Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity

Summary

Here, we present a simple and standardized method of analyzing the granulocyte-macrophage colony-stimulating factor-producing T helper subset in vivo.

Abstract

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified as a distinct subset of T helper cells (GM-CSF⁺ IFN-γ^- IL-17A^- IL-22^- effector CD4⁺ T cells) in human and mice. Contact hypersensitivity (CHS) is considered an excellent animal model for allergic contact dermatitis (ACD) in human, manifesting an intact T cell-mediated immune response. To provide a standardized and comprehensive assay to analyze the Th-GM cell subset in the T cell-dependent immune response in vivo, a murine CHS model was induced by sensitization/challenge with a reactive, low-molecular-weight, organic hapten, 2,4-dinitrofluorobenzene (DNFB). The Th-GM subset in effector CD4⁺ T cells generated upon immunization with the hapten was analyzed by flow cytometry. We found that Th-GM was mainly expanded in lesions and draining lymph nodes in the DNFB-induced CHS mouse model. This method can be applied to further study the biology of Th-GM cells and pharmacological research of therapeutic strategies centered on GM-CSF in various conditions, such as ACD.

Introduction

The granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing T helper cells-the Th-GM subset-has been emerging as a distinct subset of T helper cells in human and mice and is considered to comprise "GM-CSF-expressing only" (GM-CSF⁺ IFN-γ^- IL-17A^- IL-22^-) CD4 T cells identified by single-cell RNA analysis, mass cytometry, and GM-CSF fate mapper mice^1,2,3. In 2014, Sheng et al. reported signal transducer and activator of transcription 5 (STAT5) programming of the Th-GM subset and conceptualized the "Th-GM" subset for the first time^4,5. Th-GM cells are characterized by cytokine expression of GM-CSF, IL-2, TNF-α, IL-3, CCL20, and chemokine receptors C-X-C chemokine receptor type (CXCR) 4 or CXCR6^1,2. STAT and/or the NF-κB pathway are essential for Th-GM lineage differentiation. An in vitro method was established to differentiate naïve CD4 T cells into Th-GM cells using IL-7 in the presence of TCR stimuli⁶. Meanwhile, the cytokines IL-23 and IL-1β were shown to maintain the expression and pathogenicity of Th-GM cells ex vivo^3,7.

Elevation of Th-GM cells has been associated with several autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis^2,8,9, suggesting a potential role in the pathogenesis of autoimmunity¹⁰. Accumulating evidence suggests that GM-CSF can function as an inflammatory mediator. Mice genetically overexpressing Csf2 (gene encoding GM-CSF) in CD4⁺ T cells spontaneously developed neurological deficits accompanied by the infiltration of phagocytes into the central nervous system. In a T cell-transfer colitis model, the adoptive transfer of Csf2^−/− T cells into Rag1^−/− mice significantly reduced the clinical and histopathological features of the disease. However, there are few reports of the roles of the Th-GM subset in allergic diseases, such as ACD.

ACD is among the most common inflammatory dermatological conditions with high prevalence in work and life environments^11,12. It is a type IV delayed-type hypersensitivity response mediated by an intact immune circuit that develops in two temporally segmented phases: sensitization and elicitation. Human ACD is triggered by exposure to some chemicals (haptens or metals) that lead to sensitization. During this phase, a T cell-mediated response is primed by hapten-protein complexes presented by antigen-presenting cells. Upon subsequent exposure to the same hapten, hapten-specific effector and memory T cells are reactivated and localize to the skin, a process involving the infiltration of a variety of immune cell populations. This acute inflammatory response is known as elicitation, resulting in the full development of lesions¹³. Human ACD can be studied using animal models of contact hypersensitivity (CHS)¹⁴.

The CHS model induced by a reactive, low-molecular-weight, organic hapten, 2,4-dinitrofluorobenzene (DNFB), is a commonly used murine model that has been utilized in the study of the pathology as well as potential therapeutic interventions of ACD^15,16. Thus, this T cell-dependent model could be applied to study the generation of the Th-GM subset in allergic disease. Here, we induced a murine model of CHS with DNFB, analyzed the generation of Th-GM in lesions and draining lymph nodes, and found that the Th-GM subset was mainly expanded upon reexposure to the same hapten. This suggests that the Th-GM subset could be essential for ACD development and represents a specific therapeutic target in ACD.

Protocol

All mice utilized in this protocol were on the C57BL/6 genetic background, kept under specific pathogen-free conditions, and provided with food and water ad libitum. All experiments were approved by the animal welfare ethical review body of West China Medical Center, Sichuan University (20210302059).

1. Reagent and material preparation

1. 0.5% DNFB solution as a sensitizer
   1. For sensitization of 10 mice, prepare 1.1 mL of acetone/olive oil 4:1 (v/v) mixture: mixing 880 µL of acetone and 220 µL of olive oil to allow 0.1 mL of excess volume to account for any minor volume losses. Add 5 µL of DNFB to 1.1 mL of homogenized acetone/olive oil 4:1 mixture.
      NOTE: Prepare freshly on the day of sensitization. The acetone/olive oil mixture should be prepared in a fume hood.
2. 0.2% DNFB solution as challenger
   1. To challenge 10 mice, add 0.5 µL of DNFB in 0.25 mL of a homogenized acetone/olive oil 4:1 mixture described in step 1.1. Use the acetone/olive oil 4:1 mixture as a vehicle when challenging the mice after sensitization.
      NOTE: The acetone/olive oil mixture should be prepared in a fume hood.
3. Chloral hydrate as anesthetic
   1. Dissolve 4 g of chloral hydrate in 50 mL of phosphate-buffered saline (PBS) to make an 8% working solution. Filter and store at 4 °C for up to 3 months. Use 5 µL per g of body weight for a mouse.
4. 50x collagenase IV stock
   1. Dissolve 100 mg of collagenase IV in 1 mL of basic RPMI 1640 medium (not supplemented with antibiotics or fetal bovine serum [FBS]) to make a 100 mg/mL stock. Store at -20 °C in 100 µL aliquots for up to 6 months.
5. 1,000x DNase I stock
   1. Dissolve 5 mg of DNase I in 1 mL of 0.15 M NaCl to make a 5 mg/mL stock. Store at -20 °C in 100 µL aliquots for up to 6 months.
6. Stop buffer
   1. Dissolve 1.681 g of EDTA in 50 mL of basic RPMI 1640 medium to make a 100 mM stock. Store at 4 °C for up to 2 weeks.
7. Wash medium
   1. Dissolve 0.372 g of EDTA to a final concentration of 2 mM and 5 mL of FBS to a final concentration of 1% with 500 mL of PBS. Filter and store at 4 °C for up to 1 month.
8. Staining buffer
   1. Dissolve 0.372 g of EDTA to a final concentration of 2 mM and 10 mL of FBS to a final concentration of 2% with 500 mL of PBS. Filter and store at 4 °C for up to 1 month.
9. Restimulation reagents
   1. Dissolve 1 mg of phorbol 12-myristate 13-acetate (PMA) in 20 mL of DMSO to make a 50 µg/mL stock (500x) and store it at -20 °C in 50 µL aliquots.
   2. Dilute 250 µL of ionomycin in solution (4 mg/mL) in 7.75 mL of DMSO to make a 500 µg/mL stock (500x) and store it at -20 °C in 50 µL aliquots.
   3. Aliquot 1 mL of protein transport inhibitor solution (containing brefeldin A) in 50 µL and store the aliquots at 4 °C.

2. Induction of CHS in mice

1. On day -5, anesthetize the mice by injecting 400 mg/kg chloral hydrate intraperitoneally.
2. Using a pet shaver, shave a 2 cm × 2 cm area on the mouse abdomen.
   NOTE: Avoid scratching the mouse skin; keep the integrity of the cutaneous barrier intact.
3. Smear 100 µL of 0.5% DNFB solution onto the shaved abdomens gently and evenly with a micropipette with disposable tips. Hold the mice 5-10 s to allow some of the solvent to evaporate.
4. On day -4, repeat steps 2.1-2.3 for another sensitization.
   NOTE: Two sensitization events work better than one.
5. On day 0, challenge the right ear of the mice with 20 µL of 0.2% DNFB using a micropipettor with disposable tips, and treat the left ear with the same volume of acetone/olive oil 4:1 mixture as the vehicle.
   NOTE: Challenge the dorsal and ventral sides of the ear with equal volumes of DNFB or vehicle.
6. In the next 3 days, measure the ear thickness of the right and left ears of mice daily using a dial thickness gauge, and calculate the increase in ear thickness: right ear thickness - left ear thickness.
   ​NOTE: A gradual increase in ear thickness could be observed in 0.2% DNFB-challenged mouse ears compared with vehicle-treated ears.

3. Sample collection of CHS mice

1. On day 3, anesthetize the mice by an intraperitoneal injection of 400 mg/kg chloral hydrate. Wait for 5-10 min for the mouse to be in a state of unconsciousness and perform a gentle toe pinch on both rear feet to confirm that the mice are deeply anesthetized.
2. Take a photo of each ear per mouse using a camera. Score the incrustation (scaling on the ear) and redness of the ear (erythema on the ear) independently to evaluate the severity of inflammation on a scale from 0 to 5: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked; 5, most severe.
3. Euthanize the mouse by cervical dislocation. Cut off the whole ear and dissect the parallel draining lymph nodes of mice using sterile sharp scissors and forceps. Place the tissues into one well of a 6-well plate placed on ice containing 5 mL of prechilled PBS for enzymatic or physical dissociation to generate single-cell suspensions (Supplemental Figure S1A and Supplemental Figure S2A).
4. For histological analysis, fix the ear specimens in 4% paraformaldehyde, dehydrate, and embed them in paraffin for immunohistochemical staining, as described elsewhere¹⁷.

4. Preparation of single-cell suspension from ears

1. Split the ventral and dorsal sides of the ear by pinching and tearing the cut ends with two curved forceps (Supplemental Figure S1B). Place them in a well of a 6-well plate containing 4.5 mL of digestion buffer, ensuring that those tissues are completely immersed in the digestion buffer (Supplemental Figure S1C).
   NOTE: The digestion buffer was composed of RPMI 1640 containing 10% FBS + 25 mM HEPES + 2 mg/mL collagenase IV + 5 µg/mL DNase I.
2. Incubate the ear leaflets in a 37 °C cell culture incubator for 20 min. Cut the ear leaflets with scissors as small as possible to facilitate tissue digestion (Supplemental Figure S1D), and put the sample back in the incubator for another 40 min.
   NOTE: Efficient digestion can be achieved within 40-50 min. Overdigestion at 37 °C affects cell viability.
3. Add 0.5 mL of stop buffer per well to neutralize the enzymatic activities of collagenase IV and DNase I. Perform the next steps on ice.
4. Transfer the ear tissue fragments with a pair of curved forceps to a 50 mL centrifuge tube containing a 70 µm cell strainer and pipette the whole volume of medium into the same cell strainer.
5. Disrupt the tissues using the top end of a 1 mL syringe plunger. Press in circular movement against the 70 µm cell strainer until only white connective tissues remain (Supplemental Figure S1D).
6. Rinse the cell strainers twice with 1,000 µL of wash medium. Transfer the entire volume of the well into a 50 mL centrifuge tube through a 40 µm cell strainer. Keep the tube on ice.

5. Preparation of single-cell suspensions from draining lymph nodes (dLNs)

1. Transfer the lymph nodes with a pair of curved forceps to a 70 µm cell strainer on top of the well of a 6-well plate (Supplemental Figure S2B). Dissociate the lymph nodes using the top end of a 1 mL syringe plunger. Press in circling movements against the 70 µm cell strainer until only white debris remains (Supplemental Figure S2C).
2. Rinse the cell strainer twice with 1,000 µL of wash medium. Transfer the entire volume of the well to a 5 mL tube. Keep the tubes on ice.

6. Restimulation of the Th-GM subset with 12-myristate 13-acetate (PMA)/ionomycin in the presence of a protein transport inhibitor

1. Centrifuge the cells isolated from the ear and the draining lymph node samples for 8 min at 400 × g at 4 °C, and discard the supernatant. Resuspend the pellet with 1,000 µL of wash medium gently.
2. Collect a small portion of each sample to count the total number of cells derived from the ear and draining lymph node using a hemocytometer. Add 0.04% trypan blue to measure cell viability.
   NOTE: Cell viability should be more than 60% for subsequent T cell stimulation and analysis. Cell density could be 1-5 × 10⁶/mL.
3. Centrifuge the cells in step 6.1 and discard the supernatant. Gently resuspend the pellet with 1,000 µL of RPMI 1640 medium containing 2% FBS, and seed 1 mL of the single-cell suspension (10⁶ cells/mL) in 12-well flat-bottom tissue culture plates.
4. Add 2 µL of phorbol 12-myristate 13-acetate (PMA) (final concentration 100 ng/mL), 2 µL of ionomycin (final concentration 1,000 ng/mL), and 1 µL of protein transport inhibitor (containing Brefeldin A) to each well in the 12-well plate containing 1 mL of the single-cell suspension above and incubate at 37 °C for 4 h.

7. Analysis of Th-GM subsets generated in vivo by cell surface and intracellular staining

1. Transfer the stimulated cells to 1.5 mL centrifuge tubes using a pipette. Centrifuge the centrifuge tubes for 8 min at 400 × g at room temperature, and discard the supernatant.
2. Wash the cell pellet once with 1 mL of staining buffer and pellet the cells by centrifugation, as described in step 7.1. Resuspend the cells at 1-10 × 10⁶/mL in 0.5 mL of staining buffer.
3. Add 0.5 µL of viability dye stock solution (see the Table of Materials) to 0.5 mL of cell suspension and vortex immediately. Incubate the mixture for 10 min at room temperature in the dark. Wash the cells twice with 1 mL of staining buffer, and repeat the centrifugation in step 7.1 to pellet the cells.
4. Resuspend the pellet in 0.1 mL of staining buffer. Add 1 µL of anti-CD4 antibody (FITC-conjugated), 1 µL of anti-CD44 antibody (APC-conjugated), and 1 µL of anti-CD62L antibody (PE/Cyanine7-conjugated) to three 0.1 mL aliquots of the cell suspension. Vortex immediately and incubate at room temperature for 15 min, protected from light.
5. Repeat the washes and centrifugation in step 7.3.
6. Resuspend the pellet in 200 µL of IC fixation buffer and incubate for 20 min in the dark. Meanwhile, prepare permeabilization buffer (1x) by diluting 1 part of permeabilization buffer (10x) with 9 parts of distilled water. Wash the cells twice with 1 mL of permeabilization buffer (1x) by centrifugation at 600 × g for 5 min at room temperature.
7. Resuspend the pellet in 0.1 mL of permeabilization buffer (1x) with antibodies against GM-CSF (PE-conjugated, 1 µL/test), IFN-γ (BV711-conjugated, 1 µL/test), IL-17A (BV421-conjugated, 1 µL/test), and IL-22 (PerCP/Cyanine5.5-conjugated, 5 µL/test) and incubate for 30 min at room temperature in the dark.
8. Repeat the washes and centrifugation in step 7.6.
9. Resuspend the cell pellet in 200 µL of PBS and transfer the suspension to a round-bottom test tube. Run the stained cells in a flow cytometer.

8. Gating strategy for identifying the Th-GM subset

1. Analyze the flow cytometry data (see the Table of Materials for details about the software used) using the gating strategy illustrated in Supplemental Figure S1. For the software used here, download and set it up in the computer.
2. Import the flow cytometry data by clicking on File |Open | Import FCS Files into the software, and annotate the group and name of each sample by selecting the Rename tab.
3. Choose the lymphocytes on an FSC versus SSC plot (FSC ^low SSC ^low, P1) to exclude debris found in the lower-left corner and myeloid cells with large size and high granularity by clicking the Density Plot, and show the X-axis of this plot as FSC-A and the Y-axis as SSC-A.
4. Double-click the events in P1 to generate another density plot, and separate the single cells (shown as a correlated line, P2) from cell aggregates by selecting the cell height and area in this plot (X-axis as FSC-A vs Y-axis as FSC-H).
5. Double-click the events in P2 to generate another density plot, and show the X-axis of this plot as FSC-A and Y-axis as SSC-A. Gate the viable cell populations using FVS 780^- events (P3).
6. Double-click the events in P3 to generate the next density plot (the X-axis as CD4-A and the Y-axis as SSC-A), and distinguish the CD4 T cells by choosing the CD4⁺ population (P4).
7. Double-click the events in P4 to generate another density plot (the X-axis as CD44-A and the Y-axis as CD62L-A), and define the effector T helper cells by choosing the upper-right population of this plot (CD62L^- CD44⁺, P5).

9. Statistical analysis

1. Perform statistical analyses, comparing two groups using an unpaired t-test. *P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 (mean ± SD).

Results

DNFB-induced CHS (contact hypersensitivity) in mice
To induce CHS in mice, the mice were sensitized and challenged with DNFB applied to the ear skin, as illustrated in Figure 1A. Ear thickness, an indicator of epidermal spongiosis, was markedly increased in DNFB-challenged mice compared to vehicle-treated mice (Figure 1B, 70 vs 3 µm at day 1, 203 vs 7.5 µm at day 2, 276 vs 5 µm at day 3). Seventy-two hours after the challeng...

Discussion

This protocol provides a simple in vivo assay to analyze the generation and expansion of the Th-GM cell subset. It is essential to utilize a T cell-mediated disease model in mice initiated by haptens or antigens, mimicking that activation in human. DNFB is a small-molecule hapten that is more economical and time-saving than peptide or protein antigens for triggering the T cell immune response in vivo^18,19. During the course of the disease, we ob...

Materials

Name	Company	Catalog Number	Comments
2,4-dinitrofluorobenzene	BT REAGENT	P0001746	CAS NO: 70-34-8
Acetone	CHRON CHEMICALS	/	67-64-1
anti-CD4 antibody	Biolegend	300506	1:100 Diluted
anti-CD44 antibody	Biolegend	103012	1:100 Diluted
anti-CD62L antibody	Biolegend	104417	1:100 Diluted
anti-GM-CSF antibody	BD Bioscience	554507	1:100 Diluted
anti-IFN-γ antibody	Biolegend	505836	1:100 Diluted
anti-IL-17A antibody	BD Bioscience	563354	1:100 Diluted
anti-IL-22 antibody	Biolegend	516411	5 µL/test
CD45	Biolegend	103101	1:200 Diluted
Chloral hydrate	CHRON CHEMICALS	/	302-17-0
Dial thickness gauge (0.01 mm type)	PEACOCK	G-1A	/
DMSO	LIFESCIENCES	D8371	67-68-5
EDTA Na2	Solarbio	E8030	6381-92-6
F4/80	Biolegend	123102	1:200 Diluted
Fixable Viability Stain 780	BD Bioscience	565388	1:1,000 Diluted, viability dye
Flow cytometer	BD Bioscience	BD FACS ARIA II SORP	/
GraphPad Prism	GraphPad Software	Prism 7	Software for statistics and graphing
Intracelluar Fixtation and Permeablization Buffer Set	Thermo Fisher	88-8824-00	prepared freshly
Ionomycin	Sigma-Aldrich	407951	CAS NO: 56092-81-0
Ly6G	Biolegend	127602	1:200 Dilutied
NovoExpress	Agilent	/	Software for flow cytometry data analysis; https://www.agilent.com.cn/zh-cn/product/research-flow-cytometry/flow-cytometry-software/novocyte-novoexpress-software-1320805
Olive oil	YUANYE BIO	S30503	8001-25-0
PMA	Sigma-Aldrich	P8139	CAS NO: 16561-29-8
Protein Transport Inhibitor (Containing Brefeldin A)	BD Bioscience	555029	1 µL/mL