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

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 spleen^25,26.
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 T_FH 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 T_FH 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 T_FH 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 T_FH 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 T_FH 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 GP_61-77 peptide^11,14 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 method^27,28 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 T_FH 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.