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 0.5% DNFB solution as a sensitizer For sensitization of 10 mice, prepare 1.1 mL of acetone/olive oil 4:1 (v/v)…

Representative 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

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