In Vitro Differentiation of Naive CD4⁺ T Cells into Pathogenic Th17 Cells in Mouse

The Institutional Review Committee for Animal Studies at Southeast University approved all the animal studies that are detailed in this study, which were carried out in compliance with both local and institutional office standards. Spleen samples were taken from C57BL6/J mice. Both female and male mice, aged between 5 and 8 weeks, were included in this study. The culture medium and buffer were stored at 4 °C for up to 1 month. Surgical instruments were autoclaved before use. Wear latex gloves and masks to avoid contamination of skin, eyes, and clothing with reagents; use a lot of water or saline rinse for the skin and eyes.

1. Precoating the 24-well tissue culture plate with anti-CD3

1. Dilute anti-CD3 to a final concentration of 1 µg/mL in sterile 1x phosphate-buffered saline (PBS) or RPMI 1640 medium.
2. Fill a 24-well tissue culture plate well with 1 mL of anti-CD3 solution (1 µg/mL); then, cover the well with parafilm.
3. Place the anti-CD3 coated plate in the refrigerator (2 °C-8 °C) for 16 h or keep it at 37 °C in a cell incubator for 2-3 h.

2. Mouse spleen isolation and preparation of spleen single-cell suspension

1. Induce complete anesthesia in C57BL/6J with 3% isoflurane and euthanize by inhaling 100% carbon dioxide for 2 min. Immediately after the mice's death, soak them in 75% ethanol for disinfection to prevent contamination.
2. Place the mouse in a supine position on the clean bench, cut it open along the midline of the abdomen, and separate the abdominal structures layer by layer. Gently open the greater omentum and stomach with tweezers, gently pull out the spleen with the gastrosplenic ligament, and bluntly separate the spleen from surrounding tissues and ligaments to obtain an intact spleen (be careful to avoid spleen crushing or rupture).
   NOTE: To maintain a sterile state, the following operation is carried out in a sterile super clean bench.
3. Place a 70 µm cell filter in a 100 mm sterile culture dish, add the spleen to the cell strainer, and crush it with a syringe plunger. At the same time, add 5-8 mL of homogenate flushing fluid to push all the cells through the filter into the culture dish.
   NOTE: The composition of the lymphocyte separation liquid kit can be seen in the Table of Materials.
4. Centrifuge the filtrate at 450 × g for 5 min at room temperature. Discard the supernatant.
5. Resuspend the cell pellet with the sample diluent provided with the lymphocyte separation kit or with PBS or RPMI 1640 medium. Adjust the cell concentration of the cell suspension to 2 × 10⁸-1 × 10⁹ cells/mL.
   NOTE: Typically, 4-6 mL of sample diluent is required for one spleen.
6. In a centrifuge tube, take the same amount of lymphocyte separation solution as the tissue single-cell suspension. Carefully pipette the single-cell suspension onto the surface of the lymphocyte separation solution and centrifuge at 800 × g, 25 °C for 30 min. Set the Acceleration and Deceleration of the centrifuge to an appropriate range (e.g., set at 3 if there are the usual 1 to 9 gears).
   NOTE: The lymphocyte separation solution should not be less than 3 mL.
7. After centrifugation, observe the four layers in the centrifuge tube from the top to the bottom. The first layer corresponds to the sample diluent, followed by the annular milky white lymphocyte layer. The third layer is the separation liquid, and the fourth layer consists of red blood cells.
8. Use a pipette to carefully draw the second layer of annular milky white lymphocyte layer into another centrifuge tube and add 10 mL of cleaning solution to the centrifuge tube to mix the cells.
9. Centrifuge at 250 × g for 5 min at room temperature. Discard the supernatant. Use 10 mL of RPMI 1640 medium to resuspend the cell pellet for cell counting.

3. Purification of naïve CD4⁺ T cells based on negative selection of magnetic beads

NOTE: Isolate untouched and highly purified naïve CD4⁺ T cells (CD4⁺CD44lowCD62Lhigh) from mouse splenocytes by immunomagnetic negative selection.

1. Prepare the sample to have 1 × 10⁸ cells/mL within the volume range of 0.1-2 mL.
2. Add 50 µL/mL of rat serum and transfer the sample to a 5 mL polystyrene round-bottom tube.
   NOTE: The composition of the naive CD4 T cell sorting kit can be seen in the Table of Materials.
3. Add 50 µL/mL of Isolation Cocktail to the sample, mix well, and incubate for 7.5 min at room temperature.
4. Add 50 µL/mL of Depletion Cocktail to the sample, mix well, and incubate for 2.5 min at room temperature.
5. Vortex the magnetic beads to ensure even dispersion. Add 75 µL/mL of the magnetic beads to the sample, mix well, and incubate for 2.5 min at room temperature.
   NOTE: The particles should appear evenly dispersed; vortexing time should not be less than 30 s.
6. Top up the sample with RPMI 1640 medium to a final volume of 2.5 mL. Mix several times by gently pipetting up and down.
7. Place the tube (without the lid) into the magnet and incubate for 2.5 min at room temperature.
8. Picking up the magnet, invert the magnet and tube in one continuous motion and pour theenriched cell suspension into a new tube.
   NOTE: Keep the tube and magnet inverted for 2-3 s before turning them upright. Do not disturb any drops that may remain on the mouth of the tube.
9. Determine the cell number using a hemocytometer.
10. Perform flow cytometric analysis for naïve CD4⁺ T cells before and after isolation (Figure 1).
11. Transfer the cells to a new centrifuge tube, centrifuge at 400 × g for 5 min at room temperature, and discard the supernatant.
12. Resuspend the cells in 200-500 µL of RPMI 1640 medium (supplemented with 10% fetal bovine serum). For each 1 mL of cell culture (e.g., ~106 cells/mL), add 2 µL of leukocyte activation cocktail and mix it. Culture the cells in a CO2 incubator at 37 °C and saturated humidity for 4-6 h.
    NOTE: The cells were vortexed once every 1-2 h.
13. Centrifuge the cells at 400 × g, 4 °C for 5 min. Carefully discard the supernatant and resuspend the cells in 200 µL of PBS. Add 1 µL of Fc blocker to the cells (e.g., ~106 cells/mL). Incubate the cells for 10 min at 4 °C.
14. Wash the cells once with 1 mL of PBS by centrifuging at 400 × g for 5 min at 4 °C. Discard the supernatant and resuspend the cells in 200 µL of PBS.
15. Add antibody cocktail (Fixable Viability Dye, 1:1,000; anti-CD4, 1:200; anti-CD44, 1:200; anti-CD62L, 1:200) and incubate the cells for 15-20 min in the dark at 4 °C.
16. Wash the cells with 2 x 1 mL of PBS and then, centrifuge at 400 × g for 5 min at 4 °C.
17. Discard the supernatant, resuspend the cells in 250 µL of fixed buffer, and incubate them for 40-60 min in the dark at 4 °C.
    NOTE: The fix buffer is provided with the Foxp3/transcription factor staining buffer, which is a 4x stock solution that must be diluted with permeabilization diluent.
18. Add 1 mL of 1x permeabilization buffer to wash the cells 2x by centrifuging the cells at 300 × g, 4 °C for 5 min.
19. Discard the supernatant, resuspend the cells in 200 µL of permeabilization buffer, and add the antibody (Foxp3 [nuclear], 1:200) to the cells. After fixation and membrane rupture, incubate the cell-antibody mixtures in the dark for 40-60 min with shaking occasionally for short periods at 4 °C.
    NOTE: The Permeabilization/Wash solution is a 10x stock solution that must be diluted before use with PBS.

4. Induction of pathogenic Th17 cells in vitro

1. Remove sterile PBS from the precoated 24-well plates prior to use.
2. Use Th17 cell culture medium (Table 1) and contrast medium for culture, including Th0 cell culture medium (Table 1), classical non-pathogenic Th17 culture medium (Table 1), and classical pathogenic Th17 culture medium (Table 1). Resuspend the enriched naive CD4 + T cells and dispense into different wells in the same 24-well plate (precoated with anti-CD3), adjusting the concentration to 4 × 10⁵ cells/mL, with 1 mL of medium in each well.
3. Add 1 µL/mL of anti-CD28 solution (final concentration: diluted with medium to 2 µg/mL) to each well. Culture the cells in a 5% CO₂ incubator at 37 °C for 5 days.
4. Replace the cell culture supernatant medium with culture medium (Th0, non-pathogenic Th17, classical pathogenic Th17, and objective Th17) by carefully pipetting to distribute the cells evenly, discard half of the medium containing the cells, and then add new medium equal to the discard volume on day 2. Repeat on day 4.
5. Observe the cells under an optical microscope on day 5 (Figure 2). Collect the cell supernatants from each group and cryopreserve them at -80 °C.

5. Flow cytometric analysis for pathogenic Th17 and Th0 cell differentiation

1. Harvest all cells (per well) after 5 days of initiation of culture. Perform steps 3.12-3.15.
2. Add an antibody cocktail (Fixable Viability Dye, 1:1,000; anti-CD4, 1:200) and incubate the cells for 15-20 min in the dark at 4 °C.
3. Perform steps 3.17-3.19.
4. Discard the supernatant, resuspend the cells in 200 µL of permeabilization buffer, and add the antibody cocktail (IL-17A [intracellular], 1:200; RORγT [nuclear], 1:200) to the cells. After fixation and membrane rupture, incubate the cell-antibody mixtures in the dark for 40-60 min with shaking occasionally for short periods at 4 °C.
5. Add 1 mL of 1x Permeabilization/Wash solution to wash the cells 2x, centrifuging the cells at 400 × g for 5 min at 4 °C; then, discard the supernatant.
6. Examine the cells on a flow cytometer and analyze the data using the software (Figure 3).
   1. Turn on the flow cytometer, and turn on the self-starting cleaning and calibration process.
   2. Select the laser channel used in this experiment, and set up a blank tube, single-stained tubes, and sample tubes.
   3. Click on the blank tube option and test on the machine; adjust the voltage so that the cell event is, as far as possible, in the center of the collection box.
   4. Collect single-stained tube cells and perform spectral detection of the corresponding channels to confirm that the fluorescent antibody is added correctly.
   5. Click the unmix button and let the machine automatically adjust the spectral compensation. With other flow cytometry instruments, adjust the fluorescence compensation normally.
   6. Collect each sample tube cell, save the data format as FCS files, and export. Analyze and plot in the relevant flow cytometry data analysis software.
      1. Circle the main cell population and remove possible cell adhesions through the diagonal circle gates of FSC-H and FSC-A.
      2. Circle live cells according to the FVS negative circle area; next, circle the CD4+ cell population.
      3. Construct the cross gate with the horizontal axis as IL-17 A and the vertical axis as RORγT; the double positive selection in the Q2 area is the Th17 cell population.

6. Enzyme-linked immunosorbent assay (ELISA) for detection of IL-17A secretion induced by different media

1. Thaw the frozen cell supernatants collected in Section 4.5.
2. Add 100 µL of the diluted standard, blank, and sample into the designated wells. Use the sealer to cover the plate and incubate for 90 min at 37 °C.
   NOTE: Add the solutions to the bottom of the micro-ELISA plate well without touching the inside wall or causing any foaming.
3. Decant the liquid from each well and immediately add 100 µL of Biotinylated Detection Antibody working solution. Use a new sealer to cover the plate and incubate for 1 h at 37 °C.
4. Decant the solution and add 350 µL of wash buffer to each well. After 1 min, aspirate or decant the solution from each well and pat it dry against clean absorbent paper. Repeat this wash step 3x.
5. Add 100 µL of the horseradish peroxidase Conjugate working solution to each well. Use a new sealer to cover the plate and incubate for 30 min at 37 °C.
6. Decant the solution from each well, and repeat the wash process 5x as described in step 6.4.
7. Add 90 µL of the Substrate Reagent to each well, and incubate for about 15 min at 37 °C with a new sealer, protecting the plate from light.
   NOTE: The reaction time can be altered based on the actual color change but should not exceed 30 min. Let the microplate reader warm up for ~15 min before OD measurement.
8. Add 50 µL/well of Stop Solution in the same order as the substrate solution.
9. Determine the optical density (OD value) of each well at once with a microplate reader set to 450 nm.
10. Obtain the OD values for the standard samples and replicate wells of the samples, and subtract the OD values of the blank wells to obtain the corrected values. Then, perform linear fitting with concentration as the abscissa and OD value as the ordinate. Based on the fitted equation, calculate the IL-17A concentration values for the sample wells.
    NOTE: After 4 days of differentiation, the content of IL-17A in the supernatant of each group is shown in Figure 4. The fitting method can refer to the Orgin21 software. The standard curve obtained from this experiment has an R² value of 0.9946.

7. T-cell differentiation by signature gene expression assays via quantitative PCR (qPCR)

NOTE: To rule out the possibility of flow cytometry instability due to the effects of dyes and fixation/rupture, we detected the expression of characteristic genes by qPCR to elucidate the differentiation effect of pathogenic Th17 at the transcriptional level.

1. Measure the number of cells at the end of T cell culture. Collect the cells in 1.5 mL centrifuge tubes and centrifuge at 400 × g for 5 min. Carefully discard all the supernatants.
2. RNA extraction
   NOTE: The extraction process must be carried out in a fume hood or an ultra-clean bench to avoid RNA enzyme contamination.
   1. Add 500 µL of Lysis Buffer (e.g., to 1-2 × 10⁶ cells) to the sample tube (step 7.1), immediately shake and mix until there is no cell mass, and let it stand for 1 min.
   2. Add the mixture to the adsorption column placed in a collection tube, centrifuge at 13,400 × g for 30 s, and discard the filtrate.
   3. Add the specified amount of absolute ethanol to the Washing Buffer bottle before using it for the first time. Add 500 µL of washing buffer to the adsorption column, centrifuge at 13,400 × g for 30 s, and discard the filtrate.
   4. Repeat the washing in step 7.2.3.
   5. Place the adsorption column RA into the empty collection tube and centrifuge at 13,400 × g for 2 min to remove the rinse solution.
      NOTE: The rinse solution must be removed to prevent the residual ethanol in the rinse solution from inhibiting the downstream reaction.
   6. Remove the adsorption column RA and place it in a clean RNase-free centrifuge tube. Add 50 µL of RNase-free H₂O to the middle of the adsorption membrane, let it stand for 1 min at room temperature, and centrifuge at 9,600 × g for 1 min.
3. Measure the purity and concentration of RNA.
4. Synthesize the first strand of cDNA by following the kit manufacturer's instructions.
   1. Remove genomic DNA. Take 500 ng of the extracted template RNA in an RNase-free centrifuge tube; add RNase-free ddH₂O and 4x gDNA mix to form a mixture. Blend gently with a pipette, and place in a 42 °C water bath for 2 min.
      NOTE: The amount of the reaction mixture and the amount added are related to the RNA concentration; refer to the instructions in detail. The 20 µL reaction system was used in this experiment.
   2. Directly add a 5x reverse transcription reaction system in the reaction tube of the previous step. Take 4 µL of the reverse transcription reaction system and 16 µL of the mixture from the previous step and gently mix with a pipette.
      NOTE: The reverse transcription system includes all the required reverse transcriptase, which is a directly configured reverse transcription system.
   3. Set the reverse transcription reaction: 37 °C 15 min, 85 °C, 5 s, and finally cool to 4 °C.
5. Set up the PCR reaction following the manufacturer's instructions.
   1. Add 10 µL of 2x PCR reaction enzyme mixture in the qPCR tube, 0.4 µL of primer 1, 0.4 µL of primer 2, 0.4 µL of 50x of ROX Reference Dye 1, 2 µL of template cDNA, and 6.8 µL of ddH₂O to prepare a 20 µL mixture.
   2. Perform PCR in the real-time fluorescence quantitative PCR instrument using the following settings: stage 1, 95 °C, 30 s, Rep x 1; stage 2, first 95 °C, 10 s, then 60 °C, 30 s, Rep x 40; stage 3, first 95 °C, 15 s, next 60 °C, 60 s, finally 95 °C, 15 s, Rep x 1.
      NOTE: After 4 days of differentiation, the relative expression levels of Il17a, Il17f, Rora, and Il23r in naïve CD4⁺ T cells are shown in Figure 5.