Cd154 bv421

Peptide stimulated cells were washed and extracellularly stained with LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Thermo Fisher Scientific). Afterwards, samples were fixed and permeabilized for 30 min at 4°C using FoxP3 transcription factor staining buffer set (eBioscience). Following, intracellular staining was performed for CD3 BV650, CD4 PerCp-Cy5.5, CD8 BV510, CD137 PE, CD154 BV421, IL-2 APC, IFNγ BV605, and TNFα AF700 (Biolegend). Stained cells were then transferred into a 96-well plate and measured at a CytoflexLX (Beckman Coulter). Flow cytometry data was analyzed using FlowJo software version 10.6.2 (BD). Reactive T cells were defined as CD154+CD137+CD4+ or CD137+CD8+ T cells >0.005% within total CD4+ or CD8+ T cells and with a threshold of ≥1.2-fold signal above the background control. This threshold corresponds to the range in which 95% of all negative samples are. Unspecific activation of cells was excluded by subtracting the background signal of the DMSO stimulated negative control sample from the peptide stimulated samples. Single, double (dp), or triple (tp) cytokine producing reactive T cell subsets were analyzed using Boolean combination gates (see Supplementary Figure 1 for gating strategy).

2mM EDTA-PBS solution (to detach clumped cells) was added to the stimulated PBMC-s, followed by incubation at 37°C for 10 minutes. After washing, diluted FcR Blocking Reagent (Miltenyi Biotec) and antibody mix were added to each sample and incubated for 30 minutes at 4°C. Antibodies used for sorting were as follows: CD137 PE (Miltenyi Biotec), CD154 BV421 (BioLegend), CD3 AF488 (BioLegend), CD4 AF700 (BioLegend), CD8-BV605 (BioLegend), CD45RA PE-Cy7 (BioLegend) and CCR7 PE-Dazzle (BioLegend; Supplementary Table S6). 7-AAD (Miltenyi Biotec) was added to the samples to exclude nonviable cells. Samples were sorted with MA900 Multi-Application Cell Sorter (Sony Biotechnology; gating strategy shown in Supplementary Figures S3, S13A) and were collected at 4°C into collections tubes containing TRIzol reagent (for transcriptome analysis; Thermo Fisher Scientific) or pre-coated with 4% BSA (for epigenome analysis). Publication-grade FACS plots were generated with the help of FCS Expess 7 (De Novo Software).