Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.
This method can help answer key questions in the CD4 T-cell field such as which subset of T-helper cells is the major source of GM-CSF in pathogenesis of the inflammatory diseases such as multiple sclerosis. The main advantage of this technique is that it generates more than 50%GM-CSF expressing T-cells from naive CD4 T-cells with no or few interfering gamma OI17 expression cells which provides essential cellular source for investigation of regression and function of THGM, a novel subset of T-helper cells. Demonstrating the procedure will be Yi Lu, a graduate student from my laboratory.
Begin by using forceps to lift the skin over the abdomen of a six to eight-week-old mouse and making an about four centimeter incision below the ribcage on the left side of the animal. Open the peritoneal sac to expose the spleen and use sterile forceps to transfer the spleen onto a 70 micrometer strainer pre-wet with two milliliters of cell isolation buffer. When two to three spleens have been collected, use the end of a five milliliter syringe to macerate the spleen tissue and rinse the strainer several times with one to two milliliters of cell isolation buffer until all of the cells have been flushed into the tube.
Collect the cells by centrifugation and resuspend the pellet in five milliliters of four degree Celsius ammonium chloride potassium lysis buffer. After two minutes of gentle mixing, stop the lysis with 10 milliliters of complete RPMI medium and immediately centrifuge the cells. To isolate the CD4 positive T-cells, resuspend the white blood cell pellet in five 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 one times 10 to the seventh cells per 90 microliters of buffer concentration and add 10 microliters of anti-CD4 conjugated magnetic beads per one times 10 to the seventh cells for a 15-minute incubation at four degrees Celsius with mixing every five minutes. While the cells are incubating, place a separation column onto a magnetic stand and pre-wet the column with two milliliters of buffer. Then, wash the cells with five milliliters of buffer and resuspend the pellet in one 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 one 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 two 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 naive 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 five 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-coated with 500 microliters of buffer. Gate to select the CD4 positive CD25 negative and CD44 low CD62 ligand high cell populations. Then, sort the naive CD4 positive CD25 negative CD44 low CD62 ligand high T-cell fractions into a new 15 milliliter centrifuge tube and pellet the isolated naive 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 one times 10 to the sixth 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-coated 48-well plate. Then, seed 2.5 times 10 to the fifth cells into each well along with IL7, 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 one 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 four to six 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 one milliliter of permeabilization buffer per wash. Then, stain for intracellular cytokine expression for 30 minutes in the dark. For quantitative PCR analysis of the differentiated cell cytokine gene expression, activate the cells with plate-bound anti-CD3 epsilon for three hours.
Then, use a phenol RNA extraction reagent to isolate the total RNA to allow cDNA preparation for quantitative PCR. To assess the cytokine protein secretion by the differentiated cells, re-stimulate the cells with plate-bound anti-CD3 epsilon for 24 hours and analyze the culture supernatants for IL17 and GM-CSF by ELISA. About 55%of cells cultured under GM-CSF T-helper cell differentiation conditions expressed GM-CSF whereas only about 2%of cells differentiated under T-helper 17 conditions expressed this cytokine.
Compared to T-helper 17 differentiation conditions that result in IL17 production by about 8%of cells, GM-CSF T-helper cell differentiation conditions induced IL17 expression in only about 1%of cells. Notably, only a fraction of cells expresses IFN gamma or IL4 under either culture condition. GM-CSF T-helper differentiated cells demonstrate a significantly higher expression of CSF2 mRNA, but a much lower expression of IL17 compared to the T-helper 17 cells.
This correlates with the GM-CSF protein expression observed in culture supernatants from T-helper GM-CSF and T-helper 17 cells, with three times the concentration of the cytokine detected in T-helper GM-CSF cell cultures and no measurable IL17 at all. T-helper GM-CSF cells also exhibit a slightly lower expression of Rorc, a higher Stat5 expression, and a slightly lower expression of Stat3 than T-helper 17 cells indicating that although T-helper cell GM-CSF and IL17 differentiation are governed by different transcriptional factors, they may share some common features. While attempting this procedure, it's important to remember to perform all the steps in biological safety cabinet to keep the cells and the reagents sterile.
Following this procedure, other methods like transcriptome analysis can be performed to answer additional questions such as, what are the important genes expressed by THGM cells? After its development, this technique paved the way for researchers in the field of immunology to explore how T-cell derived GM-CSF contributes to inflammation in rodents. Don't forget that working with beta-mercaptoethanol and RNA extraction reagent can be extremely hazardous and precautions such as using fume hoods should always be taken while using these reagents.
