Apc anti mouse ifn γ

Single-cell suspensions prepared from mouse spleens were incubated with a cell activation cocktail (BioLegend, San Diego, CA, USA) at 37°C and 5% CO₂ for 6 h. Then, cells were stained for cell surface and intracellular markers with the following conjugated monoclonal antibodies: FITC anti-mouse CD3 (cat# 100203), PerCP/Cyanine5.5 anti-mouse CD8a (cat# 100734), APC anti-mouse IFN-γ (cat# 505810), and PE anti-mouse IL-17A (cat# 506904). All antibodies were purchased from BioLegend. CD3⁺CD8a⁻IL-17A⁺ cells were identified as Th17 cells, and CD3⁺CD8a⁻IFN-γ⁺ cells were identified as Th1 cells. Cells were analyzed with a BD Accuri C6 instrument (BD Biosciences) and FlowJo software v10.

Single-cell suspensions of peripheral blood or these prepared from lung tissue were stained with the following directly labeled antibodies: PerCP-Cy5.5-anti-mouse CD11b (Clone M1/70, BD Pharmingen, 561114, 1: 100); APC-anti-mouse Gr1(Clone RB6-8C5, BD Pharmingen, 553129, 1: 100); BV421-anti-mouse Ly6G (Clone 1A8, Biolegend 127628, 1: 100); FITC-anti-mouse Ly6G (Clone 1A8, eBioscience, 11-9668-82, 1:100); PE- anti-mouse Ly6C (Clone HK1.4, Biolegend, 128008, 1:100); APC-anti-mouse CD8 (Clone 53-6.7, Biolegend, 100712, 1:100); BV421-anti-mouse CD4 (Clone GK1.5, Biolegend, 100438, 1:100); APC-anti-mouse IFNγ (Clone XMG1.2, Biolegend, 505810, 1:100); PE-anti-mouse IL-17A (Clone ebio17B7, Biolegend, 559502). Detailed antibody information was summarized in Supplementary Table 3. Stained cells were collected and analyzed by FACSVerse device (BD Biosciences) and data was analyzed by Flowjo v10.

Splenocytes (10⁷ cells/well) were cultivated in the presence of RBD (10 μg/mL) as a specific antigen or Con A (20 μg/mL) as a positive control. Unstimulated samples were used as a negative control. The stimulated cells were grown at 37 °C for 72 h and then incubated for an additional 5 h with Brefeldin A (BioLegend, San Diego, CA, USA). After incubation, the stimulated cells were harvested, treated with TruStain FcX (Anti-mouse CD16/32 antibody, BioLegend, San Diego, CA, USA) and subjected to surface staining with PE-Cy7 anti-mouse CD3, PE anti-mouse CD4 and FITC anti-mouse CD8 (BD Biosciences) antibodies. Subsequently, cells were processed with a fixation/permeabilization kit (BD Biosciences, San Diego, CA, USA), followed by staining with APC anti-mouse IFN-γ (BioLegend, San Diego, CA, USA). The percentages of positive cells were enumerated by flow cytometry. Simultaneously, aliquots of the culture supernatant were harvested at 12, 24, 48 and 72 h of treatment, and were then subjected to IFN-γ, IL-2 and IL-4 detection using ELISA (BioLegend, San Diego, CA, USA)

Splenocytes were transferred into wells at 500,000 cells/well and stimulated at 37°C for 8 h with Ad5 containing entire structural proteins C-E3-E2-6K-E1. Brefeldin A (BioLegend) was then added to block cytokine secretion and incubation continued for 4 h. Following two washes with PBS, splenocytes were incubated with the following antibodies against lineage markers: PE anti-mouse CD3e, FITC anti-mouse CD4, and PerCP/Cy 5.5 anti-mouse CD8a (all from BioLegend). After two washes with PBS, cells were fixed, permeabilized with Cytofix/Cytoperm (BD Biosciences), and incubated with the following antibodies against intracellular markers: APC anti-mouse IFN-γ and PE/Cy7 anti-mouse IL-2 (all from BioLegend). Cells were analyzed using a FACS Lyric analyzer (BD Biosciences).

Fresh mouse splenocytes were plated in a 96 round-bottom well plate (Costar, Corning Inc., Corning, NY, USA) at a density of 2 × 10⁶ cells/well and incubated overnight with either 1 µM/peptide of the ZIKV E peptide pool, or 5 µM of YFV-17D NS3 ATLTYRML or 50 µg/ml of Vero E6 cell lysate. After treatment with 5 µg/ml brefeldin A (Biolegend, San Diego, CA, USA) for 2 h, the splenocytes were stained for viability with Zombie Aqua™ (Biolegend) (1:200 dilution in PBS) for 15 min. Subsequently, extracellular markers CD3 (4 µg/ml eFluor® 450 anti-mouse CD3 antibody, ThermoFisher, Waltham, MA, USA) and CD8 (2 µg/ml APC/Cy7 anti-mouse CD8a antibody, Biolegend) were stained and cells were fixed in 2% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA). Finally, splenocytes were permeabilized with 0.1% saponin before intracellular staining for IFN-γ (2 µg/ml APC anti-mouse IFN-γ, Biolegend), TNF-α (6.5 µg/ml PE anti-mouse TNF-α, Biolegend) and granzyme B (5 µg/ml FITC anti-human/mouse granzyme B, Biolegend). Samples were analyzed on a BD LSRFortessa™ X-20 (Becton Dickinson, Franklin Lakes, NJ, USA). Percentages of responding CD4⁺ or CD8⁺ T cells were calculated by subtracting the percentage of responders from non-stimulated samples (incubated with non-infected Vero E6 cell lysates) from corresponding stimulated samples.