Anti mouse ifn γ pe

APC anti-mouse CD8, APC anti-mouse CD4, APC anti-mouse CD3, PE anti-mouse IFN-γ, and PE anti-mouse PD-1 antibodies (Abs) were obtained from BD Biosciences (San Jose, CA). Intracellular cytokine staining (ICS) of IFN-γ was performed as previously described^{23 (link)} using a Cytofix/Cytoperm Plus (with GolgiStop) kit from BD Biosciences. Foxp3 staining was performed using the Foxp3 staining buffer set from eBioscience (San Diego, CA). Appropriate isotype controls were used for the flow cytometric analysis. Flow cytometric data were collected using the BD FACSCalibur flow cytometer (BD Biosciences). Data were analyzed using the Cell Quest (BD Biosciences).

Flow cytometry analysis was performed to evaluate CD4⁺ and CD8⁺ T cell depletion and ZIKV-specific CD8⁺ T cell responses in the challenged mice (Guerrero et al., 1986 (link); Zhang et al., 2016 (link)). For analysis of CD4⁺ and CD8⁺ depletion in whole blood and splenocytes, peripheral blood cells and splenocytes were treated with 1 × Red Blood Cell Lysis Buffer (Biolegend), and stained with FITC-anti-mouse CD4 or PerCP-Cy5.5-anti-mouse CD8 antibody (BD Biosciences), followed by flow cytometry analysis using BD LSRFortessa 4 system. For analysis of ZIKV-specific CD8⁺ T cell responses, the above-treated splenocytes (2 × 10⁶ cells/well) were incubated with ZIKV NS3 overlapping peptides (0.25 nM/peptide, final concentration 5 μg/ml) in the presence of 5 μg/ml brefeldin A (Biolegend), and cultured at 37°C for 5 h. After stimulation, the cells were washed with PBS and stained for surface marker using PerCP/Cy5.5 anti-mouse CD8a. After fixation and permeabilization, the cells were stained for intracellular markers using FITC-anti-mouse IL-2, PE-anti-mouse IFN-γ, and Brilliant Violet 421-anti-mouse TNF-α antibodies (BD Biosciences), followed by analysis using flow cytometry as described above.

Purified naïve T cells were cultured in 96-well plates (2 x 10⁵/well) alone or stimulated with recombinant murine IL-12 (500pg/ml: PeproTech) and/or IL-18 (5pg/ml – 1000pg/ml; R&D systems) for 24h at 37°C. IFN-γ levels in the supernatants of triplicate cultures were determined using a sandwich ELISA kit (R&D Systems). For intracellular IFN-γ response, cells were pre-stimulated as described above for 16–20h before addition of 10μg/ml brefeldin A solution (Sigma-Aldrich) and further incubated for 4h (intracellular protein accumulation period). For PMA plus ionomycin stimulation condition, brefeldin A solution and PMA (0.05μg/ml final) plus ionomycin (0.75 μg/ml final) were added simultaneously and T cells incubated for 4h. Cells from triplicate wells were pooled, washed and stained with LIVE/DEAD^® Fixable Aqua Dead Cell dye (Life Technologies) for 30 min at 4°C. Subsequently cells were surface stained with fluorochrome-conjugated anti-mouse CD3ε, CD4, CD8α and Vβ6/8.1–2 for 30 min at 4°C then were treated with Cytofix/Cytoperm (BD Biosciences) and stained with anti-mouse IFN-γ-PE (BD Pharmingen). Data were acquired on BD LSR II flow cytometer and analyzed using FlowJo analysis software.

Myelin oligodendrocyte glycoprotein 35–55 (MOG_35–55) peptide (MEVGWYRSPFSRVVHLYRNGK) and myelin basic protein (MBP) Ac_1–11 peptide (Ac-ASQKRPSQRSK) were generated by the Protein Core Laboratory of the Blood Research Institute, BloodCenter of Wisconsin. KYC was synthesized by Biomatik (Wilmington, DE). The 2.4G2 hybridoma was obtained from the American Tissue Culture Collection. Anti-mouse CD4-APC-eFluor 780 CD11b-PE, CD11b-biotin, IL-17-Alexa Fluor 647, Foxp3-PE and streptavidin-PE Cy5.5 were purchased from eBioscience (San Diego, CA). Anti-mouse IFN-γ-PE was purchased from BD Biosciences (San Diego, CA). Anti-mouse Ly-6C-APC and Ly-6G-APC-Cy7 were purchased from Biolegend (San Diego, CA). The SMI-32 antibody, which detects nonphosphorylated neurofilament-H was purchased from Covance (Emeryville, CA). Streptavidin Alexa 405 and goat anti-mouse Alex 456 (H + L) were purchased from Life Sciences Advanced Technologies (St. Petersburg, FL). Anti-MPO heavy chain was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Anti-rabbit IgG (H + L)-HRP was purchased from Jackson ImmunoResearch (West Grove, PA). Anti-β-actin-HRP was purchased from Sigma-Aldrich (St. Louis, MO). For all experiments, the mice were age (6–8 wk) and gender matched with both sexes being utilized.