title: 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.affiliation: Department of Microbiology & Immunology, The University of Texas Medical Branch.
authors.forename: Guorui
authors.initials: G
authors.lastname: Xie
authors.affiliation: Department of Pathology, The University of Texas Medical Branch.
authors.forename: Melissa C
authors.initials: MC
authors.lastname: Whiteman
authors.affiliation: Department of Pathology, The University of Texas Medical Branch.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Wicker
authors.affiliation: Department of Microbiology & Immunology, The University of Texas Medical Branch; Department of Pathology, The University of Texas Medical Branch; Center for Biodefense and Emerging Infectious Diseases, Sealy Center for Vaccine Development, The University of Texas Medical Branch.
authors.forename: Alan D T
authors.initials: AD
authors.lastname: Barrett
authors.affiliation: Department of Microbiology & Immunology, The University of Texas Medical Branch; Department of Pathology, The University of Texas Medical Branch; Center for Biodefense and Emerging Infectious Diseases, Sealy Center for Vaccine Development, The University of Texas Medical Branch; ti1wang@utmb.edu.
authors.forename: Tian
authors.initials: T
authors.lastname: Wang
mesh_terms: D000818:Animals; D017951:Antigen Presentation; D018920:Coculture Techniques; D016207:Cytokines / Q000276:immunology; D003713:Dendritic Cells / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D007111:Immunity, Cellular / Q000276:immunology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D053594:Myeloid Differentiation Factor 88 / Q000276:immunology*; D015398:Signal Transduction / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*; D014901:West Nile Fever / Q000276:immunology*; D014902:West Nile virus / Q000276:immunology*
chemical_list: D016207:Cytokines; D018984:Epitopes, T-Lymphocyte; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88