Expansion and Polarization of Human γδT17 Cells in vitro from Peripheral Blood Mononuclear Cells
外周血单核细胞体外扩增和极化人 γδT17 细胞
发布: 2024年01月05日第14卷第1期 DOI: 10.21769/BioProtoc.4914 浏览次数: 2318
评审: Vivien J. Coulson-ThomasAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
May 2022
Abstract
γδ T cells play a critical role in homeostasis and diseases such as infectious diseases and tumors in both mice and humans. They can be categorized into two main functional subsets: IFN-γ-producing γδT1 cells and IL-17-producing γδT17 cells. While CD27 expression segregates these two subsets in mice, little is known about human γδT17 cell differentiation and expansion. Previous studies have identified γδT17 cells in human skin and mucosal tissues, including the oral cavity and colon. However, human γδ T cells from peripheral blood mononuclear cells (PBMCs) primarily produce IFN-γ. In this protocol, we describe a method for in vitro expansion and polarization of human γδT17 cells from PBMCs.
Key Features
• Expansion of γδ T cells from peripheral blood mononuclear cells.
• Human IL-17A-producing γδ T-cell differentiation and expansion using IL-7 and anti-γδTCR.
• Analysis of IL-17A production post γδ T-cell expansion.
Keywords: γδT17 cell (γδT17 cell)
γδ T cell (γδ T cell)
IL-17 (IL-17)
Peripheral blood mononuclear cells (外周血单核细胞)
Background
γδ T cells are a group of lymphocytes consisting of a γ and δ chain, being considered as a bridge linking innate and adaptive immunity (Melandri et al., 2018). γδ T cells are mainly found in cutaneous and mucosal tissues such as the skin, gut, and oral mucosa (Cai et al., 2011; Wu et al., 2014; Hovav et al., 2020), but they also circulate in peripheral blood (Davey et al., 2018). γδ T cells are commonly classified based on their TCR chains and cytokine production. In mice, γδ T cells bear different Vγ chains ranging from Vγ1 to Vγ7, according to Heilig and Tonegawa nomenclature (Heilig and Tonegawa, 1986). Murine γδ T cells are heterogenous, secrete different cytokines, and can be divided into two main functional subsets: IFN-γ-producing γδT1 cells and IL-17-producing γδT17 cells (Ribot et al., 2009). Surface markers such as CD27 and CCR6 can be used to define these two subsets (Haas et al., 2009; Ribot et al., 2009). In humans, γδ T cells can be distinguished by the δ chain, including Vδ1, Vδ2, Vδ3, and Vδ5 (Ling et al., 2022). While previous studies have reported methods for in vitro expansion of human γδ T cells (Ness-Schwickerath et al., 2010; Caccamo et al., 2011; Hur et al., 2023), the majority of peripheral blood γδ T cells in humans are Vδ2+ subsets that mainly produce IFN-γ. It is difficult to investigate human γδT17 cells as they are scarce, and little has been done to expand human γδT17 cells in vitro. A previous report from Michel et al. demonstrated that IL-7 promotes the expansion of human IL-17-producing γδ T cells (Michel et al., 2012). In this study, we describe a modified method for in vitro polarization of human γδT17 cells from peripheral blood mononuclear cells (PBMCs) (Chen et al., 2022).
Materials and reagents
24-well plate (Falcon, catalog number: 353047)
6-well plate (NEST, catalog number: 703001)
Blood-drawing tubes containing EDTA-K2 (Improve Medical, catalog number: 101680720)
12 × 75 mm plastic tubes (Falcon, catalog number: 352052)
Anti-human γδTCR Ab (Beckman, clone: IMMU510, IM1349)
Human IL-7 (R&D system, catalog number: 207-IL-005/CF)
RPMI-1640 medium (Sigma-Aldrich, catalog number: R8758-500ML)
2-Mercaptoethanol (Gibco, catalog number: 21985023)
Sterile PBS (Sangon Biotech, catalog number: E607008-0500)
Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11150)
Penicillin-Streptomycin liquid (100×) (Solarbio, catalog number: P1400)
Trypan Blue (STEMCELL Technologies, catalog number: 07050)
Lympholyte® cell separation media (Ficoll) (Cedarlane Laboratories, catalog number: CL5020)
Anti-human IL-17A (BioLegend, catalog number: 512306, referred to as anti-human IL-17 later in this protocol)
Anti-human CD3 (BioLegend, catalog number: 300328)
Anti-human γδTCR (Miltenyi Biotec, catalog number: 130-113-508)
Anti-human CCR6 (BioLegend, catalog number: 353412)
Viability dye (Invitrogen, catalog number: 65-0865-14)
GolgiPlug (Brefeldin A solution 1,000×) (BioLegend, catalog number: 420601)
Phorbol 12-myristate 13-acetate (PMA) (Millipore Sigma, catalog number: P8139)
Ionomycin (Millipore Sigma, catalog number: I0634)
Human TruStain FcXTM (BioLegend, catalog number: 422302)
Fixation buffer (BioLegend, catalog number: 420801)
Intracellular staining perm 10× wash buffer (BioLegend, catalog number: 421002, referred to as wash buffer later in this protocol)
Equipment
CO2 incubator (Thermo Fisher Scientific, model number: 3543 or 3111)
Centrifuge (Beckman Coulter, model: Allegra® X-15R, catalog number: 392934)
Laminar flow hood (Scitech Equipments Ltd., model: EVL-5S, catalog number: ZX0907-04)
Flow cytometry (BD Bioscience, model: FACSCantoTM II, catalog number: 338962)
Pipettes (multi-channel, Eppendorf)
Software and Datasets
FlowJo v10.8.1 (BD Biosciences)
Procedure
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文章信息
版权信息
© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Chen, X., Hu, X., Chen, F. and Yan, J. (2024). Expansion and Polarization of Human γδT17 Cells in vitro from Peripheral Blood Mononuclear Cells. Bio-protocol 14(1): e4914. DOI: 10.21769/BioProtoc.4914.
- Chen, X., Cai, Y., Hu, X., Ding, C., He, L., Zhang, X., Chen, F. and Yan, J. (2022). Differential metabolic requirement governed by transcription factor c-Maf dictates innate γδT17 effector functionality in mice and humans. Sci. Adv. 8(21): eabm9120.
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分类
免疫学 > 免疫细胞分化 > T 细胞
免疫学 > 免疫细胞分离 > 淋巴细胞
细胞生物学 > 细胞分离和培养 > 细胞分化
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