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Immunology and Infection

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

Published: September 10th, 2018

DOI:

10.3791/58087

1Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 2Immunology Programme, Life Sciences Institute, National University of Singapore, 3Cancer Science Institute of Singapore, Yong Loo Lin School of Medicine, National University of Singapore

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)γ or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFNα or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFNα, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

CD4+ T helper (TH) cells are essential components of the immune system, having crucial roles in the host defense against microbial pathogens, in cancer surveillance, and in autoimmunity1,2,3. Upon T cell receptor (TCR) activation, naive CD4+ T cells can be differentiated into TH1, TH2, TH 17, or regulatory T (Treg) cells under the influence of different cytokine milieus2,4,5. Recently, a new subset of TH cells, which predominantl....

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All mice used in this protocol were on the C57BL/6 genetic background and housed under specific pathogen-free conditions at the National University of Singapore. All experiments were performed using protocols approved by the Institutional Animal Care and Use Committee of the National University of Singapore.

1. Reagent and Material Preparation

  1. Prepare 500 mL of phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) and 1 mM ethylenediaminetetraacetic acid (EDTA).

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Naive CD4+ T cells isolated from two 8-week-old male C57BL/6 mice were divided into three portions. One portion of the cells was differentiated into THGM cells following the protocol described. Another portion was cultured under a THGM condition in the presence of anti-IL-4 antibody (10 µg/mL) to test the influence of an IL-4 blockade in the differentiation of THGM. The last portion was cultured under a TH17 differentiation condition (3 .......

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Here we described a protocol of an in vitro THGM differentiation from mouse naive CD4+ cells, followed by an analysis of the differentiated cells to validate the method. Of note, both spleen and lymph nodes can be used for naive CD4+ T cell purification and THGM differentiation. The cytokine expression determined by intracellular cytokine staining combined with flow cytometry showed that about 55% of the cells were induced to become GM-CSF-expressing cells under the THGM conditio.......

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This study was supported by grants from the National University Health System of Singapore (T1-2014 Oct-12 and T1-2015 Sep-10).

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Name Company Catalog Number Comments
RPMI 1640 Biowest L0500-500
FBS Heat Inactivated Capricorn FBS-HI-12A
Penicillin-Streptomycin Gibco 15140122
10x PBS 1st Base BUF-2040-10 Diluted to 1x PBS in sterile water
EDTA 1st Base BUF-1053-100ml-pH8.0
10X ACK buffer Biolegend 420301 diluted in sterile water
cell strainer SPL Life Sciences 93070
CD4 microbeads Miltenyi Biotec 130-049-201
2-Mercaptoethanol Sigma M7522
LS column Miltenyi Biotec 130-042-401
magnetic stand Miltenyi Biotec 130-042-303
1ml syringe Terumo SS+01T
Centrifuge Eppendorf Eppendorf 5810R
Sony SY3200 cell sorter Sony Sony SY3200 cell sorter
50ml conical centrifuge tube Greiner Bio-One 210261
15m conical centrifuge tube Greiner Bio-One 188271
FACS tube Corning 352054
24 well cell culture plate Greiner Bio-One 662160
anti-mouse CD4 PerCP-eFluor 710 eBioscience 46-0041-82
PE conjugated anti-mouse CD25 (IL-2Ra, p55) eBioscience 12-0251-82
anti-human/mouse CD44 APC eBioscience 17-0441-82
anti-mouse CD62L FITC eBioscience 11-0621-85
Neubauer-improved counting chamber Marienfeld 640010
Hyclone Trypan Blue Solution GE healthcare Life Sciences SV30084.01
microscope Nikon Nikon Elipse TS100
Purified anti-mouse CD3e antibody Biolegend 100314
Purified hamster anti-mouse CD28 BD Biosciences 553295
Purified Rat anti-mouse IFNγ   eBioscience 16-7312-85
Purified anti-mouse IL-4 Antibody Biolegend 504102
Recombinant Mouse IL-7 Protein R&D system 407-ML
Recombinant Mouse IL-6 R&D system 406‑ML
recombinant human TGF-beta R&D system 240-B-010
PMA Merck Millipore 19-144
Ionomycin Sigma I0634
GolgiPlug protein transport inhibitor BD Biosciences 51-2301KZ
Intracellular Fixation&Permeabilization buffer set eBioscience 88-8824-00
anti-mouse GM-CSF PE eBioscience 12-7331-82
FITC anti-mouse IL-17A Biolegend 506908
APC anti-mouse IFN-gamma Biolegend 505810
GO Taq qPCR master mix Promega A6002
mouse GM-CSF ELISA Ready-SET-Go! invitrogen 88-7334-88
Mouse IL-17A Uncouted ELISA invitrogen 88-7371-22

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