Immunology and Infection
Published: September 10th, 2018
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....
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
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 .......
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.......
|FBS Heat Inactivated
|Diluted to 1x PBS in sterile water
|10X ACK buffer
|diluted in sterile water
|SPL Life Sciences
|Sony SY3200 cell sorter
|Sony SY3200 cell sorter
|50ml conical centrifuge tube
|15m conical centrifuge tube
|24 well cell culture plate
|anti-mouse CD4 PerCP-eFluor 710
|PE conjugated anti-mouse CD25 (IL-2Ra, p55)
|anti-human/mouse CD44 APC
|anti-mouse CD62L FITC
|Neubauer-improved counting chamber
|Hyclone Trypan Blue Solution
|GE healthcare Life Sciences
|Nikon Elipse TS100
|Purified anti-mouse CD3e antibody
|Purified hamster anti-mouse CD28
|Purified Rat anti-mouse IFNγ
|Purified anti-mouse IL-4 Antibody
|Recombinant Mouse IL-7 Protein
|Recombinant Mouse IL-6
|recombinant human TGF-beta
|GolgiPlug protein transport inhibitor
|Intracellular Fixation&Permeabilization buffer set
|anti-mouse GM-CSF PE
|FITC anti-mouse IL-17A
|APC anti-mouse IFN-gamma
|GO Taq qPCR master mix
|mouse GM-CSF ELISA Ready-SET-Go!
|Mouse IL-17A Uncouted ELISA
Copyright © 2024 MyJoVE Corporation. All rights reserved