Cd25 pc61.5

Single cell suspensions were stained with Near-IR Dead Cell Stain Kit (ThermoFisher) followed by surface, cytoplasmic and/or nuclear staining with the following fluorochrome-conjugated monoclonal antibodies: CD25 (PC61.5) purchased from ThermoFisher; H3K4me3 (C42D8), H3K27me3 (C3B11) purchased from Cell Signaling Technology; 4-1BB/CD137 (1AH2), TNF-α (MP6-XT22) purchased from BD Biosciences; CD44 (IM7), CD62L (MEL-14), CD8 (53-6.7), Fc Block (93), Granzyme B (QA16A02), ICOS (C398.4A), IFN-γ (XMG1.2) purchased from Biolegend. Staining of cytoplasmic and nuclear antigens was performed using the Fixation/Permeabilisation kit (BD Biosciences) and the Transcription Factor buffer set (Invitrogen/eBiosciences), respectively. To measure intracellular cytokines, cells were re-stimulated with PMA, Ionomycin and Brefeldin (all Sigma) for 4 hours prior to staining. Samples were analysed on a FACSCanto II flow cytometer (BD Biosciences).

Single cell suspensions were stained with Near-IR Dead Cell Stain Kit (ThermoFisher) followed by surface, cytoplasmic and/or nuclear staining with the following fluorochorome- conjugated monoclonal antibodies: CD25 (PC61.5) purchased from ThermoFisher; CD44 (IM7), CD62L (MEL-14), CD8 (53-6.7), Fc Block (93), Granzyme B (QA16A02), ICOS (C398.4A), IFNγ (XMG1.2) and TNF⍺ (MP6-XT22) purchased from Biolegend. Staining of cytoplasmic and nuclear antigens was performed using the Fixation/Permeabilization kit (BD Biosciences) and the Transcription Factor buffer set (Invitrogen/eBiosciences), respectively. For staining of intracellular cytokines CD8+ T cells were re-stimulated with PMA, Ionomycin and Brefeldin A (all Sigma) for 4 hours. Samples were analysed on a FACSCanto II flow cytometer (BD Biosciences) and using FlowJo version 10 software (BD Biosciences). The gating strategy was as follows: FSC-A, SSC-A lymphocytes >> FSC-A, FSC-H singlets >> CD8 positive, LIVE/DEAD Negative.