Daan Pieren

101 Co-expression of TIGIT and Helios by CD8+ T cells Invitrogen). A Fixable Viability Stain 780 (BD Horizon) was added to the labeling to identify viable cells. Cells were then fixed and permeabilized with buffers for intracellular labeling (eBioscience) at 4°C for 30 minutes with fluorescent labeled antibodies targeting: Helios-PE-Cy7 (clone 22F6, Biolegend) and γ H2AX (clone N1-431, BD Horizon). Samples were measured on an LSRFortessa™ X-20 (BD Biosciences) and data were analyzed using FlowJo software (v10.6.1, TreeStar). The gating strategy used to define T-cell subsets is shown in Supplementary Figure 2b . T-cell stimulation assays The number of viable cells in thawed PBMC samples was determined manually using trypan blue staining and Bürker-Türk. Cells were then washed with PBS and labeled with 0.5 µ M CellTrace TM Violet (Invitrogen) in PBS per milliliter of cell suspension (10 6 cells/mL) for 20 minutes at 37 °C to 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. Part of the CellTrace-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 in U-bottom plates (2*10 5 cells/well), whereas another part was directly labeled with fluorescent labeled antibodies (day zero). For each donor, expression of activation markers was assessed at day zero, day one, and day three after culturing. T-cell proliferation was assessed by dilution of CellTrace after three days of culturing. At each time point, cells were labeled for surface markers with the following fluorescent labeled antibodies: CD3-FITC (clone UCHT1, at day zero), CD4-PerCP-Cy5.5 (clone RPA-T4), CD69-BV785 (clone FN50), CD25-PE (clone M-A251) all from Biolegend, CD8-BUV395 (clone RPA-T8, BD Horizon), TIGIT-PE-eFluor610 (clone MBSA43, eBioscience), and CD226-BV785 (clone DX11, BD Optibuild). A Fixable Viability Stain 780 (BD Horizon) was added to the labeling to identify viable cells. Cells were labeled for intracellular markers with the following fluorescent labeled antibodies: CD3- FITC (clone UCHT1, Biolegend, at days one and three) and Helios-PE-Cy7 (clone 22F6, Biolegend). Dimensionality reduced analyses (viSNE) Dimensionality reduced analysis (viSNE) of flow cytometry data was performed in Cytobank (www.cytobank.com ) [23]. Fingerprint color dot plots generated by viSNE indicate expression of each of the indicated markers within the CD8 + T-cell population as measured by flow cytometry. These plots were generated 4