小鼠BMDC依赖性T淋巴细胞的极化试验

Vijaya Satchidanandam

doi:10.21769/BioProtoc.1721

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Peer-reviewed

Mouse BMDC-dependent T Cell Polarization Assays

小鼠BMDC依赖性T淋巴细胞的极化试验

Vijaya Satchidanandam email

发布: 2016年02月05日第6卷第3期 DOI: 10.21769/BioProtoc.1721 浏览次数: 14581

评审: Fanglian HeMeenal SinhaMarco Di Gioia

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Abstract

In response to exposure to antigen, T cells whose T cell receptor (TCR) are capable of recognizing the self MHC-antigen derived peptide complex, respond to the antigen and differentiate into one of several subsets, namely TH1, TH2, TH17, Treg, etc. characterized by the signature cytokine they secrete, namely IFN-γ, IL-4, IL-17 or IL-10, respectively, referred to as syngeneic polarization as the MHC presenting the foreign antigen/epitope is self-derived.

T cell responses following incubation for defined periods, usually 3 days for mouse splenocytes, are routinely measured by assessing the antigen-stimulated proliferation of T cells by measuring the radiolabeled precursor thymidine incorporated into the genomic DNA of the dividing T cell; the direction of polarization is assessed by measuring the cytokine produced by the proliferating or non-proliferating responding T cells using ELISA of culture supernatants or by intracellular cytokine staining followed by flow cytometry.

In the protocols detailed below, we describe the use of syngeneic mouse bone marrow-derived primary dendritic cells (BMDC) as APC to stimulate spleen derived T cells. The proliferative response of the T cells is measured by incorporation of radiolabeled precursor thymidine into the genomic DNA and their direction of polarization is assessed by measuring the cytokines they secrete, namely IFN-γ, IL-4 and IL-17 over a 72 h period using ELISA. In addition, we used flow cytometry after intracellular cytokine staining to detect IL-17 positive T cells within the CD3⁺/CD4⁺/CD25low population. Prior live infection of BMDC with strains of Mycobacterium bovis- Bacille Calmette Guerin (BCG) was used as antigen to pre-condition the BMDC that presented antigens derived therefrom to T cells. We also measured cytokines secreted within 6 to 8 h of BCG infection by BMDC in order to correlate the BMDC cytokine profile with subsequent direction of T cell polarization.

Keywords: Dendritic cells (树突状细胞)

GM-CSF (GM-CSF)

Primary bone marrow cells (原发性骨髓细胞)

Cytokines (细胞因子)

T cell polarization (T细胞极化)

Materials and Reagents

10 cm Petri dishes, non-treated (Thermo Fisher Scientific, Falcon^TM, catalog number: 08-757-100D )
6 well 35 mm dia plastic cell culture treated dishes (Thermo Fisher Scientific, Falcon^TM, catalog number: 353046 )
0.22 µm filter
BALB/c mice (6 to 8 weeks old female)
Iscove’s modification of Dulbecco’s minimum essential medium (IMDM) (Life Technologies, catalog number: 12200069 )
Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 12200069”.
Sterile PBS
Dulbecco’s Phosphate buffered saline (Sigma-Aldrich, catalog number: D5773 )
Saponin from quillaja bark (wash buffer for flow cytometry staining) (Sigma-Aldrich, catalog number: S7900 )
Murine rGM-CSF (recombinant mouse GM-CSF) (PEPROTECH, catalog number: 315-03 )
Note: Recombinant GM-CSF from Peprotech Reconstitute powder (recommended by manufacturer).
ELISA antibodies
Note: We use R&D Systems Duoset capture and detection antibodies or BD paired ELISA antibody sets as detailed below:
Mouse IFN-γ DuoSet ELISA (R&D Systems, catalog number: DY485-05 )
Mouse IL-17 DuoSet ELISA (R&D Systems, catalog number: DY421-05 )
Mouse IL-6 DuoSet ELISA (R&D Systems, catalog number: DY406-05 )
Mouse TNF-α DuoSet ELISA (R&D Systems, catalog number: DY410-05 )
Mouse IL-10 DuoSet ELISA (R&D Systems, catalog number: DY417-05 )
Mouse IL-2 DuoSet ELISA (R&D Systems, catalog number: DY402-05 )
Mouse IL-12p40 DuoSet ELISA (R&D Systems, catalog number: DY499-05 )
Mouse IFN-gamma DuoSet ELISA (R&D Systems, catalog number: DY1679-05 )
1x PBS-1% BSA-0.05% sodium azide
Concanavalin A from Canavalia ensiformis (Jack bean) (Sigma-Aldrich, catalog number: C5275 )
CD11c MicroBeads (Miltenyi Biotech, catalog number: 130-052-001 )
Glutamax-100x (Thermo Fisher Scientific, Gibco^TM, catalog number: 35050-061 )
Mitomycin C from Streptomyces caespitosus (Sigma-Aldrich, catalog number: M4287 )
Penicillin-Streptomycin, 100x (Thermo Fischer Scientific, Gibco^TM, catalog number: 15070-063 )
Penicillin-Streptomycin (10,000 U/ml) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140-122 )
IL-17-APC (eBioscience, clone: eBio17B7 )
10% FBS
[methyl-³H]
70% and 100% ethanol
Brefeldin A (BFA) (Sigma-Aldrich, catalog number: B7651 )
Ammonium chloride (NH₄Cl) (Sigma-Aldrich, catalog number: A9434 )
Potassium bicarbonate (KHCO₃) (Sigma-Aldrich, catalog number: 60339 )
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
Tris (pH 7.5)
IMDM complete (see Recipes)
Thymidine (see Recipes)
Brefeldin A (BFA) (Sigma-Aldrich, catalog number: B7651) (see Recipes)
Monensin sodium salt (Sigma-Aldrich, catalog number: M5273 ) (see Recipes)
RBC lysis buffer (100 ml) (see Recipes)
RBC lysis buffer (see Recipes)

Equipment

Sterile scissors and forceps
Cell scrapers (Corning, Costar^®, model: 3010 Small Cell Scraper )
Centrifuges (Table top) (Eppendorf)
Water bath set at 37 °C
Flow cytometer [FACS-Canto-II flow cytometer (BD) or any machine with Argon ion Blue laser)
Biosafety cabinet with laminar air flow

Procedure

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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:

Satchidanandam, V. (2016). Mouse BMDC-dependent T Cell Polarization Assays. Bio-protocol 6(3): e1721. DOI: 10.21769/BioProtoc.1721.
Satchidanandam, V., Kumar, N., Jumani, R. S., Challu, V., Elangovan, S. and Khan, N. A. (2014). The glycosylated Rv1860 protein of Mycobacterium tuberculosis inhibits dendritic cell mediated TH1 and TH17 polarization of T cells and abrogates protective immunity conferred by BCG. PLoS Pathog 10(6): e1004176.