed daily for 10 days (a) and mLNs were isolated on d.p.i 10 (b). The error bars in (a) represent the mean ± SD. n = 4 mice per group. Please click here to view a larger version of this figure.

Figure 2: Gating strategy of Tfh cells and GC B cells. (a) Lymphocytes are defined by FSC-A and SSC-A, and cell singlets are gated with FSC-A, FSC-H and SSC-A, SSC-W. (b) After gating in CD4⁺ T cells, surface markers CD62L and CD44 are used to distinguish the naïve T cells (CD44^loCD62L^hi) and activated T cells (CD44^hiCD62L^lo). Polyclonal Tfh cells can be gated from activated T cells as PD-1^hi CXCR5^hi population, conversely, non-Tfh cells as PD-1^lowCXCR5^low. PR8 virus-specific Tfh cells are defined as CD4⁺CD44⁺ NP_311-325 tetramer⁺PD-1^hi CXCR5^hi cells. (c,e) Kinetics of Tfh frequency in activated cells (c) and GC B frequency in B220⁺ cells (e). (d) GC B cells are gated as B220⁺ PNA⁺FAS⁺ cells, and plasma cells are IgD^-CD138⁺ cells. Please click here to view a larger version of this figure.

Figure 3: Analysis of Tfh differentiation in PR8 virus-infected mice. Mice were sacrificed on d.p.i 10 and mLNs and spleens were isolated for Tfh differentiation analysis. (a) Tfh percentage in mLNs and spleens in PR8 virus-infected mice and PBS-treated mice (upper panel). The statistics of Tfh cells (lower panel). (b) The intracellular staining of Bcl6 in CD4⁺CD44^hi T cells (upper panel). The statistics of Bcl6⁺CXCR5⁺ cells (lower panel). (c) Bcl6 and (d) ICOS expression in Tfh (line-red) and non-Tfh cells (solid-gray). (e) Gating of NP_311-325-specific CD4⁺ T cells in mLNs and spleens of PR8 virus-infected and PBS-treated mice (left panel). The percentage of PR8 virus-specific Tfh cells in mLNs and spleens (middle panel). “Isotype” indicates staining with irrelevant tetramer control. The statistics of NP_311-325-specific CD4⁺ T cells (right panel). (f) Intracellular staining of IL-21 in splenic CD4⁺ T cells from PR8 virus-infected and PBS-treated mice, the unstimulation shown as control (left). The statistics of IL-21 staining (right). **P < 0.01, ***P < 0.001 and **** P < 0.0001 (two-tailed Student’s t-test). The error bars represent the mean ± SD. n = 3 mice per group. Please click here to view a larger version of this figure.

Figure 4: Analysis of GC B cell-associated response in PR8 virus-infected mice. Mice were sacrificed on d.p.i 10 and the mLNs and spleens were isolated for analysis. (a) The percentage of GC B cells (upper panel). The statistics of GC B cells (lower panel). (b) The percentage of plasma cells (upper panel). The statistics of plasma cells (lower panel). (c) Quantification of PR8 virus HA-specific IgG, IgM, IgG1, IgG2b and IgG2c in the serum (d.p.i 14) of PR8 virus-infected mice and PBS-treated mice. (d) Confocal microscopy of B cell follicles (IgD⁺, Red) and GCs (PNA⁺, Green) in the spleen samples of PR8 virus-infected mice and PBS-treated mice (d.p.i 10). *P < 0.5, **P < 0.01, and ***P < 0.001 (two-tailed Student's t-test). The error bars represent the mean ± SD. n = 3 mice per group. Please click here to view a larger version of this figure.

surface marker	fluorochrome	clone	volume per sample(ul)
CD4	Percp-eFluor 710	GK1.5	0.2
CD44	eVolve 605	IM7	0.2
CD62L	FITC	MEL-14	0.2
ICOS	BV421	7E.17G9	0.2
PD1	PE/Cy7	29F.1A12	0.3
Streptavidin	PE		0.2

Table 1: Surface marker (except for CXCR5) antibodies panel for staining Tfh cells (PD-1^hiCXCR5^hi).

surface marker	fluorochrome	clone	volume per sample(ul)
CD4	Percp-eFluor 710	GK1.5	0.2
CD44	FITC	IM7	0.2
PD1	PE/Cy7	29F.1A12	0.3
Streptavidin	BV421		0.5

Table 2: Surface marker antibodies (except for CXCR5) panel for staining Bcl6 in Tfh cells.

surface marker	fluorochrome	clone	volume per sample(ul)
CD4	Percp-eFluor 710	GK1.5	0.2
CD44	FITC	IM7	0.2

Table 3: Surface marker antibodies panel for intracellular staining of IL21.

surface marker	fluorochrome	clone	volume per sample(ul)
B220	APC	RA3-6B2	0.2
IgD	eFluor 450	11-26c	0.2
CD95	PE/Cy7	Jo2	0.3
PNA	FITC		0.3
CD138	PE	281-2	0.2

Table 4: Surface marker antibodies panel for staining GC B and plasma B cells.

Discussion

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, immunofluorescence and ELISA. These assays will facilitate detection of Tfh differentiation, GC B development and influenza virus-specific antibodies and help researchers explore and identify new crucial molecules in the immune response.

In studies with influenza virus-infected mice models, weight loss is a commonly used indicator of mouse morbidity. The expected weight change kinetics in influenza virus-infected mice is as described in Figure 1a, which reflects the appropriate immune response induced in the mice. However, abnormal cases would regularly occur, in which the mice lose their weight or do not show any weight decline all through the observation period. According to our experiences, these mice would mostly bear abnormal lower or higher immune response, thus disrupting the experiment results. To avoid such variations, firstly mice used in the experiment should be sex and age-matched to guarantee the similar responsive ability to virus. Consistent virus titer for each mouse is also important²¹. The virus titer used in this protocol is 40 PFU. However, the virus titer to induce appropriate weight change kinetics in each lab could be variable due to the inconsistency in virus titer evaluation procedure and mouse strains used in the experiment. So we advise titration of virus titer for infection is necessary before immune response-relevant study.

In this protocol, we identified Tfh cells with frequently used markers PD-1, CXCR5 and the essential transcription factor Bcl6. Although both PD-1^hiCXCR5^hi and Bcl6⁺CXCR5⁺ cells could be denoted as Tfh cells, they represent different population and do not have the precursor-progeny relationship based on the fact that not all the PD-1^hiCXCR5^hi cells are Bcl6⁺ and not all the Bcl6⁺CXCR5^hi cells are PD-1^hiCXCR5^hi. This phenotype could be explained by the heterogeneity of Bcl6 expression in Tfh cells²². ICOS, a critical molecule for both Tfh differentiation and migration should also be included in analysis of Tfh differentiation. In addition, other function-associated co-stimulatory molecules, such as OX40 and CD40L should also be detected for their expression level, though not included in this protocol. IL21 and IL4 are both Tfh-secreted cytokines playing roles in inducing IgG1 class switching. Protocol of detecting IL21 expression is described in this paper. However, due to the difficulty in detection of IL4 in Tfh cells, IL4-GFP reporter mice were used in previous studies²³. In this protocol, we also used fluorochrome-labeled NP tetramers to detect NP_311-325-specific Tfh cells. Nevertheless, the limit in the amount of NP_311-325-specific Tfh cells confers the difficulty in further analysis. Therefore, adoptive transfer experiment of influenza hemagglutinin specific-TCR transgenic (Tg) CD4⁺ (TS-1) T cells, which could be isolated from TS-1 mice, is an alternative strategy in solving this problem²⁴.

Here we identified GC B as B220⁺PNA⁺Fas⁺ cells in flow cytometry staining. An alternative marker combination strategy to define GC B as GL7^hiFas^hi cells is also used in other papers^14,16. We also use immunofluorescence to visualize GCs with combination of anti-IgD and PNA. Herein addition of CD3 antibody can help visualize Tfh cells, thus enabling study of the interaction between these two cell types¹⁰.

Given differentiation of Tfh cells is a multistage and multifactorial process, additional assay of other significant molecules at multiple time points is necessary to elucidate more detailed mechanism in Tfh differentiation. In addition, parameters detected here are also commonly used in other models¹⁸. Therefore, besides in influenza virus infection, protocols described here, especially the immunostaining part, can also provide instructions in Tfh-associated study with other models.

Materials

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