In vitro analysis of MyD88-mediated cellular immune response to West Nile virus mutant strain infection.

Abstract

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods - an in vitro T cell priming assay and an intracellular cytokine staining (ICS) - were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4(+) T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4(+) T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

• Authors: Guorui Xie, Melissa C Whiteman, Jason A Wicker, Alan D T Barrett, Tian Wang
• Journal: Journal of visualized experiments : JoVE (J Vis Exp) Issue 93 Pg. e52121 (Nov 27 2014) ISSN: 1940-087X [Electronic] United States
• PMID: 25489855 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Video-Audio Media)

Chemical References

• Cytokines
• Epitopes, T-Lymphocyte
• Myd88 protein, mouse
• Myeloid Differentiation Factor 88

Topics

• Animals
• Antigen Presentation
• Coculture Techniques
• Cytokines (immunology)
• Dendritic Cells (immunology)
• Epitopes, T-Lymphocyte (immunology)
• Female
• Flow Cytometry (methods)
• Immunity, Cellular (immunology)
• Male
• Mice
• Mice, Inbred C57BL
• Mice, Transgenic
• Myeloid Differentiation Factor 88 (immunology)
• Signal Transduction (immunology)
• T-Lymphocytes (immunology)
• West Nile Fever (immunology)
• West Nile virus (immunology)