*2.5. Flow Cytometry*

Two million splenocytes were cultured for 5–6 hours with the peptide pools used above, as previously described [44], and with eBioscience protein transport inhibitor cocktail (Invitrogen). Surface (for CD4 and CD8) and intracellular (for remaining markers) staining followed. Biolegend anti-mouse antibodies conjugated to fluorophores used in this experiment included CD3ε-PE/Cy5 (145-2C11), CD4-FITC (RM4-5), CD8a-APC/Cy7 (53-6.7), IFNγ-APC (XMG1.2), TNFα-BV605 (MP6-XT22), and IL-2-PE-Cy7 (JES6-5H4). Live-dead exclusion was performed using violet fluorescent reactive dye (Invitrogen). Data was collected using a BD Biosciences LSRII flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using FlowJo v10 (FlowJo LLC, Ashland, OR, USA).
