JoVE Logo
Autorzy
Skontaktuj Się z Nami

Zaloguj się

Aby wyświetlić tę treść, wymagana jest subskrypcja JoVE. Zaloguj się lub rozpocznij bezpłatny okres próbny.

W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 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 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.

Access restricted. Please log in or start a trial to view this content.

Protokół

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 studies15,17.

  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 (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.
  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.

Access restricted. Please log in or start a trial to view this content.

Wyniki

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...

Access restricted. Please log in or start a trial to view this content.

Dyskusje

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...

Access restricted. Please log in or start a trial to view this content.

Ujawnienia

The authors have nothing to disclose.

Podziękowania

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&D Program of China (2016YFA0502202), the National Natural Science Foundation of China (31570886).

Access restricted. Please log in or start a trial to view this content.

Materiały

NameCompanyCatalog NumberComments
Immunostaining of Tfh cells, NP-specific Tfh cells and Bcl-6
37% formaldehydeSigmaF1635
Anti-CD16/32 mouseThermo Fisher Scientific14-0161-86
APC-conjugated-IAbNP311-325 MHC class II tetramerNIH
Bcl-6 PEBiolegend358504clone:7D1
Biotin-CXCR5Thermo Fisher Scientific13-7185-82clone: SPRCL5
CD4 Percp-eFluor 710Thermo Fisher Scientific46-0041-82clone:GK1.5
CD44 eVolve 605Thermo Fisher Scientifi83-0441-42clone:IM7
CD44 FITCThermo Fisher Scientifi11-0441-82clone:IM7
CD62L FITCBD Pharmingen553150clone:MEL-14
ICOS BV421Biolegend564070clone:7E.17G9
PD1 PE/Cy7Biolegend135216clone:29F.1A12
Streptavidin BV421BD Pharmingen563259
Streptavidin PEBD Pharmingen554081
Intracelluar staining of IL21
37% formaldehydeSigmaF1635
anti-human IgGJackson ImmunoResearch Laboratories109-605-098
Brefeldin ASigmaB6542
human FCc IL-21 receptorR&D System
ionomycinSigmaI0634
Live/Dead Fixable Aqua Dead Cell staining kitThermo Fisher ScientificL34966
PMASigmaP1585
SaponinMP102855
GC B and plasma cells staining
B220 APCThermo Fisher Scientific17-0452-81clone:RA3-6B2
CD138 PEBD Pharmingen561070clone:281-2
CD95 (FAS) PE/Cy7BD Pharmingen557653clone:Jo2
IgD eFluor 450Thermo Fisher Scientific48-5993-82clone:11-26c
PNA FITCSigmaL7381
Assay of HA-specific antibody titer with ELISA
PR8-HASino Biological11684-V08H
BSASSBC
Goat anti mouse Ig (SBA Clonotyping System-HRP)SouthernBiotech5300-05
Goat anti mouse IgM (SBA Clonotyping System-HRP)SouthernBiotech5300-05
Goat anti mouse IgG1 (SBA Clonotyping System-HRP)SouthernBiotech5300-05
Goat anti mouse IgG2b (SBA Clonotyping System-HRP)SouthernBiotech5300-05
Goat anti mouse IgG2c (SBA Clonotyping System-HRP)SouthernBiotech5300-05
TMB Substrate Reagent SetBD Pharmingen555214
Histology
Alexa Fluor 555-Goat-anti rat IgGLife TechnologyA21434
anti-mouse IgDBiolegend405702
biotinylated PNAVector laboratoriesB-1075
dilute Alexa Fluor 488-streptavidinLife TechnologyS11223
normal goat serumSouthernBiotech0060-01
Pro-long gold antifade reagentThermo Fisher ScientificP3630
STREPTAVIDIN/BIOTIN blocking kitVector laboratoriesSP-2002

Odniesienia

  1. Johnston, R. J., et al. Bcl6 and Blimp-1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation. Science. 325 (5943), 1006-1010 (2009).
  2. Nurieva, R. I., et al. Bcl6 mediates the development of T follicular helper cells. Science. 325 (5943), 1001-1005 (2009).
  3. Yu, D., et al. The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment. Immunity. 31 (3), 457-468 (2009).
  4. Pedros, C., et al. A TRAF-like motif of the inducible costimulator ICOS controls development of germinal center TFH cells via the kinase TBK1. Nature Immunology. 17 (7), 825-833 (2016).
  5. Xu, H., et al. Follicular T-helper cell recruitment governed by bystander B cells and ICOS-driven motility. Nature. 496 (7446), 523-527 (2013).
  6. Lee, S. K., et al. B cell priming for extrafollicular antibody responses requires Bcl-6 expression by T cells. The Journal of Experimental Medicine. 208 (7), 1377-1388 (2011).
  7. Zotos, D., et al. IL-21 regulates germinal center B cell differentiation and proliferation through a B cell-intrinsic mechanism. The Journal of Experimental Medicine. 207 (2), 365-378 (2010).
  8. Vogelzang, A., et al. A fundamental role for interleukin-21 in the generation of T follicular helper cells. Immunity. 29 (1), 127-137 (2008).
  9. Ansel, K. M., et al. A chemokine-driven positive feedback loop organizes lymphoid follicles. Nature. 406 (6793), 309-314 (2000).
  10. Shi, J., et al. PD-1 Controls Follicular T Helper Cell Positioning and Function. Immunity. 49 (2), 264-274 (2018).
  11. Methot, S. P., Di Noia, J. M. Molecular Mechanisms of Somatic Hypermutation and Class Switch Recombination. Advanced ImmunoChemical. 133, 37-87 (2017).
  12. Crotty, S. Follicular helper CD4 T cells (TFH). Annual Review of Immunology. 29, 621-663 (2011).
  13. Calame, K. L. Plasma cells: finding new light at the end of B cell development. Nature Immunology. 2 (12), 1103-1108 (2001).
  14. Yoo, J. K., Fish, E. N., Braciale, T. J. LAPCs promote follicular helper T cell differentiation of Ag-primed CD4+ T cells during respiratory virus infection. The Journal of Experimental Medicine. 209 (10), 1853-1867 (2012).
  15. Leon, B., Bradley, J. E., Lund, F. E., Randall, T. D., Ballesteros-Tato, A. FoxP3+ regulatory T cells promote influenza-specific Tfh responses by controlling IL-2 availability. Nature Communications. 5, 3495(2014).
  16. He, L., et al. Extracellular matrix protein 1 promotes follicular helper T cell differentiation and antibody production. Proceedings of the National Academy of Sciences of the United States of America. 115 (34), 8621-8626 (2018).
  17. León, B., et al. Regulation of TH2 development by CXCR5+ dendritic cells and lymphotoxin-expressing B cells. Nature Immunology. 13 (7), 681-690 (2012).
  18. Wang, H., et al. The transcription factor Foxp1 is a critical negative regulator of the differentiation of follicular helper T cells. Nature Immunology. 15 (7), 667-675 (2014).
  19. Bouvier, N. M., Lowen, A. C. Animal Models for Influenza Virus Pathogenesis and Transmission. Viruses. 2 (8), 1530-1563 (2010).
  20. Miyauchi, K., et al. Protective neutralizing influenza antibody response in the absence of T follicular helper cells. Nature Immunology. 17 (12), 1447-1458 (2016).
  21. Rodriguez, L., Nogales, A., Martinez-Sobrido, L. Influenza A Virus Studies in a Mouse Model of Infection. Journal of Visualized Experiments. (127), (2017).
  22. Kitano, M., et al. Bcl6 protein expression shapes pre-germinal center B cell dynamics and follicular helper T cell heterogeneity. Immunity. 34 (6), 961-972 (2011).
  23. Yusuf, I., et al. Germinal center T follicular helper cell IL-4 production is dependent on signaling lymphocytic activation molecule receptor (CD150). The Journal of Immunology. 185 (1), 190-202 (2010).
  24. Sun, J., Dodd, H., Moser, E. K., Sharma, R., Braciale, T. J. CD4+ T cell help and innate-derived IL-27 induce Blimp-1-dependent IL-10 production by antiviral CTLs. Nature Immunology. 12 (4), 327-334 (2011).

Access restricted. Please log in or start a trial to view this content.

Przedruki i uprawnienia

Zapytaj o uprawnienia na użycie tekstu lub obrazów z tego artykułu JoVE

Zapytaj o uprawnienia

Przeglądaj więcej artyków

T Follicular Helper CellsGerminal Center ResponseInfluenza A VirusCD4 T CellsB CellsVirus TiterPR8 VirusC57 Black 6 MiceBiosafety Level 2Dissection TechniquesCell CollectionSpleen ExtractionLymph Node AnalysisDMEM Supplemented With FBSCentrifugation

This article has been published

Video Coming Soon

JoVE Logo

Prywatność

Warunki Korzystania

Zasady

Skontaktuj Się z Nami

Poleć Bibliotece

BIULETYNY JoVE

Badania

Edukacja

Autorzy

Bibliotekarze

O JoVE

Copyright © 2025 MyJoVE Corporation. Wszelkie prawa zastrzeżone