In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

Summary

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (T_HGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of T_HGM, and analysis of differentiated T_HGM cells. This method can be applied to studies of the regulation and function of T_HGM cells.

Abstract

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (T_HGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)γ or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and T_HGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into T_HGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the T_HGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFNα or IL-17. The predominant expression of GM-CSF by T_HGM cells was further confirmed by the analysis of the expression of GM-CSF, IFNα, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to T_HGM cells in vitro, which will be useful in the study of T_HGM cell biology.

Introduction

CD4+ T helper (T_H) cells are essential components of the immune system, having crucial roles in the host defense against microbial pathogens, in cancer surveillance, and in autoimmunity^1,2,3. Upon T cell receptor (TCR) activation, naive CD4+ T cells can be differentiated into T_H1, T_H2, T_H 17, or regulatory T (Treg) cells under the influence of different cytokine milieus^2,4,5. Recently, a new subset of T_H cells, which predominantly produces GM-CSF, was identified and named T_HGM⁶. The differentiation of T_HGM cells is driven by IL-7 through the activation of a signal transducer and an activator of transcription 5 (STAT5). These cells express a large amount of GM-CSF while having a low expression of other T_H-cell signature cytokines such as IFNγ and IL-17⁶. GM-CSF was found to play a critical role in the development of CD4+ T cell-mediated neuroinflammation^7,8. Compared to IFNγ- or IL-17-expressing autoreactive T cells, GM-CSF-expressing T cells transferred to wild-type (WT) mice caused an earlier disease onset and higher disease severity. In addition, Csf2-/- T cells failed to induce experimental autoimmune encephalomyelitis (EAE) after being adoptively transferred to WT recipients, whereas T cells lacking IFNγ or IL-17A retained the ability to mediate EAE⁷. Moreover, a blockade of GM-CSF using neutralizing antibodies ameliorated EAE disease severity⁸. Furthermore, a deficiency of STAT5 in T cells in mice resulted in a diminished T_HGM generation and, hence, in a resistance of the mice to EAE development⁶. These findings underscore the importance of GM-CSF-expressing T_H cells in autoimmune neuroinflammatory disease. Thus, establishing a method to differentiate GM-CSF-expressing T_H cells from naive CD4+ T cells would be important in the study of the pathogenesis of autoimmune neuroinflammation and T cell-mediated immune responses. However, a protocol that efficiently generates T_HGM cells from murine naive CD4+ has not been established.

Here we present a method that differentiates murine T_HGM cells from naive CD4+ T cells. This protocol describes the whole procedure, including the extraction of the spleen from the mouse, the preparation of a single-cell suspension, the CD4 positive selection, the fluorescence-activated cell sorting (FACS), and the T_H cell differentiation and analysis. The differentiated T helper cells are analyzed by intracellular cytokine staining combined with flow cytometry to determine the cytokine expression at the single-cell level, by a quantitative real-time PCR to determine the cytokine expression at mRNA level, and by ELISA to assess the cytokine expression at protein level. This method can be applied to studies of T_HGM cell biology under various conditions, such as EAE, where GM-CSF plays an important role in pathogenesis.

Protocol

All mice used in this protocol were on the C57BL/6 genetic background and housed under specific pathogen-free conditions at the National University of Singapore. All experiments were performed using protocols approved by the Institutional Animal Care and Use Committee of the National University of Singapore.

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 in 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 (of 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 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 a restimulation and analysis.
   NOTE: Intracellular cytokine staining and ELISA assays require ≥0.5 million cells, and qPCR analysis requires ≥ 1 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.
4. For a qPCR analysis of the cytokine gene expression by differentiated cells, activate the cells with plate-bound anti-CD3e (3 μg/mL) (precoating the plate as described in step 1.3). At 3 h after the stimulation, harvest the cells to isolate total RNA using a phenol RNA extraction reagent to prepare cDNA for the qPCR. The primers and program used for the qPCR are listed in Tables 2 and 3, respectively.
5. For the analysis of the cytokine protein secretion by differentiated cells, restimulate the cells with plate-bound anti-CD3e (3 μg/mL) for 24 h (precoating the plate as described in step 1.3). Harvest the cell culture supernatant for IL-17 and GM-CSF ELISA to determine their concentrations.

Results

Naive CD4+ T cells isolated from two 8-week-old male C57BL/6 mice were divided into three portions. One portion of the cells was differentiated into T_HGM cells following the protocol described. Another portion was cultured under a T_HGM condition in the presence of anti-IL-4 antibody (10 µg/mL) to test the influence of an IL-4 blockade in the differentiation of T_HGM. The last portion was cultured under a T_H17 differentiation condition (3 µg/mL an...

Discussion

Here we described a protocol of an in vitro T_HGM differentiation from mouse naive CD4+ cells, followed by an analysis of the differentiated cells to validate the method. Of note, both spleen and lymph nodes can be used for naive CD4+ T cell purification and T_HGM differentiation. The cytokine expression determined by intracellular cytokine staining combined with flow cytometry showed that about 55% of the cells were induced to become GM-CSF-expressing cells under the T_HGM conditio...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI 1640	Biowest	L0500-500
FBS Heat Inactivated	Capricorn	FBS-HI-12A
Penicillin-Streptomycin	Gibco	15140122
10x PBS	1st Base	BUF-2040-10	Diluted to 1x PBS in sterile water
EDTA	1st Base	BUF-1053-100ml-pH8.0
10x ACK buffer	Biolegend	420301	diluted in sterile water
Cell strainer	SPL Life Sciences	93070
CD4 microbeads	Miltenyi Biotec	130-049-201
2-Mercaptoethanol	Sigma	M7522
LS column	Miltenyi Biotec	130-042-401
magnetic stand	Miltenyi Biotec	130-042-303
1 mL syringe	Terumo	SS+01T
Centrifuge	Eppendorf	Eppendorf 5810R
Sony SY3200 cell sorter	Sony	Sony SY3200 cell sorter
50 mL conical centrifuge tube	Greiner Bio-One	210261
15 mL conical centrifuge tube	Greiner Bio-One	188271
FACS tube	Corning	352054
24 well cell culture plate	Greiner Bio-One	662160
anti-mouse CD4 PerCP-eFluor 710	eBioscience	46-0041-82
PE conjugated anti-mouse CD25 (IL-2Ra, p55)	eBioscience	12-0251-82
anti-human/mouse CD44 APC	eBioscience	17-0441-82
anti-mouse CD62L FITC	eBioscience	11-0621-85
Neubauer-improved counting chamber	Marienfeld	640010
Hyclone Trypan Blue Solution	GE healthcare Life Sciences	SV30084.01
microscope	Nikon	Nikon Elipse TS100
Purified anti-mouse CD3e antibody	Biolegend	100314
Purified hamster anti-mouse CD28	BD Biosciences	553295
Purified Rat anti-mouse IFNγ	eBioscience	16-7312-85
Purified anti-mouse IL-4 Antibody	Biolegend	504102
Recombinant Mouse IL-7 Protein	R&D system	407-ML
recombinant human TGF-beta	R&D system	240-B-010
PMA	Merck Millipore	19-144
Ionomycin	Sigma	I0634
GolgiPlug protein transport inhibitor	BD Biosciences	51-2301KZ
Intracellular Fixation&Permeabilization buffer set	eBioscience	88-8824-00
anti-mouse GM-CSF PE	eBioscience	12-7331-82
FITC anti-mouse IL-17A	Biolegend	506908
APC anti-mouse IFN-gamma	Biolegend	505810
GO Taq qPCR master mix	Promega	A6002
mouse GM-CSF ELISA Ready-SET-Go!	invitrogen	88-7334-88
Mouse IL-17A Uncouted ELISA	invitrogen	88-7371-22