In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection |
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Authors: | Guorui Xie Melissa C. Whiteman Jason A. Wicker Alan D.T. Barrett Tian Wang |
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Affiliation: | 1.Department of Microbiology & Immunology, The University of Texas Medical Branch;2.Department of Pathology, The University of Texas Medical Branch;3.Center for Biodefense and Emerging Infectious Diseases, Sealy Center for Vaccine Development, The University of Texas Medical Branch |
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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. |
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Keywords: | Immunology Issue 93 West Nile Virus Dendritic cells T cells cytokine proliferation in vitro |
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