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JoVE Journal Immunology and Infection

Immunology and Infection

Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice

Published: April 26, 2024 doi: 10.3791/66752
*1,2, *3, *4, 5, 6, 5, 7
1Department of Urology, South China Hospital, Medical School, Shenzhen University, 2Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Shenzhen University Medical School, 3Cancer Center, Daping Hospital & Army Medical Center of PLA, Third Military Medical University, 4Guangdong Provincial Key Laboratory of Immune Regulation and Immunotherapy, School of Laboratory Medicine and Biotechnology, Southern Medical University, 5Institute of Immunology, Third Military Medical University, 6Dermatology Hospital, Southern Medical University, 7Institute of Immunological Innovation and Translation, Chongqing Medical University
* These authors contributed equally

Abstract

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, thus establishing long-term humoral immunity. During acute viral infection, the fate commitment of virus-specific TFH cells is determined in the early infection phase, and investigations of the early-differentiated TFH cells are crucial in understanding T cell-dependent humoral immunity and optimizing vaccine design. In the study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection and the TCR-transgenic SMARTA (SM) mouse with CD4+ T cells specifically recognizing LCMV glycoprotein epitope I-AbGP66-77, we described procedures to access the early fate commitment of virus-specific TFH cells based on flow cytometry stainings. Furthermore, by exploiting retroviral transduction of SM CD4+ T cells, methods to manipulate gene expression in early-differentiated virus-specific TFH cells are also provided. Hence, these methods will help in studies exploring the mechanism(s) underlying the early commitment of virus-specific TFH cells.

Introduction

Encountering different pathogens or threats, naïve CD4+ T cells tailor their immune responses by differentiating into various helper T (TH) cell subsets with specialized functions1. In the scenario of acute viral infection, a large portion of naïve CD4+ T cells differentiate into follicular helper T (TFH) cells that provide help to B cells2,3. Distinct from other CD4+ TH cell subsets (e.g., TH1, TH2, TH9, and TH17 cells), TFH cells express a substantial level of CXCR5, which is the chemokine receptor for the B cell homing chemokine CXCL13, enabling TFH cells to migrate into B cell follicles. In the B cell follicles, TFH cells assist cognate B cells in initiating and maintaining germinal center reactions, thus enabling rapid high-affinity antibody production and long-term humoral memory2,3.

Upon acute viral infection, the early fate commitment of virus-specific TFH cells occurs within 72 h4,5and is controlled by the transcriptional repressor B cell lymphoma-6 (Bcl-6)5,6,7,8, which acts as the "master regulator" governing TFH cell fate decisions. Deficiency of Bcl-6 severely blunts TFH cell differentiation, while ectopic Bcl-6 expression substantially promotes TFH cell fate commitment. In addition to Bcl-6, multiple molecules are involved in instructing early TFH cell fate commitment. Transcription factors TCF-1 and LEF-1 initiate TFH cell differentiation via the induction of Bcl-69,10,11. The inhibition of Blimp1, by both Bcl-6 and TCF-1, is required for early TFH cell fate commitment11,12. STAT1 and STAT3 are also required for early TFH cell differentiation13. Besides, epigenetic modifications by histone methyltransferase EZH214,15and m6A methyltransferase METTL316 help to stabilize TFH cell transcriptional programs (especially Bcl6 and Tcf7) and thus prime early TFH cell fate commitment. While advances, including the aforementioned molecules and others, summarized elsewhere3, have been made in understanding the transcriptional and epigenetic regulations of early TFH cell fate commitment, previously unknown molecules remain to be learned.

In the mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection, adoptively transferred congenic TCR-transgenic SMARTA (SM) CD4+ T cells, which specifically recognize the LCMV glycoprotein epitope I-AbGP66-77, undergo either TFH or TH1 cell differentiation during viral infection. This TFH/TH1 bifurcation differentiation pattern supports the advancement of the SM/acute LCMV infection model in studying the biology of virus-specific TFH cells. Indeed, the SM/acute LCMV infection model has been widely used in the TFH cell research field and has played a crucial role in milestone discoveries in TFH cell biology. This includes the identification of the aforementioned Bcl-6 as the lineage-defining transcription factor of TFH cells5,6, as well as other important transcriptional factors (e.g., Blimp-16, TCF-1/LEF9,10,11, STAT1/STAT313, STAT517, KLF218, and Itch19) guiding TFH cell differentiation, post-transcriptional regulations (e.g., METTL316 and miR-17~9220) of TFH cell differentiation, TFH cell memory and plasticity21,22, and rational vaccination strategies targeting TFH cells (e.g., selenium23).

The current study describes reproducible methods for accessing the early fate commitment of virus-specific TFH cells, including (1) establishing an acute LCMV-infected SM chimera mouse model suitable for accessing early-differentiated TFH cells, (2) conducting flow cytometry stainings of molecules related to early-differentiated TFH cells, and (3) performing retroviral vector-based gene manipulation in SM CD4+ T cells. These methods will be useful for studies investigating the early fate commitment of virus-specific TFH cells.

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Protocol

All animal experiments were conducted following procedures approved by the Institutional Animal Care and Use Committees of the Third Military Medical University. The following mouse strains were used in the present study: C57BL/6J (B6) mouse (both genders), aged 6 to 8 weeks, weighing 25-30 g; CD45.1+SM TCR transgenic mouse (B6 CD45.1 × SM TCR transgenic), both genders, aged 6 to 8 weeks, weighing 25-30 g; and CXCR5-GFP CD45.1+SM TCR transgenic mouse (B6 CD45.1 × SM TCR transgenic × CXCR5-GFP knock-in), both genders, aged 6 to 8 weeks, weighing 25-30 g. The transgenic species were generated following a previously published report24. The details of the reagents, buffers, and equipment used in the study are listed in the Table of Materials.

1. Harvesting CD45.1+ SM CD4+ T cells for adoptive transfer

  1. Euthanize (following institutionally approved protocols) the required number of 6- to 8-week-aged CD45.1+ TCR-transgenic SM mice24. Make a 1-inch incision at the left of the peritoneal wall of the euthanized mice by surgical scissor, dissect the spleens from the peritoneum, and tear the connective tissue behind the spleen25,26.
  2. Place each spleen into a 70 µm cell strainer within a Petri dish (60 mm × 15 mm) and grind the spleen with 3 mL of red blood cell (RBC) lysis buffer using a 1 mL syringe plunger.
    NOTE: Use the round side of the syringe plunger to grind the spleen until only connective tissue remains.
  3. Add 6 mL of RPMI + 2% FBS (RPMI 2%) into each dish to dilute 3 mL of RBC lysis buffer. Pipette the cell suspensions into a 15 mL conical tube from the dish. Rinse the dish with an additional 3 mL of 2% RPMI and transfer to the same conical tube.
  4. Centrifuge the cell suspensions at 800 × g for 5 min at 4 °C and discard the supernatant by decantation. Resuspend the cells with an isolation buffer and adjust the cell density to ~1 × 108 cells per mL. Place the tube on ice.
  5. Prepare a 1x biotinylated antibody cocktail against CD8a, CD19, NK1.1, F4/80, CD11c and Ter119 in isolation buffer for the enrichment of CD4+ T cells.
    NOTE: Add biotinylated antibodies into the isolation buffer at the indicated dilution as mentioned in the Table of Materials. Keep the volume of the antibody cocktail the same as that of the isolation buffer in step 1.4.
  6. Centrifuge the cell suspensions at 800 × g for 5 min at 4 °C and discard the supernatant by decantation.
  7. Resuspend cell pellet with indicated 1x biotinylated antibody cocktail in the conical tube.
  8. Lay the conical tube on ice in a shaker for 30 min at 45 rpm.
  9. During the incubation with biotinylated antibodies, prepare the required volume of streptavidin-coated beads according to the manufacturer's instructions.
    NOTE: Total splenocytes from one naïve mouse need 200 µL of streptavidin-coated beads.
  10. Wash the cells by centrifuging at 800 x g with 8-10 mL of isolation buffer for 5 min at 4 °C and discard the supernatant. Repeat this step for another two times.
  11. Resuspend cell pellet with prepared streptavidin-coated beads.
  12. Lay the tube on ice in a shaker for 40 min at 45 rpm.
  13. Then, put the tube on a magnetic stand for 3-4 min until all the beads are assembled on the magnetic side.
  14. Carefully collect the supernatant and transfer it into a new 15 mL conical tube.
  15. Centrifuge the cell suspensions at 800 × g for 5 min at 4 °C and discard the supernatant by decantation.
    NOTE: There are other commercial kits available for CD4+ T cell enrichment, which can be used as substitutions for the CD4+ T cell enrichment procedure described in steps 1.5-1.15.
  16. Resuspend the cell pellet with 2-3 mL of 2% RPMI. Aspirate 50 µL of cell suspension for flow cytometry analysis of the efficiency of CD4+ T cell enrichment (CD4 and Vα2) and cell viability.
  17. Centrifuge the cell suspensions at 800 × g for 5 min at 4 °C and discard the supernatant by decantation. Resuspend the cell pellet with RPMI and adjust cell density to 1 × 107 CD4+Vα2+ SM cells per mL. Put the tube on ice and move forward to adoptive cell transfer in the next step.

2. Establishing the acute LCMV-infected SM chimera mouse model for accessing early-differentiated SM TFH cells

  1. Warm the 6- to 8-week-aged C57BL/6 recipient mice with an infrared lamp to dilate the tail vein for intravenous injection.
  2. Aspirate 100 µL of cell suspension containing 1 × 106 SM cells prepared in step 1.17 with an insulin syringe.
    NOTE: The injection volume should be modified by each researcher and should not exceed the maximum injection volume allowed by institutional and local law.
  3. Hold the mice in place with a mouse fixator, straighten the tail, and wipe the tail with 75% ethanol using a cotton ball to make the tail vein visible.
  4. Insert the insulin syringe needle into the tail vein and inject the cell suspensions smoothly and slowly. Then, let the recipient mice rest overnight.
  5. The following day, dilute the LCMV Armstrong virus stock with RPMI medium to achieve a final concentration of 1 × 107 plaque-forming units/mL.
  6. Inject 100 µL of diluted LCMV Armstrong solution (containing 1 × 106 plaque-forming units) into the tail vein of the recipient mice.
    NOTE: Each researcher should be cautious about whether the recommended infectious titer is allowed by institutional and local law.

3. Flow cytometry stainings of early-differentiated SM TFH cells

  1. Sacrifice the mice (following institutionally approved protocols) from step 2.6 on day 3-post infection, and process lymphocytes from spleens as described in step 1. Adjust the cell density to approximately 2 × 107 cells per mL.
  2. Load 50 µL of cell suspensions (~1 × 106 cells) to a round-bottom 96-well plate and top up with 150 µL of staining buffer per well. Keep the plate on ice.
  3. Prepare the purified rat anti-mouse CXCR5 antibody cocktail (i.e., CXCR5 primary antibody) at a 1:50 dilution in 50 µL of TFH cell staining buffer for each sample in a tube. Thoroughly vortex the tube and centrifuge it at 15,000 × g for 3 min at 4 °C to aggregate the particles. Keep the tube on ice.
  4. Centrifuge the 96-well plate at 800 × g for 1 min at 4 °C, discard the supernatant by decantation, briefly vortex the plate, and add 200 µL of staining buffer per well.
  5. Repeat step 3.4 and resuspend the cells with 50 of µL the CXCR5 primary antibody per well. Mix well by pipetting up and down and incubate on ice for 1 h.
  6. Prepare the biotinylated-goat anti-rat antibody cocktail (i.e., CXCR5 secondary antibody) at a 1:200 dilution in 50 µL of TFH cell staining buffer for each sample in a new tube. Vortex the tube and centrifuge it at 15,000 × g for 3 min at 4 °C to aggregate the particles. Keep the tube on ice.
  7. Centrifuge the plate at 800 × g for 1 min at 4 °C, discard the supernatant by decantation, briefly vortex the plate, and add 200 µL of staining buffer per well.
  8. Repeat step 3.7 twice and keep the plate on ice.
  9. Add 50 µL of CXCR5 secondary antibody per well to the plate. Mix well by pipetting up and down and incubate on ice for 30 min.
  10. Prepare fluorescence-conjugated streptavidin (i.e., CXCR5 tertiary antibody) at a 1:200 dilution in 50 µL of TFH cell staining buffer for each sample. Meanwhile, add other antibodies against surface markers (e.g., CD45.1, CD4, PD-1, CD25, and other interested molecules) and the dead cell stain along with the CXCR5 tertiary antibody.
  11. Vortex the tube and centrifuge it at 15,000 × g for 3 min at 4 °C to aggregate the particles. Keep the tube on ice.
  12. Centrifuge the plate at 800 × g for 1 min at 4 °C, discard the supernatant by decantation, briefly vortex the plate, and add 200 µL of staining buffer per well.
  13. Repeat step 3.12 twice and keep the plate on ice.
  14. Add 50 µL of CXCR5 tertiary antibody cocktail per well to the plate. Mix well by pipetting up and down and incubate on ice for 30 min in the dark.
  15. Prepare 200 µL of fixation/permeabilization buffer for each well according to the manufacturer's instructions.
  16. Centrifuge the plate at 800 × g for 1 min at 4 °C, discard the supernatant by decantation, briefly vortex the plate, and add 200 µL of staining buffer per well.
  17. Repeat step 3.16 twice and keep the plate on ice in the dark.
  18. Add 200 µL of fixation/permeabilization buffer into each well. Mix well by pipetting up and down and incubating at room temperature for 30 min (in the dark).
    NOTE: Prepare permeabilization buffer (1x) according to the manufacturer's instructions.
  19. Prepare the antibody cocktail against transcriptional factors, including Bcl-6, TCF-1, T-bet, and other interested molecules, in 50 µL of permeabilization buffer (1x) for each well in a new tube.
  20. Vortex the tube and centrifuge at 15,000 x g for 3 min at 4 °C to aggregate the particles.
  21. Centrifuge the plate at 800 × g for 1 min at 4 °C, discard the supernatant by decantation, briefly vortex the plate, and add 50 µL of transcriptional factor antibody cocktail per well. Mix well and incubate on ice for 30 min in the dark.
  22. Centrifuge the plate at 800 × g for 1 min at 4 °C, discard the supernatant, briefly vortex the plate by decantation, and add 200 µL of permeabilization buffer (1x) per well.
  23. Repeat step 3.22.
  24. Add 200 µL of staining buffer into each well, centrifuge the plate at 800 × g for 1 min at 4 °C, discard the supernatant by decantation, and briefly vortex the plate.
  25. Resuspend the cell pellet in each well with 200 µL of staining buffer and transfer the cells into flow cytometry tubes for immediate flow cytometer analysis.
    NOTE: Alternatively, the samples can be detected within 3 days in the conditions of adding 200 µl of 2% PFA into each tube and being stored at 4 °C in the dark.

4. Retrovirus production

  1. Cultivate the 293T cells (with less than ten passages) with DMEM 10% medium in a 10 cm culture dish for a duration of 2-3 passages.
  2. When the cells reached 90% confluency, discard the old medium and gently wash the cells with PBS twice, followed by brief trypsinization of 293T cells using 1 mL of 0.25% Trypsin-EDTA at room temperature for 1 min.
  3. Stop the digestion process by resuspending cells in 2 mL of pre-warmed 10% DMEM medium, transferring them to a 15 mL conical tube, and centrifuging at 200 × g for 5 min at 4 °C.
  4. Discard the supernatant and resuspend the cell pellet by pipetting up and down culture media with 2 mL of pre-warmed 10% DMEM medium.
  5. Quantify the number of cells and plate 5 × 105 cells in 2.5 mL 10% DMEM medium per well in a 6-well plate.
    NOTE: Swirl the dish gently to allow the 293T cells to spread evenly throughout the plate.
  6. When the 293T cell confluence exceeds 80%, prepare the transfection reagents as described in steps 4.7 to 4.9.
  7. For each well of a 6-well plate, add 250 µL of pre-warmed Opti-MEM medium into a sterile 1.5 mL tube. Then, add 3 µg of plasmid DNA (containing 2 µg of retroviral vector and 1 µg of pCL-Eco) to the Opti-MEM medium, followed by gentle pipetting up and down to ensure complete mixing.
    NOTE: The retroviral vectors employed as examples in this study are MigR1 and modified MigR1 vector with Bcl6 CDS region at the upstream of IRES-GFP (MigR1-Bcl6).
  8. Next, add 7.5 µL of pre-warmed transfection reagent (commercially obtained) to the mixture and gently pipette up and down to ensure thorough mixing.
  9. Incubate the mixture at room temperature for 20 min.
  10. Dispense the mixture drop-wise into distinct regions of the 293T cell-cultured well, followed by gently rocking the culture vessel in a back-and-forth and side-to-side motion to ensure even distribution of the mixture.
  11. Incubate the 293T cells for 72 h in 37 °C incubator containing 5% CO2.
  12. Detect the tag of the retroviral vector as the proxy for transfection efficacy in the transfected 293T cells using a fluorescence microscope or flow cytometry.
    NOTE: For example, we confirmed the GFP fluorescence signal in a significant proportion of MigR1 vector-transfected 293T cells, suggesting high-quality transfection.
  13. Collect cell culture medium and centrifuge at 2,500 × g for 10 min at 4 °C to eliminate cellular debris. The resulting supernatant should be carefully collected and stored either for long-term preservation at -80 °C or temporarily kept at 4 °C.

5. In vivo activation, retrovirus transduction and adoptive transfer of SM CD4+ T cells

  1. Inject one CD45.1+ SM mouse (6- to 8-week-aged) intravenously with 200 µg of LCMV GP61-77 peptide11,14 in a volume of 100 µL of RPMI medium.
    NOTE: The injection volume should be modified by each researcher and not exceed the maximum injection volume allowed by institution and local law.
  2. Euthanize the SM mouse at 16-18 h after peptide injection (following institutionally approved protocols). Acquire the splenocytes following the aforementioned procedures in step 1.
    NOTE: Alternatively, in vitro anti-CD3/anti-CD28 stimulation method27,28 can be used to activate SM CD4+ T cells in place of in vivo peptide stimulation described in steps 5.1 and 5.2.
  3. Suspend the splenocytes with 2% RPMI and adjust the cell density to approximately 1 × 108 cells per mL in a 15 mL conical tube. Keep the tube on ice.
  4. Dispense 50 µL of cell suspension into a round-bottom 96-well plate and supplement with 150 µL of staining buffer per well.
  5. Centrifuge the 96-well plate at 800 × g for 1 min at 4 °C to pellet the cells, carefully remove the supernatant by decantation, briefly vortex the plate, and add 200 µL of staining buffer per well.
  6. Repeat step 5.5 and resuspend the cells with 50 µL of surface antibody cocktail, including CD4, CD25, and CD69, to detect the activation status of SM CD4+ T cells. Thoroughly mix by pipetting up and down and incubate on ice for 30 min in the dark.
  7. Top up each well with staining buffer to 200 µL, followed by centrifugation of the plate at 800 × g for 1 min at 4 °C. Discard the supernatant by decantation and briefly vortex the plate.
  8. Repeat step 5.7 for two times.
  9. Resuspend the cells in 200 µL of staining buffer and transfer them into a flow cytometry tube.
  10. Analyze the fluorescence-conjugated antibody-stained cells with a flow cytometry to assess the activation status of SM CD4+ T cells.
    NOTE: Activated SM CD4+ T cells exhibited abundant CD25 and CD69 expressions as compared to naïve cells, thus enabling subsequent transduction procedures.
  11. Add 1 × 106 SM CD4+ T cells (from step 5.3) per well into a 24-well plate. Spin down cells at 800 × g for 3 min at 4 °C, then carefully remove the medium by decantation.
  12. Add 1 mL of retrovirus medium as described in step 4.13 and 8 µg of polybrene to each well.
  13. Spin-transduce the cells at 800 × g for 2 h at 37 °C.
  14. Carefully discard the retrovirus medium by pipetting and add 1 mL of pre-warmed 10% RPMI supplemented with 10 ng/mL recombinant murine IL-2 to each well.
  15. Incubate the cells for 48 h in a 37 °C incubator containing 5% CO2.
  16. Transfer cell suspension into a 15 mL conical tube and spin down the cells at 800 × g for 6 min at 4 °C.
  17. Remove the supernatant by decantation and resuspend the cells with an appropriate volume of 2% RPMI to make a cell density of 1 × 108 cells per mL in a 15 mL conical tube. Keep the tube on ice.
  18. Dispense 50 µL of cell suspension into a round-bottom 96-well plate and supplement with 150 µL of staining buffer per well.
  19. Centrifuge the 96-well plate at 800 × g for 1 min at 4 °C to pellet the cells, carefully remove the supernatant by decantation, briefly vortex the plate, and add 200 µL of staining buffer per well.
  20. Repeat step 5.19 and resuspend the cells with 50 µL of surface antibody cocktail, including CD4, Vα2, and vector tag-associated antibody. Thoroughly mix by pipetting up and down and incubate on ice for 30 min in the dark.
  21. Top up each well with staining buffer to 200 µL, followed by centrifugation of the plate at 800 × g for 1 min. Discard the supernatant by decantation and briefly vortex the plate.
  22. Repeat step 5.21 for two times.
  23. Resuspend the cells in 200 µL of staining buffer and transfer them into a flow cytometry tube.
  24. Analyze the fluorescence-conjugated antibody-stained cells with flow cytometry to assess the transduction efficiency in SM CD4+ T cells.
    NOTE: Efficiently-transduced SM CD4+ T cells exhibited high expression of the tag of retrovirus, such as GFP, in the study.
  25. Centrifuge the cell suspensions from step 5.17 at 800 × g for 5 min at 4 °C and discard the supernatant by decantation. Resuspend cell pellet with RPMI and adjust cell density to 1 × 107 CD4+Vα2+ SM cells/mL.
  26. Adoptively transfer a total number of 1 × 106 CD45.1+ SM CD4+ T cells to each C57BL/6 recipient mouse (6- to 8-week-aged) by an intravenous injection of 100 µL of cell suspension in step 5.25.
  27. On the next day, infect C57BL/6 recipient mice with 1 × 106 plaque-forming units LCMV Armstrong through intravenous injection.

6. Flow cytometry analysis of retrovirus-transduced SM TFH cells at early acute LCMV infection stage

  1. Acquire the splenocytes from C57BL/6 mice from step 5.25 on day 3-post infection.
  2. Perform flow cytometry staining of surface markers, including CD4, CD45.1, CXCR5, CD25, and other interested molecules, in splenocytes as described in step 3.
  3. For the detection of transcriptional factors in SM CD4+ T cells transduced with retrovirus tagged by fluorescent proteins, add 200 µL of 2% PFA to each well and incubate at room temperature for 20 min in the dark.
  4. Perform fixation/permeabilization, flow cytometry staining of the interested transcriptional factors, and perform flow cytometry analysis as described in step 3.

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Representative Results

Characteristics of early-differentiated virus-specific TFH cells during acute LCMV infection
To probe the early fate commitment of virus-specific TFH cells, naïve congenic SM CD4+ T cells that specifically recognize LCMV GP epitope I-AbGP66-77 were adoptively transferred into CD45.2+ C57BL/6 recipients. The next day, these recipients were intravenously infected with a high dosage of the acutely resolved LCMV Armstrong (Figure 1A). On day 3 after infection, splenocytes of C57BL/6 recipients were analyzed by flow cytometry, and the results indicated a large quantity of transferred CD45.1+ SM CD4+ T cells (~10% of total CD4+ T cells) (Figure 1B), which provided sufficient number of virus-specific CD4+ T cells for further investigation. Further analyses of transferred SM CD4+ T cells showed co-expression of CXCR5 and Bcl-6/TCF-1 and reciprocal expression of CXCR5 and CD25/T-bet, thus defining TFH (CXCR5+Bcl-6+ or CXCR5+TCF-1+) and TH1 (CXCR5-CD25+ or CXCR5-T-bet+) lineages (Figure 1C). To evaluate the specificity and sensitivity of CXCR5 antibody staining presented in the study, we further crossed the aforementioned CD45.1+ SM mouse line with the CXCR5-GFP knock-in mouse line, which was generated by the insertion of an IRES-GFP construct after the open reading frame of Cxcr529. Then, CD45.1+ CXCR5-GFP reporter SM cells were transferred into C57BL/6 recipients. These recipients were infected with LCMV Armstrong, and their splenocytes were analyzed on day 3-post infection. Indeed, the fluorescence signal by the CXCR5 antibody staining method in the study finely overlapped with the GFP signal in transferred CXCR5-GFP reporter29 SM CD4+ T cells on day 3-post infection (Figure 1D), indicating the high specificity of the three-step CXCR5 antibody staining method.

Retrovirus-based Bcl-6 overexpression in SM CD4+ T cells
Bcl-6 is one of the most important molecules in fostering TFH cell differentiation6,7,8. To ascertain the reliability of the current protocol in accessing early-differentiated TFH cells, CD45.1+ SM CD4+ T cells were in vivo activated and transduced with retrovirus supernatant from MigR1 plasmid- or MigR1-Bcl6 overexpression plasmid-transfected 293T cells (Figure 2 and Figure 3A). Retrovirus-transduced SM CD4+ T cells were next adoptively transferred into C57BL/6 recipients, which were then infected with LCMV Armstrong (Figure 3B). On day 3 after infection, we found a balanced bifurcation of TFH and TH1 cells in MigR1-SM CD4+ T cells, while a predominant TFH cell-directed differentiation in MigR1-Bcl6-SM CD4+ T cells (Figure 3C). Consistently, the Bcl-6 expression level was enhanced in MigR1-Bcl6-SM CD4+ T cells as compared to that in MigR1-SM CD4+ T cells (Figure 3D). Therefore, the phenotype that ectopic Bcl-6 expression-driven TFH cell differentiation6 was finely reproduced following the current protocol.

Figure 1
Figure 1: Flow cytometry analyses of early fate commitment of SM TFH cells during LCMV Armstrong infection. (A) Experimental design. Splenic CD4+ T cells were harvested from CD45.1+ SM mice and then adoptively transferred into C57BL/6 recipients (CD45.2+). The next day, these recipients were infected with LCMV Armstrong. On day 3 after infection, transferred CD45.1+ SM cells from the spleens of recipients were analyzed by flow cytometry. (B) Representative flow cytometry data showing the gating strategy of transferred CD45.1+ SM CD4+ T cells from the spleens of recipients on day 3-post LCMV Armstrong infection. (C) Representative flow cytometry data showing co-stainings of CXCR5 and other TFH cell-associated molecules (Bcl-6, TCF-1, CD25, and T-bet) in transferred CD45.1+ SM cells from the spleens of recipients on day 3-post LCMV Armstrong infection. (D) Representative flow cytometry data showing co-stainings of fluorescence-conjugated antibody against CXCR5 and GFP signal in transferred CD45.1+ CXCR5-GFP reporter SM cells from the spleens of recipients on day 3-post LCMV Armstrong infection. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Retroviral transduction in SM CD4+ T cells. (A) Schematic diagram of retrovirus production and retroviral transduction in SM CD4+ T cells. For retrovirus production, 293T cells seeded in a 6-well plate were transfected with target DNA plasmids when cell density reaches ≥80% confluence. Then, cell culture supernatant containing retroviruses was collected 72 h after transfection and immediately used or stored at -80 °C. For retroviral transduction in SM CD4+ T cells, a CD45.1+ SM mouse was intravenously injected with 200 µg of LCMV GP61-77 peptide. Then, the spleen was dissected from SM mouse 16-18 h after peptide injection, and the activation status of splenic CD4+ T cells was detected. Activated SM CD4+ T cells were transduced with culture supernatant containing retroviruses and cultured in vitro for 48 h before accessing the transduction efficiency. (B) Fluorescence microscopy imaging of GFP in plasmid DNA-transfected 293T cells. Scale bar: 400 µm. (C) Flow cytometry analysis of CD25 and CD69 expression levels in splenic naïve CD4+ T cells from naïve mouse (naïve, left) or splenic SM CD4+ T cells from LCMV GP61-77 peptide-injected SM mouse (activated, right). (D) Flow cytometry analysis of GFP expression in retrovirus-transduced SM CD4+ T cells (transduced, left) or retrovirus-non-transduced SM CD4+ T cells (non-transduced, left). Please click here to view a larger version of this figure.

Figure 3
Figure 3: Effects of Bcl-6 overexpression in SM TFH cell fate commitment. (A) Linear maps of retroviral MigR1 backbones (up panel) and modified MigR1 vector with Bcl6 CDS region at the upstream of IRES-GFP (MigR1-Bcl6) (bottom panel). (B) Experimental design. MigR1-transduced or MigR1-Bcl6-transduced SM CD4+ T cells were adoptively transferred into C57BL/6 recipients (CD45.2+) one day prior to LCMV Armstrong. On day 3 after infection, transferred CD45.1+ SM CD4+ T cells from recipients' spleens were analyzed by flow cytometry. (C) Flow cytometry analysis of CXCR5 and CD25 in MigR1-transduced or MigR1-Bcl6-transduced GFP+ SM CD4+ T cells. The numbers adjacent to the gate indicate the frequencies of TFH cells in SM CD4+ T cells. (D) Flow cytometry analysis of Bcl-6 expression level in MigR1-transduced or MigR1-Bcl6-transduced SM CD4+ T cells. MFI, mean fluorescence intensity. Please click here to view a larger version of this figure.

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Discussion

The research in the field of TFH cells has been highlighted since the discovery of the specialized function of TFH cells in helping B cells. Accumulating studies indicated that TFH cell differentiation is a multistage and multifactorial process30, wherein the TFH cell fate commitment is determined in the early stage5. Therefore, a better understanding of the mechanisms underlying early-differentiated TFH cells is crucial for TFH cell biology and rational vaccine design. Herein, we provided methods to access the early fate commitment of virus-specific TFH cells and to manipulate gene expression in early-differentiated TFH cells by exploiting the LCMV Armstrong viral infection model, TCR-transgenic SM mouse, and retrovirus-mediated transduction.

The chemokine receptor CXCR5 is an important surface marker that distinguishes TFH cells from other CD4+ TH cell subsets6,7,8,31, thus necessitating the CXCR5 staining method of high specificity and sensitivity in TFH cell research. In the study, a three-step streptavidin-biotin CXCR5 staining method was showcased in identifying the TFH cell subset (Figure 1C). Though loss of specificity is one sticky problem for two- or three-strep antibody staining approaches of high sensitivity, it was found that the fluorescence signal by three-step CXCR5 antibody staining almost completely overlapped with the GFP signal in CXCR5-GFP29 reporter SM CD4+ T cells on day 3-post infection (Figure 1D), suggesting the achievement of both specificity and sensitivity by the three-step CXCR5 antibody staining method. Indeed, this three-strep CXCR5 staining strategy is tried-and-trusted and has helped the researchers to better investigate TFH cell biology11,14,15,21 as well as other CXCR5-expressing T cell types29,32.

Upon infection and immunization, the fate-commitment of TFH cells is generally modeled by a bifurcated differentiation of naïve CD4+ T cells into either TFH cells or non-TFH effector TH cells2,3,30. This study showcased the balanced bifurcation of TFH and TH1 cells in transferred SM CD4+ T cells. Moreover, these transferred SM CD4+ T cells are of adequate cell quantity in the early infection stage, thus acting as a suitable model for studying TFH cell differentiation. Assuredly, retrovirus-mediated ectopic Bcl-6 expression in transferred SM CD4+ T cells was strongly biased towards TFH cell differentiation as compared to that in control SM CD4+ T cells (86.6% vs. 53.8%) (Figure 3C), which is aligned with previous report6 and further proves the accuracy of the protocols described in the study. Alternatively, the aforementioned protocols can also be applied to retrovirus-mediated knock-down of interested genes (e.g., bcl6, Tcf7) in early-activated SM CD4+ T cells11 (data not shown).

One limitation in the protocol is the timing issue (more than 2 h in total) and the multiple-step process of the three-step CXCR5 staining method. Alternatively, one-step18,33 or two-step4,9,34 CXCR5 staining methods were also applied in TFH cell research. A comparison of the specificity/sensitivity of these CXCR5 staining methods is warranted.

In summary, the current study provided protocols to access early-differentiated virus-specific TFH cells and to investigate interested gene(s) that potentially regulate TFH cell differentiation. These protocols will fuel the investigations of early TFH cell fate commitment.

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Disclosures

The authors declare no competing interests.

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (No. 32300785 to X.C.), the China National Postdoctoral Program for Innovative Talents (No. BX20230449 to X.C.), and the National Science and Technology Major Project (No. 2021YFC2300602 to L.Y.).

Materials

Name Company Catalog Number Comments
0.25% Trypsin-EDTA Corning 25-052-CI
4% Paraformaldehyde Fix Solution, 4% PFA Beyotime P0099-500mL
70 μm cell strainer Merck CLS431751
Alexa Fluor 647 anti-mouse TCR Vα2 (clone B20.1) Biolegend 127812 1:200 dilution
Alexa Fluor 700 anti-mouse CD45.1 (clone A20) Biolegend 110724 1:200 dilution
APC anti-mouse CD25 (clone PC61) Biolegend 101910 1:200 dilution
B6 CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ) mouse The Jackson Laboratory 002014
BeaverBeads Streptavidin Beaver 22321-10
Biotin anti-mouse F4/80 Antibody (clone BM8) Biolegend 123106 1:200 dilution
Biotin Rat anti-mouse CD11c (clone N418) Biolegend 117304 1:200 dilution
Biotin Rat anti-Mouse CD19 (clone 6D5) Biolegend 115504 1:200 dilution
Biotin Rat anti-Mouse CD8a (clone 53-6.7) Biolegend 100704 1:200 dilution
Biotin Rat anti-mouse NK-1.1 (clone PK136) Biolegend 108704 1:200 dilution
Biotin Rat anti-mouse TER-119/Erythroid Cells (clone TER-119) Biolegend 116204 1:200 dilution
bovine serum albumin, BSA Sigma A7906
Brilliant Violet 421 anti-T-bet (clone 4B10) Biolegend 644816 1:100 dilution
Brilliant Violet 605 anti-mouse CD279 (PD-1) (clone 29F.1A12) Biolegend 135220 1:200 dilution
C57BL/6J (B6) mouse The Jackson Laboratory 000664
CXCR5-GFP knock-in reporter mouse In house; the CXCR5-GFP knock-in mouse line was generated by the insertion of an IRES-GFP construct after the open reading frame of Cxcr5.
DMEM 10% medium DMEM medium containing 10% FBS
DMEM medium Gibco 11885092
EDTA Sigma E9884
FACSFortesa BD Biosciences
Fetal bovine serum, FBS Sigma F8318
FlowJo (version 10.4.0) BD Biosciences
Foxp3/Transcription Factor Staining Buffer Set Invitrogen 00-5523-00 The kit contains three reagents: a. Fixation/Permeabilization Concentrate (4X); b. Fixation / Permeabilization Diluent; c. Permeabilization Buffer.
Goat Anti-Rat IgG Antibody (H+L), Biotinylated Vector laboratories BA-9400-1.5 1:200 dilution
Invitrogen EVOS FL Auto Cell Imaging System ThermoFisher Scientific
Isolation buffer FACS buffer containing 0.5% BSA and 2mM EDTA
LCMV GP61-77 peptide (GLKGPDIYKGVYQFKSV) Chinese Peptide Company
LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm excitation Life Technologies L10199 1:200 dilution
MigR1 addgene #27490
NaN3 Sigma S2002
Opti-MEM medium Gibco 31985070
pCL-Eco addgene #12371
PE anti-mouse CD69 (clone H1.2F3) Biolegend 104508 1:200 dilution
PE Mouse anti-Bcl-6 (clone K112-91) BD Biosciences 561522 1:50 dilution
Phosphate buffered saline, PBS Gibco 10010072
Polybrene Solarbio H8761
Purified Rat Anti-Mouse CXCR5 (clone 2G8) BD Biosciences 551961 1:50 dilution
Rat monoclonal PerCP anti-mouse CD4 (clone RM4-5) Biolegend 100538 1:200 dilution
recombinant murine IL-2 Gibco 212-12-1MG
Red Blood Cell Lysis Buffer Beyotime C3702-500mL
RPMI 1640 medium Sigma R8758
RPMI 2% RPMI 1640 medium containing 2% FBS
SMARTA (SM) TCR transgenic mouse SM TCR transgenic line in our lab is a gift from Dr. Rafi Ahmed (Emory University). Additionally, this mouse line can also be obtained from The Jackson Laboratory (stain#: 030450).
Staining buffer PBS containing 2% FBS and 0.01% NaN3
Streptavidin PE-Cyanine7 eBioscience 25-4317-82 1:200 dilution
TCF1/TCF7 (C63D9) Rabbit mAb (Alexa Fluor 488 Conjugate)  Cell signaling technology 6444S 1:400 dilution
TFH cell staining buffer FACS buffer containing 1% BSA and 2% mouse serum
TransIT-293 reagent Mirus Bio MIRUMIR2700

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Cite this Article

Lin, Y., Yue, S., Yang, Y., He, J.,… More

Lin, Y., Yue, S., Yang, Y., He, J., Yang, X., Ye, L., Chen, X. Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice. J. Vis. Exp. (206), e66752, doi:10.3791/66752 (2024).

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