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

Granulocyte-macrophage colony-stimulating factor, GM-CSF is secreted by GM-CSF-producing T helper or T_HGM cells. These cells show a low expression of other T helper cell-specific cytokines.

To begin in vitro differentiation of T_HGM cells, take a T helper cell suspension — isolated from the mouse spleen — containing antigen-activated mature cells and undifferentiated naïve cells.

Add fluorescently-labeled antibodies targeting characteristic surface markers of the naïve and mature cells. Perform fluorescence-activated cell sorting to isolate the naïve T cells.

Plate the cells in a well coated with antibodies that bind to the T cell receptor complex. Add an antibody that binds to the co-stimulatory receptor. The synergistic binding activates the cells.

Add interleukin 7 — a cytokine — inducing differentiation into T_HGM cells and GM-CSF production. Add phorbol myristate acetate, PMA — a pro-inflammatory compound, ionomycin — a calcium ionophore, and a protein transport inhibitor.

PMA enters the cell and activates a protein kinase. The membrane-permeable ionomycin translocates the calcium ions inside the cell — raising the intracellular calcium concentration.

The protein kinase activation and a high calcium level initiate signals that restimulate the T_HGM cells — resulting in higher GM-CSF production. The protein transport inhibitor blocks GM-CSF secretion, aiding intracellular detection.

Perform immunofluorescence staining, detecting intracellular cytokines, and analyze the cells using flow cytometry. A majority of cells showing high GM-CSF levels but a low expression of other cytokines indicate successful differentiation into T_HGM cells.

To isolate the CD4-positive T cells, resuspend the white blood cell pellet in 5 milliliters of cell isolation buffer, and filter the cell suspension through a 30-micrometer strainer into a new 15-milliliter tube to remove any debris. After counting, resuspend the cells at a 1 x 10⁷ cells per 90 microliters of buffer concentration, and add 10 microliters of anti-CD4 conjugated magnetic beads per 1 x 10⁷ cells for a 15-minute incubation at 4 degrees Celsius, with mixing every 5 minutes.

While the cells are incubating, place a separation column onto a magnetic stand, and pre-wet the column with 2 milliliters of buffer. Then, wash the cells with 5 milliliters of buffer and resuspend the pellet in 1 milliliter of buffer. Load the sample onto the column, and collect the CD4-negative fraction in a 15-milliliter conical tube.

When all of the cells have passed through the column, wash the reservoir three times with 1 milliliter of buffer per wash before it dries. After the last wash, transfer the column to a new 15-milliliter conical tube, and plunge the column with 2 milliliters of cell isolation buffer to expel the positive bead fraction. Then, collect the CD4-positive cells by centrifugation.

For fluorescence-activated cell sorting of the naïve T cells, resuspend the bead-sorted CD4-positive cell pellet in 500 microliters of buffer, and mix the cells with an appropriate fluorescence-conjugated antibody cocktail. After 20 to 30 minutes on ice, protected from light, wash the cells in 5 milliliters of fresh buffer, and resuspend the pellet in 500 microliters of fresh buffer.

Filter the cells through a nylon mesh, and transfer the cells to a FACS tube pre-coded with 500 microliters of buffer. Gate to select the CD4-positive, CD25-negative, and CD44 low CD62 ligand high cell populations. Then, sort the naïve CD4-positive, CD25-negative, CD-44 low, CD-62 ligand high T cell fractions into a new 15-milliliter centrifuge tube and pellet the isolated naïve CD4-positive T cells by centrifugation.

For granulocyte-macrophage colony-stimulating factor, or GM-CSF, T helper cell differentiation, resuspend the sorted T cells at a 1 x 10⁶ cells per milliliter concentration in complete RPMI medium containing 50 micromolar beta-mercaptoethanol, and aspirate the anti-CD3 epsilon antibody from each well of a pre-coded 48-well plate.

Then, seed 2.5 x 10⁵ cells into each well along with IL-7, anti-CD28, and anti-IFN gamma, and incubate the cells at 37 degrees Celsius and 5% CO₂ for three days. On the third day of culture, use a microscope to check the cell differentiation, and harvest the cells for centrifugation.

Resuspend the pellet in fresh complete RPMI medium for two washes, resuspending the cells in 1 milliliter of fresh complete medium after the second wash for counting. Split the cells into three equal volumes, and re-stimulate the first aliquot of cells with PMA and ionomycin in the presence of a protein transport inhibitor for 4 to 6 hours.

At the end of the stimulation, harvest the cells for staining with anti-CD4 antibody. After 30 minutes, fix the cells with 200 microliters of fixation buffer for 20 to 60 minutes, followed by two washes in 1 milliliter of permeabilization buffer per wash. Then, stain for intracellular cytokine expression for 30 minutes in the dark.