An In Vitro Technique to Differentiate GM-CSF Producing T Helper Cells from Naïve T Cells

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Reagent and Material Preparation

1. Prepare 500 mL of phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) and 1 mM ethylenediaminetetraacetic acid (EDTA).
   NOTE: Keep the buffer on ice throughout the whole procedure for optimal results.
2. Prepare 50 mL of complete RPMI media containing RPMI 1640, 10% FBS, and 1% penicillin-streptomycin. Use complete RPMI media containing 50 µM β-mercaptoethanol for the T_HGM cell differentiation. Add the β-mercaptoethanol to a fume hood.
3. Prepare 7.5 mL of an anti-CD3e antibody mixture by diluting anti-CD3e concentrated stock to 3 μg/mL in PBS. Coat 48-well plates by adding 150 μL of the antibody mixture to each well and incubate the plate at 37 °C for at least 1 h, or at 4 °C overnight.
4. Sterilize a pair of scissors and forceps and keep them in 70% ethanol between dissections.

2. Preparation of Murine Splenocytes

1. Euthanize the mouse (6–8 weeks old) using an institutionally-approved CO₂ asphyxiation or cervical dislocation. Spray the mouse with 70% ethanol and mount it on a polystyrene block on its back. Transfer the mouse to a biological safety cabinet to proceed with the following steps.
2. Hold the skin using a pair of forceps and cut the skin below the rib cage on the left side for about 4 cm, using a pair of scissors. Open the peritoneal sac to expose the spleen and use sterile forceps to extract the spleen.
3. Place a 70 μm cell strainer on a 50 mL tube and pre-wet it with 2 mL of buffer. Place the spleens on the cell strainer (2–3 spleens for one cell strainer) and macerate them using the end of a 5 mL syringe plunger.
4. Rinse the strainer and tube several times with 1–2 mL of cell isolation buffer until all cells are flushed into the tube. Discard the cell strainer and centrifuge the cells at 350 x g for 5 min at 4 °C. Discard the supernatant.
5. Resuspend the cell pellet with 5 mL of cold (4 °C) ammonium-chloride-potassium (ACK) lysing buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA; pH 7.2–7.4) and gently mix them for 2 min. Add 10 mL of complete RPMI media to neutralize the ACK buffer and immediately centrifuge the cells at 350 x g for 5 min at 4 °C. Discard the supernatant after the centrifugation.

3. Isolation of CD4⁺ T Cells Using Magnetic CD4 Microbeads and a Cell Separation Column

1. Resuspend the cell pellet in 5 mL of cell isolation buffer. Filter the cell suspension using a pre-separation filter (30 μm) into a new 15-mL tube to remove any debris.
2. Take 10 μL of the cell suspension and make a 10x dilution by adding 90 μL of the buffer. Take 10 μL of the diluted cell suspension and mix it with 10 μL of trypan blue for cell counting. Count the cells in a hemocytometer to determine the yield of viable cells.
3. Centrifuge the cells at 350 x g for 5 min at 4 °C and discard the supernatant.
4. Resuspend the cells in 90 μL of buffer per 10⁷ cells. Add 10 μL of magnetic CD4 microbeads per 10⁷ cells. Incubate the cells for 15 min at 4 °C. For optimal results, mix the cell suspension gently every 5 min during incubation.
5. While the cells are incubating, place a separation column on the magnetic stand. Pre-wet the column with 2 mL of buffer.
6. At the end of the cell/bead incubation, wash the cells with 5 mL of buffer and centrifuge them at 350 x g for 5 min at 4 °C. Discard the supernatant.
7. Resuspend the cells in 1 mL of buffer, load the sample into the cell separation column, and let it flow through. Use a 15-mL centrifuge tube to collect the CD4- cell fraction. Add 1 mL of buffer to wash the reservoir before it is dry. Repeat the washing step 2x.
8. Remove the cell separation column from the magnetic stand and place it on a new 15 mL centrifuge tube. Add 2 mL of cell isolation buffer to the cell separation column and apply the plunger firmly to force the cells out of the column.
9. Centrifuge the fraction at 350 x g for 5 min at 4 °C to obtain CD4⁺ cells. Discard the supernatant.

4. Purification of the Naive CD4⁺ T cells (CD4⁺CD25^–CD44^loCD62L^hi) by Fluorescence-activated Cell Sorting and T_HGM differentiation

1. Resuspend the obtained CD4⁺ cell pellet in 500 µL of buffer. Mix the cells with a fluorescent-conjugated antibodies mixture containing CD4-PerCp (2.8 µg/mL), CD44-APC (2.4 µg/mL), CD25^–PE (2 µg/mL), and CD62L-FITC (10 µg/mL) (Table 1). Incubate the cells on ice for 20–30 min, while protecting them from light.
   NOTE: The concentrations of the antibodies were optimized previously in the lab. The number of antibodies listed is for cells from 1–2 mice.
2. After the incubation, wash the cells with 5 mL of buffer and centrifuge the cells at 350 x g for 5 min at 4 °C.
3. Discard the supernatant and resuspend the cells in 500 µL of buffer. Filter the cells again using a nylon mesh.
4. Transfer the cell suspension to a FACS tube for cell sorting. Precoat the collection FACS tubes with 500 µL of buffer.
5. Obtain a CD4⁺CD25^–CD44^loCD62L^hi population (of naive CD4⁺ T cells) by FACS sorting. Gate on CD4⁺CD25^– first, and then select CD44^loCD62L^hi cells from this population.
6. Collect the naive CD4⁺ T cell fractions into a new 15-mL centrifuge tube. Centrifuge the cells at 350 x g for 5 min at 4 °C and discard the supernatant.
7. Resuspend the naive CD4⁺ T cells at a concentration of 106 cells/mL in complete RPMI media containing 50 µM β-mercaptoethanol.
8. Aspirate the anti-CD3e antibodies used for precoating in the 48-well plate. Seed 0.25 million (250 µL) cells in each well with IL-7 (2 ng/mL), anti-CD28 (1 µg/mL), and anti-IFNγ (10 µg/mL) (Table 1).
   NOTE: The concentrations of cytokine and antibodies were optimized previously in the lab for a T_HGM cell differentiation.
9. Incubate the cells at 37 °C with 5% CO₂ for 3 days. Do not change the medium during the incubation.

5. Analysis of the Mouse T_HGM Cells Generated In Vitro

1. Using a microscope, check the cell differentiation 3 d after the differentiation. Harvest the cells and centrifuge them at 350 x g for 5 min at 4 °C. Wash the cells 2x with complete RPMI media.
   NOTE: Differentiated cells are larger in size than naive CD4⁺ T cells.
2. Resuspend the cells in 1 mL of complete RPMI media. Take 10 μL of cell suspension and mix it well with 10 μL of trypan blue to determine the cell number with a hemocytometer. Divide differentiated cells into three potions for restimulation and analysis.
   NOTE: Intracellular cytokine staining requires ≥0.5 million cells.
3. For intracellular cytokine staining, restimulate the cells with phorbol 12-myristate 13-acetate (PMA) (100 ng/mL) and ionomycin (1 μg/mL) in the presence of a protein transport inhibitor (1 µL/mL) for 4–6 h.
   1. Harvest the cells and stain them with anti-CD4 antibody (PerCP-conjugated, 0.2 mg/mL, 1:100 dilution; or FITC-conjugated, 0.5 mg/mL, 1:100 dilution) for 30 min.
   2. Fix the cells with 200 μL of fixation buffer for 20–60 min. After the fixation, wash the cells 2x with 1 mL of permeabilization buffer.
   3. Perform intracellular staining with antibodies against GM-CSF (PE-conjugated, 0.2 mg/mL, 1:100 dilution), IL-17A (FITC-conjugated, 0.5 mg/mL, 1:100 dilution; APC-conjugated, 0.2 mg/mL, 1:100 dilution), IL-4 (APC-conjugated, 0.2 mg/mL, 1:100 dilution), and IFNγ (APC-conjugated, 0.2 mg/mL, 1:100 dilution; PE-conjugated, 0.2 mg/mL, 1:100 dilution) for 30 min in the dark.

Table 1: Used cytokines and antibodies.