In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Summary

Two flow cytometry-based methods – an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

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.

Introduction

West Nile virus (WNV), a neurotropic, plus-sensed flavivirus, is an emerging public health threat. Currently, no vaccines have been approved for human use¹. An attenuated WNV strain, which has a P38G substitution in the nonstructural (NS)4B protein, is known to induce no lethality in mice but higher innate cytokines and T cell responses in mice than wild-type WNV NY99 strain². Mice immunized with the NS4B-P38G mutant were all protected from a secondary challenge with lethal wild-type WNV. This suggests that the NS4B-P38G mutant has suitable features for an ideal vaccine candidate. The mechanisms by which the NS4B-P38G mutant induces high protecti....

Protocol

All animal experiments were approved by the Animal Care and Use Committee at the University of Texas Medical Branch.

1. Isolation of DCs from Non-infected and WNV-infected Mice

1. Age- and sex- match 6-10 week-old, wild-type C57BL/6 (B6) and MyD88^−/−mice. Inoculate intraperitoneally (i.p.) with 500 plaque forming unit (PFU) of WNV NS4B- P38G mutant. At day 3 post-infection, euthanize B6 and MyD88^−/− mice with CO₂. Use non-infected mice of both groups as controls. Use three mice from each group.
2. Wet fur on left side of sacrificed mouse using 70....

Results

In the T cell priming assay, CFSE labeled CD4⁺ T cells were cultured with purified DCs from the NS4B-P38G mutant-infected wild-type and MyD88^−/− mice in the presence or absence of OVA peptides. Labeled T cells cultured alone with or without OVA for 5 days were used as negative controls. As shown in Figure 1A, total T cells were gated for analysis of fluorescence intensity on the FL1 channel. The marker was set up based on the freshly labeled CD4⁺ T c.......

Discussion

WNV is a BL3 pathogen. Due to safety regulations, immunological assays with WNV-infected samples are often restricted by the availability of equipment at BL3 facilities or more lengthy and tedious to perform. In a recent study, we used two flow cytometry-based methods to study cell mediated immune responses during WNV infection⁵. In both assays, WNV-infected cells were treated with 1-2% PFA directly or with fixation/permeabilization solution containing 4% PFA. It is known that 1% PFA fixation of virus-infected.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI 1640	Invitrogen	11875	warm up at 37 °C
anti-CD11c magnetic beads	Miltenyi Biotec	130-052-001	follow the manufacturer’s instructions
anti-CD4 magnetic beads	Miltenyi Biotec	130-095-248	follow the manufacturer’s instructions
CFSE	Invitrogen	C34554
OVA residue 323-339	Genscript Corporation	RP10610
Peptides	Proimmune	PC0AD-D
Brefeldin A solution	BD Bioscience	555029
Mouse Fc Blocker	e-Bioscience	14-0161-85
APC-conjugated CD4	e-Bioscience	17-0041-81
FITC-conjugated CD8	e-Bioscience	11-0081-82
Fixation/Permeabilization Solution	BD-Bioscience	554722
Permeabilization/wash buffer	BD-Bioscience	554723
anti-IFNγ-PE	e-Bioscience	12-7311-82
Accuri flow cytometer	BD Bioscience