Intracellular Cytokine Staining of PBMCs

Intracellular cytokine staining was performed as previously described [13 (link)]. Briefly, PBMCs were resuspended in warmed media and rested overnight at 37°C. The following day, cells were adjusted to 1 × 10⁶ cells/mL in media containing anti-CD28, CD49d, and CD107a-PerCP cy5.5 (1 µg/mL). Cells were then untreated or stimulated with EBOV GP peptide pool, containing 187 × 15-mer overlapping peptides at 2.5 µg/peptide or 1 µg/mL Staphylococcal Enterotoxin B peptide for 16–18 hours. After 2 hours, brefeldin A and monensin (1 µg/mL) were added to block cytokine secretion. The following day, samples were washed and LIVE/DEAD dye added. Samples were washed, incubated with cell surface antibodies (CD3-APC 750, CD4-BV786, CD8-AF700, CD19-BV510, and then CD14-BV510, CCR7-APC, CD95-BV395, and CD45RO-BV605); then washed, fixed, and permeabilized; then stained for intracellular cytokines using IFNγ-AF488, TNFα-BV421, and IL-2-PE. Samples were analyzed using a BD Fortessa machine and FACS Diva, FlowJo, Pestle, and SPICE software (see Supplementary Information).