Anti cd4 brilliant violet 650

All cells were labeled with live/dead dye near infra red (Life Technologies) for dead cell exclusion and treated with Fc Block (Miltenyi, San Diego, CA, USA) prior to staining with fluorescently labeled antibodies. Anti-human antibodies used for DC staining were anti-CD83 PE (Biolegend, San Diego, CA, USA), anti-CD86-PE-Cy7 (Biolegend), anti-HLA-DR V450 (BD), and anti-CD11c APC (BD). Antibodies used in the T cell characterization were anti-LAG-3 FITC (Novus, Littleton, CO, USA), anti-PD-1 PerCP-Cy5.5, anti-CD3 Alexa700, anti-CD4 Brilliant Violet 650, anti-CD8 Brilliant Violet 570, anti-IFN-γ PE, anti-IL-5, anti-CD62L PE, and anti-CD45RA Alexa700 (all, Biolegend), anti-CD25 PE, and anti-IFN-γ PE-Cy7, anti-IL-4 PE-Cy7 (all BD), and anti-TIM-3 APC (R&D Systems). Surface staining and intracellular staining was performed using Cytofix/Cytoperm™ Plus kit (BD) according to the manufacturer’s instructions. Data acquisition was performed using the LSRFortessa flow cytometer (BD). Data analysis was performed with FlowJo version 9 (Tree Star, Inc., Ashland, OR, USA).

Spleens of mice were digested and passed using a 40 μm nylon mesh. Then, peripheral blood was collected and suspended in PBS. Splenocytes were stimulated with a cell stimulation cocktail (00-4975-93, eBioscience, San Diego, CA, USA) and Th cells subtypes were identified using the following antibodies (BioLegend, San Diego, CA, USA): anti-CD3-FITC (100204), anti-CD4-Brilliant Violet 650™ (100545), anti-IFN-γ-APC (505810), anti-IL-4-PE-Cy7 (504118), anti-IL-17A-Brilliant Violet 421™ (506926) and anti-Foxp3-PE (320008). Flow cytometry was carried out with a FACSCalibur cytometer and data were analyzed with CellQuest software (Beckman Coulter, Brea, CA, USA).

Splenocytes were isolated for analysis via IFN-γ ELISpot, surface and intracellular cytokine staining (ICS) and flow cytometry as previously described^{69 (link)–71 (link)}. Splenocytes were restimulated with 2 μg/mL of the appropriate antigenic peptide pool (comprised of individual peptides that were 20 amino acids in length with 10 amino acids of overlap) spanning the full-length LASV GPC sequence (Mimotopes). Reference sequences for peptide synthesis are as follows: NP_694870.1 (Josiah), AIT17836.1 (Pinneo), AAF86703.1 (803213), CAA36645.1 (GA391). Cells were restimulated for 18–20 h or 6 h (at 37 °C and 5% CO₂) for IFN-γ ELISpot and ICS, respectively. Surface staining and ICS were carried out using the following antibodies: LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Thermo Fischer Scientific); anti-CD8a-PerCP/Cy5.5, anti-CD62L-PeCy7, anti-IFN-γ-eFluor 450, anti-TNF-α-Alexa Fluor 488, anti-IL-2-PE (eBioscience); anti-CD4-Brilliant Violet 650, anti-CD44-Alexa Fluor 700 (BioLegend); and anti-CD127-eFluor 660 (Invitrogen). Antigen-specific T-cell responses were quantified by subtracting the response (SFU for IFN-γ ELISpot and the percentage of cytokine-positive cells for ICS) measured without stimulation from that observed after restimulation.