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

Guorui Xie, Melissa C. Whiteman, Jason A. Wicker, Alan Barrett, Tian Wang

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)e52121
JournalJournal of visualized experiments : JoVE
Issue number93
DOIs
StatePublished - Nov 27 2014

Fingerprint

Myeloid Differentiation Factor 88
West Nile virus
T-cells
Viruses
Cellular Immunity
T-Lymphocytes
Assays
Infection
Cytokines
Flow cytometry
Staining and Labeling
Dendritic Cells
Flow Cytometry
Tissue culture
In Vitro Techniques
Cell proliferation
Virus Diseases
Coculture Techniques
Cell culture
Transgenic Mice

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

In vitro analysis of MyD88-mediated cellular immune response to West Nile virus mutant strain infection. / Xie, Guorui; Whiteman, Melissa C.; Wicker, Jason A.; Barrett, Alan; Wang, Tian.

In: Journal of visualized experiments : JoVE, No. 93, 27.11.2014, p. e52121.

Research output: Contribution to journalArticle

@article{c8433525c8324a8892f37634b568774a,
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.",
author = "Guorui Xie and Whiteman, {Melissa C.} and Wicker, {Jason A.} and Alan Barrett and Tian Wang",
year = "2014",
month = "11",
day = "27",
doi = "10.3791/52121",
language = "English (US)",
pages = "e52121",
journal = "Journal of visualized experiments : JoVE",
issn = "1940-087X",
publisher = "MYJoVE Corporation",
number = "93",

}

TY - JOUR

T1 - In vitro analysis of MyD88-mediated cellular immune response to West Nile virus mutant strain infection

AU - Xie, Guorui

AU - Whiteman, Melissa C.

AU - Wicker, Jason A.

AU - Barrett, Alan

AU - Wang, Tian

PY - 2014/11/27

Y1 - 2014/11/27

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84969372202&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84969372202&partnerID=8YFLogxK

U2 - 10.3791/52121

DO - 10.3791/52121

M3 - Article

C2 - 25489855

AN - SCOPUS:84969372202

SP - e52121

JO - Journal of visualized experiments : JoVE

JF - Journal of visualized experiments : JoVE

SN - 1940-087X

IS - 93

ER -