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, GM-CSF, Primary bone marrow cells, Cytokines, T cell polarization
Materials and Reagents
Equipment
Procedure
Note: The protocol outlined below has been adapted from Satchidanandam et al. (2014).
Representative data
Table 1. The table shows the levels of IFN-γ and IL-17 detected by ELISA in the culture supernatants of splenocytes co-cultured with the indicated BMDC samples. The cytokines are shown as pg/ml culture volume secreted by one million non-adherent splenocytes.
Notes
Recipes
Acknowledgments
This work was funded under the grant BT/PR7240/INF/22/52/2006 from Department of Biotechnology, Ministry of Science and Technology, Government of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Shoba Elangovan and Rajni Nyodu for help with producing the videos. This protocol was adapted from Satchidanandam et al. (2014).
References
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