Daan Pieren

126 Chapter 5 780 (BD Horizon) was used to identify viable cells. Samples were measured on an LSRFortessa™ X-20 (BD Biosciences) and acquired flow cytometry data were analyzed using FlowJo software (TreeStar). Suppression of responder proliferation by suppressor cells was calculated as follows: ((% proliferation responder only - % proliferation with suppressor cells) / (% proliferation responder only))*100%. Intracellular cytokine assay PBMCs were added to U-bottom plates (1*10 6 cells/well) in RPMI-1640 medium (+10% FCS, +P/S/G) in the presence of anti-CD107a labeling (Biolegend), and exposed to phorbol-12-myristate-13-acetate (PMA, 25 ng/mL, Sigma-Aldrich) with ionomycin (250 ng/mL, Sigma-Aldrich) for 6 hours at 37 °C or to medium only as control. After two hours of incubation, Brefeldin A (1:100, Sigma-Aldrich) and Monensin (1:150, GolgiStop, BD Biosciences) were added and present during the final four-hour incubation period. After incubation, cells were labeled for surface makers for 30 minutes at 4 °C with saturating concentrations of antibodies targeting: CD8, CD27, CD45RA, KIR2D, KIR3DL1, and NKG2A in FACS buffer, followed by intracellular labeling of CD3 and the cytokines IL-2, IFN- γ , and TNF- α ( 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). RNA sequencing Cell sorting for RNA sequencing The number of viable cells in thawed PBMC samples was determined manually using trypan blue staining and Bürker-Türk as described above. Cells were labeled for surface markers at 4°C for 30 minutes with saturating concentrations of antibodies targeting: CD3, CD4, CD8, CD19, CD27, CD45RA, CD56, KIR2D, KIR3DL1, and NKG2A in FACS buffer ( listed in Supplementary Table 4 ). Cells were suspended in FACS buffer +25mM HEPES. First, viable single CD8 + T cells were selected as CD56 - CD4 - CD19 - CD3 + CD8 + cells. These CD8 + T cells were then subdivided into T NAIVE cells (CD8 + CD45RA + KIR - NKG2A - CD27 + ), T EMRA cells (CD8 + CD45RA + KIR - NKG2A - CD27 - ), KIR + RA + T cells (CD8 + CD45RA + KIR + NKG2A - ), and NKG2A + RA + T cells (CD8 + CD45RA + KIR - NKG2A + ), which were sorted separately into tubes containing RPMI-1640 medium (+20% FCS, +P/S/G). Cell sorting was performed using the FACSMelody™ (BD Biosciences).