一种简单稳定的用小鼠CD4+ T细胞体外分化非致病性T辅助17细胞的方法

Siwen Kang; Ruohan Wu; Ruoning Wang

doi:10.21769/BioProtoc.4029

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Peer-reviewed

A Simple and Robust Protocol for in vitro Differentiation of Mouse Non-pathogenic T Helper 17 Cells from CD4⁺ T Cells

一种简单稳定的用小鼠CD4+ T细胞体外分化非致病性T辅助17细胞的方法

SK Siwen Kang email

RW Ruohan Wu

RW Ruoning Wang

发布: 2021年05月20日第11卷第10期 DOI: 10.21769/BioProtoc.4029 浏览次数: 4498

评审: Guangyong PengRAMESH KUDIRAMichael EnosAnonymous reviewer(s)

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Abstract

Functional and mechanistic studies of CD4⁺ T cell lineages rely on robust methods of in vitro T cell polarization. Here, we report an optimized protocol for in vitro differentiation of a mouse non-pathogenic T helper 17 (T_H17) cell lineage. Most of the previously established protocols require irradiated splenocytes as artificial antigen presenting cells (APC) for TCR activation. The protocol described here employs plate-bound antibodies and a T_H17-polarizing cytokine cocktail to activate and differentiate naïve CD4⁺ T (T_nai) cells, reflecting a simple and robust protocol for in vitro T_H17n differentiation. Using T cells that are genetically engineered with an IL-17 reporter, this protocol may enable the rapid production of a pure population of IL17-expressing CD4⁺ T cells for system biology studies and high-throughput functional screening.

Keywords: T_H17 (T_H17)

IL-17 (IL-17)

Plate-bound (板结式)

FACS (荧光活化细胞分选)

T cell polarization (T细胞极化)

Background

Naive CD4⁺ T cells circulate through secondary lymphoid tissues after emerging from the thymus. The activation of naïve CD4⁺ T cells, by antigen-presenting cells that offer cognate antigens, initiates various differentiation programs that lead to the development of highly specialized T helper (T_H) cell lineages (Bhaumik and Basu 2017). T_H17 cells are a subset of pro-inflammatory T_H cells defined by the production of interleukin 17 (IL-17) (Hartigan-O'Connor et al., 2011). To date, at least two different types of T_H17 have been described. Type 1, pathogenic or disease-inducing T_H17 (T_H17p) cells, are induced by the combined IL-6, IL-1β, and IL-23 cytokine stimulation, and produce IL-17 together with IFN-γ, which results in tissue inflammation and autoimmunity (Cua et al., 2003; Awasthi et al., 2009; Jager et al., 2009; Lee et al., 2012; Lee et al., 2014). Type 2, non-pathogenic T_H17 (T_H17n) cells, are induced by TGF-β and IL-6 in vitro, and produce IL-17 but are not able to induce effective tissue inflammation or autoimmunity. T_H17n cells play a protective role in mucosal tissues, promoting tissue homeostasis as well as maintaining barrier function (Bettelli et al., 2008; Ouyang et al., 2008; Korn et al., 2009; Guglani and Khader 2010; Gaffen et al., 2011). Here, we report a simple and robust protocol for generating non-pathogenic T_H17 (T_H17n) cells in vitro.

Materials and Reagents

48-well cell culture plates (ASi Alkali Scientific Inc., catalog number: TP9048)
CellPro^TM 70 μm cell strainers (ASi, Falcon^®, catalog number: MT4070)
1 ml syringe (BD Tuberculin Slip Tip, catalog number: 9189520)
50 ml centrifuge tubes (ASi, catalog number: C5602)
15 ml centrifuge tubes (ASi, catalog number: C5600)
C57BL/6J mice (The Jackson Laboratory, catalog number: 000664)
C57BL/6-IL17a^tm1Bcgen/J mice (The Jackson Laboratory, catalog number: 018472)
RBC lysis buffer 10× (Biolegend^®, catalog number: 420302)
Cell culture grade water (HyPure^TM, catalog number: SH30529.02)
MojoSort^TM Mouse CD4 Naïve T Cell Isolation Kit (Biolegend^®, catalog number: 480040)
Coating antibodies:
1. Anti-mouse CD3ϵ (clone 145-2C11, Bio X Cell, catalog number: BE0001-1)
2. Anti-mouse CD28 (clone 37.51, Bio X Cell, catalog number: BE0015-1)
Staining antibodies:
1. APC anti-mouse CD4 (clone RM4-5, Biolegend, catalog number: 100516)
2. PE/Cy7 anti-mouse IL-17A (clone TC11-18H10.1, Biolegend, catalog number: 506922)
Cell Stimulation Cocktail Plus Protein Transport Inhibitors (Invitrogen eBioscience^TM, catalog number: 00-4975-93)
FoxP3/Transcription Factor Staining Buffer Set (Fixation Diluent, Fixation Reagent, Permeabilization kit, eBioscience^TM, catalog number: 00-5523-00)
RPMI 1640 1× with L-glutamine (Corning, catalog number: 10-040-CV)
Fetal bovine serum (FBS) (ATLANTA biologicals^TM, R&D SYSTEMS, catalog number: S12450)
L-glutamine 200 mM (Corning, catalog number: 25-005-CI)
Penicillin-streptomycin solution (PS) 100× (Corning, catalog number: 30-002-CI)
Sodium pyruvate 100 mM (Corning, catalog number: 25-000-CI)
2-mercaptoethanol (ME) 1000× (Life Technologies, Gibco^®, catalog number: 21985-023)
T_H17 polarization cytokines and antibodies:
1. Recombinant Human TGF-β1 (PeproTech, catalog number: 100-21C)
2. Recombinant Murine IL-6 (PeproTech, catalog number: 216-16)
3. Anti-mouse IFN-γ (clone XMG1.2, Bio X Cell, catalog number: BE0055)
4. Anti-mouse IL-2 (clone JES6-1A12, Bio X Cell, catalog number: BE0043)
5. Anti-mouse IL-4 (clone 11B11, Bio X Cell, catalog number: BE0045)
Phosphate-buffered saline (PBS) 1× (Corning, catalog number: 21-040-CV)
Bovine serum albumin (BSA) (Fisher BioReagents^TM, catalog number: BP1600-100)
Ethylenediamine tetraacetate acid (EDTA) 0.5 M (Fisher BioReagents^TM, catalog number: BP2482-500)
Carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen^TM, catalog number: C1157)
Culture medium (see Recipes)
TCR-stimulation antibody mixture (see Recipes)
RBC lysis buffer (1×) (see Recipes)
MACS buffer (see Recipes)
T_H17 polarization medium (see Recipes)
Staining buffer (see Recipes)

Equipment

NovoCyte (ACEA Biosciences)

Software

FlowJo software (BD Biosciences)

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Kang, S., Wu, R. and Wang, R. (2021). A Simple and Robust Protocol for in vitro Differentiation of Mouse Non-pathogenic T Helper 17 Cells from CD4⁺ T Cells. Bio-protocol 11(10): e4029. DOI: 10.21769/BioProtoc.4029.
Ruohan Wu*, Xuyong Chen*, Siwen Kang*, Tingting Wang, JN Rashida Gnanaprakasam, Yufeng Yao, Lingling Liu, Gaofeng Fan, Mark R. Burns, Ruoning Wang†. (2020). De novo synthesis and salvage pathway coordinately regulate polyamine homeostasis and determine T cell proliferation and function. Science Advances 6 (51): eabc4275.