Tnfα af700

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).