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Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity
In This Article
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 mice1,2,3. 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&#....
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 vivo18,19. During the course of the disease, we ob.......
Acknowledgements
This work was supported by the National Natural Science Foundation of China (No. 81602763, 81803142, 82003347), the Excellent Researcher Program of China Postdoctoral Science Foundation (No. 2017T100700), and the Regular Researcher Program of China Postdoctoral Science Foundation (No. 2016M592673). The authors would like to thank Yan Wang and Meng-Li Zhu (Core Facilities of West China Hospital, Sichuan University) for technical support of flow cytometry in this study.
....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 |
References
- Rasouli, J., et al. A distinct GM-CSF(+) T helper cell subset requires T-bet to adopt a TH1 phenotype and promote neuroinflammation. Science Immunology. 5 (52), (2020).
- Galli, E., et al.
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