Daan Pieren

125 Regulatory KIR+RA+ CD8+ T cells accumulate with age track their proliferation. Ice-cold RPMI-1640 medium (+10% FCS, +P/S/G) was added and cells were rested at room temperature for 5 minutes. Cells were centrifuged at 400 g for 5 minutes and washed with RPMI-1640 medium (+10% FCS, +P/S/G) three times. Cells were then labeled for surface markers at 4 °C for 30 minutes with saturating concentrations of antibodies targeting: CD4, CD8, CD45RA, CD56, KIR2D, KIR3DL1, and NKG2A ( listed in Supplementary Table 4 ), before suspension in FACS buffer +25mM HEPES. Cell sorting was performed using the FACSMelody™ (BD Biosciences). CD4 - CD56 - CD8 + single cells were sorted into the following subsets: KIR + RA + T cells (CD8 + CD45RA + KIR + NKG2A - ), NKG2A + RA + T cells (CD8 + CD45RA + KIR - NKG2A + ), and KIR - NKG2A - T cells (CD8 + KIR - NKG2A - ). T-cell stimulation: proliferation and activation assays Total CellTrace™ Violet (Invitrogen) labeled PBMCs were cultured in the presence of 0.005 µ g/mL plate-bound purified mouse anti-human CD3 (Clone HIT3 α , BD Biosciences) in RPMI-1640 medium (+10% FCS, +P/S/G) in U-bottom plates (2*10 5 cells/well). FACS-sorted and CellTrace™ Violet-labeled KIR + RA + , NKG2A + RA + and KIR - NKG2A - T-cell subsets were cultured in the presence of anti-CD3/CD28-coupled beads (Dynabeads™, Gibco) at a 1:12 bead/cell ratio in RPMI-1640 medium in U-bottom plates for three days. After three days of culturing, cells were first labeled for surface markers at 4 °C for 30 minutes with saturating concentrations of a combination of antibodies targeting: CD4, CD8, CD45RA, CD69, KIR2D, KIR3DL1, NKG2A, and TIGIT in FACS buffer, before intracellular labeling of CD3 and Helios ( listed in Supplementary Table 4 ). A Fixable Viability Stain 780 (BD Horizon) was used to identify viable cells. Samples were measured on an LSRFortessa™ X-20 (BD Biosciences) and data were analyzed using FlowJo software (v10.6.1, TreeStar). T-cell suppression assays Suppression assays were performed by culturing FACS-sorted and CellTrace™ Violet-labeled KIR - NKG2A - CD8 + T cells (responder cells) with KIR + NKG2A - CD8 + T cells (suppressor cells) that were either CellTrace™ Violet-labeled or not. Cells were cultured together in a range of suppressor-to-responder ratios from 1:1 to 1:16 in a total amount of 10.000 cells/well. During culture, cells were stimulated with anti-CD3/CD28-coupled beads (Dynabeads™, Gibco) at a 1:12 bead/ cell ratio in U-bottom plates. After three days of culturing, cells were labeled for surface markers at 4°C for 30 minutes with saturating concentrations of antibodies targeting: CD3, CD8, CD45RA, CD69, KIR2D, KIR3DL1, NKG2A, and TIGIT in FACS buffer ( listed in Supplementary Table 4 ). A Fixable Viability Stain 5