Daan Pieren

100 Chapter 4 among TIGIT + T cells. For example, Helios co-expression with TIGIT has been described in CD4 + T cells, in which it defines a functionally distinct cell subset that is characterized by regulatory properties [16,17]. Additionally, a significant proportion of CD8 + T cells also expresses Helios and a recent study hinted towards Helios as marker that might indicate immunosenescence of CD8 + T cells [18]. However, the value of Helios as marker of CD8 + T-cell immunosenescence remains to be defined. Here, we aimed to define the potency of Helios combined with TIGIT as marker to more precisely pinpoint CD8 + T-cell immunosenescence and how these markers relate to CD8 + T-cell differentiation status during aging. MATERIALS AND METHODS Study design Blood was obtained from healthy individuals (n=50, 21-82 years of age) from three different sources: blood donors (Sanquin Blood Supply Foundation), healthy participants of the NVI-255 study at the RIVM (Netherlands Trial Register NTR2070) [19], or the ILI-3 study at the RIVM (Netherlands Trial Register NTR4818) ([20], Van Kaaijk et al. submitted). Only CMV-seronegative participants were included in the study, as determined by ELISA [21] or our in- house Multiplex Immunoassay [22]. PBMC isolation and flow cytometry Peripheral Blood Mononuclear cells (PBMCs) were isolated by density gradient (Ficoll-Hypaque, Amersham Biosciences) from heparinized blood or buffy coats and stored at -135 °C in 10% dimethyl sulfoxide (DMSO, Sigma Aldrich) and 10% fetal calf serum (FCS) until further use. For flow cytometric analyses, frozen PBMCs were thawed at 37°C and were transferred to RPMI-1640 medium (GIBCO, Thermo Fisher Scientific) supplemented with 10% FCS and Penicillin- Streptomycin-Glutamine (P/S/G) and washed twice. Thawed cells were then rested in medium for 30 minutes at room temperature after which the number of viable cells was determined on a Coulter Counter (Beckman). 4*10 5 PBMCs of each individual were labeled for surface markers at 4°C for 30 minutes in FACS buffer (1x PBS +0.5% BSA +2mM EDTA) with saturating concentrations of the following fluorescent labeled antibodies: CD3-FITC (clone UCHT1), CD4- PerCP-Cy5.5 (clone RPA-T4), CD28-BV711 (clone CD28.2), CD27-BV510 (clone O323), and CD57 (clone HCD57) all from Biolegend, CD8-BUV395 (clone RPA- T8, BD Horizon), TIGIT-PE-eFluor610 (clone MBSA43, eBioscience), CD226- BV785 (clone DX11, BD Optibuild), and KLRG1-PerCP-eFluor710 (clone 13F12F2,