Anti cd19 pe cf594 clone 1d3

Mouse splenocytes were stimulated ex vivo with SFTSV OLP pools. Brefeldin A (GolgiPlug, BD Biosciences) and monensin (GolgiStop, BD Biosciences) were added to the culture 1 h after stimulation and the culture was maintained for 5 h. The Live/Dead Fixable Red Dead Cell stain kit (Invitrogen), anti-CD19–PE-CF594 (clone 1D3, BD Biosciences, 562291, 1:100), anti-CD3–BV510 (clone 145-2C11, BD Biosciences, 563024, 1:100), anti-CD4–Alexa Flour 700 (clone RM4-5, BD Biosciences, 557956, 1:100), and anti-CD8–APC-H7 (clone 53-6.7, BD Biosciences, 560182, 1:100) were used for immunostaining at 4 °C for 20 min. For intracellular staining, cells were permeabilized and fixed using the BD Cytofix/Cytoperm kit (BD Biosciences) at 4 °C for 20 min, which was followed by staining with anti-IFN-γ–APC (clone XMG1.2, BioLegend, 505809, 1:100), anti-TNF–PE (clone MP6-XT22, BD Biosciences, 554419, 1:100), and anti-IL-2–PE-Cy7 (clone JES6-5H4, BD Biosciences, 560538, 1:100) at 4 °C for 20 min. All data were collected using a LSRΙΙ flow cytometer (BD Biosciences), and Boolean gating was performed using FlowJo software to analyze the polyfunctionality of T cells. Charts were visualized using SPICE software (https://niaid.github.io/spice/).

T-cell epitopes were mapped within NSs antigen. IFN-γ ELISpot assays were performed with splenocytes from vaccinated mice by stimulation with a single peptide (15-mer; total 41 peptides). Peptides with high numbers of IFN-γ spots were identified, and the epitope sequence corresponding to high-affinity binding to mouse MHC class Ι, H-2L^d was identified using NetMHC 4.0 (Technical University of Denmark). The epitope peptide YPYLMAHYL was used to synthesize the H-2L^d dextramer (H-2L^d NSs_247–255 dextramer, Immunedex). Splenocytes of vaccinated mice were stained with PE–conjugated H-2L^d NSs_247–255 dextramer at 4 °C for 20 min, followed by washing with FACS buffer (PBS containing 1% FBS and 0.05% sodium azide). For live/dead and surface staining, the following reagent and antibodies were used: Live/Dead Fixable Red Dead Cell stain kit (Invitrogen), anti-CD19–PE-CF594 (clone 1D3, BD Biosciences, 562291, 1:100), anti-CD8–BV510 (clone 53-6.7, BD Biosciences, 560776, 1:100), anti-CD62L–BV605 (clone MEL-14, BD Biosciences, 563252, 1:50), anti-CD44–BV650 (clone IM7, BioLegend, 103049, 1:50), and anti-CD3–Alexa Fluor 700 (clone 500A2, eBioscience, 56-0033-82, 1:100).