Live dead fixable blue dead cell kit

2 × 10⁶ cells from LN cell suspensions were stained with LIVE/DEAD Fixable Blue Dead Cell kit according to manufacturer’s protocol (Invitrogen). Samples were then incubated with a titrated amount of H10-tetramer probe for 20 min at 4°C. H10-tetramer probes were prepared beforehand by mixing biotinylated H10 protein with streptavidin-BV421 (Biolegend) at a 4:1 M ratio. Samples were washed and stained with a surface antibody cocktail (Table S2 in Supplementary Material). Before intracellular staining (Table S2 in Supplementary Material), samples were fixed and permeabilized using the Transcription Factor Buffer Set (BD Biosciences). Samples were resuspended in 1% paraformaldehyde before acquisition using a Fortessa flow cytometer (BD Biosciences). Results were analyzed using FlowJo version 9.7.6.

1.5 × 10⁶ cells from indicated time points were stained with LIVE/DEAD Fixable Blue Dead Cell kit according to manufacturer’s protocol (Invitrogen). Samples were surfaced stained with a panel of fluorescently labeled antibodies (Table S1 in Supplementary Material) to identify specific cell subsets. Additionally, 1.5 × 10⁶ PBMCs were rested for 3 h and then stimulated overnight in complete media (10% FCS, 1% penicillin/streptomycin/glutamine in RPMI, all from Gibco, Stockholm, Sweden) in U-bottom 96-well plates with H10 peptides (15mers overlapping by 11 amino acids, 2 µg/ml) and Brefeldin A at 10 µg/ml. Cells were stained with surface-specific antibodies (Table S1 in Supplementary Material), fixed, and permeabilized using fixation and permeabilization solution (BD Biosciences) before being stained for intracellular cytokines (Table S1 in Supplementary Material). Samples were resuspended in 1% paraformaldehyde before acquisition using a Fortessa flow cytometer (BD Biosciences). Results were analyzed using FlowJo version 9.7.6. Background cytokine staining was subtracted, as defined by staining in samples incubated without peptide.