Various amounts of whole blood were obtained by venipuncture or leukapheresis using vacutainer collection tubes at the time points indicated in (Fig. 1 ). In order to achieve processing within 6 h, vacutainer tubes were transported within the Radboudumc at room temperature to the Department of Tumor Immunology. PBMCs were isolated via gradient centrifugation using Lymphoprep (Axis-Shield) following the manufacturer's instructions. Cells were frozen in Xvivo 15 (Lonza) supplemented with 40% Albuman 200 g/L (Sanquin) and 10% CryoSure-DMSO (WAK Chemie Medical GMBH) in Cryo.S vials (greiner bio-one) at 10 to 100 × 106 cells per vial using Cryo 1°C Freezing Container (NALGENE) following the manufacturer's instructions. The freezing container was placed at −80°C, and vials were transferred into the vapor phase of liquid nitrogen after about 72 h for long-term storage.
For intracellular cytokine secretion testing, 1 to 2 vials per sample with varying cell numbers depending on experimental setup and availability were thawed. In case of several time points measured for a single patient, we attempted to include all samples in one experiment which was however not always possible.
For intracellular cytokine secretion testing, 1 to 2 vials per sample with varying cell numbers depending on experimental setup and availability were thawed. In case of several time points measured for a single patient, we attempted to include all samples in one experiment which was however not always possible.