Leucosep tube

Venous blood was drawn into Monovette^® lithium heparin blood collection tubes (Sarstedt, Newton, USA). Peripheral blood mononuclear cells (PBMC) were isolated using Leucosep™ tubes (Greiner Bio-One, Frickenhausen, Germany) and Biocoll^® separation solution (1.077 g/mL, Merck, Darmstadt, Germany). After separation, PBMCs were washed with 45 mL of CTL Wash™ (CTL Europe, Bonn, Germany) and suspended in 1 mL of CTL Wash™. Cells were counted with a Vi-Cell XR counter (Beckman Coulter, Brea, USA) and divided for ELISPOT and flow cytometry. After centrifugation, PBMCs for flow cytometry were resuspended at a concentration of 1 × 10⁷ cells per mL in RPMI 1640 Glutamax™ (Gibco, Thermo Fisher, Waltham, USA) supplemented with 5% heat-inactivated, sterile-filtered autologous serum. PBMCs for ELISPOT were diluted in CTL Test™ medium (CTL Europe, Bonn, Germany) at a concentration of 2 × 10⁶ cells per mL.

Peripheral whole blood was harvested in K2 EDTA tubes. PBMCs were separated from whole blood by density gradient centrifugation using Ficoll-Paque (GE Healthcare) and Leucosep tubes (Greiner Bio-One GmbH) according to the manufacturer’s protocol (MACS; Miltenyi Biotec GmbH). After PBMC isolation, cells were either stimulated with antibodies or rested overnight before COMP fusion protein/FACS staining. For antibody stimulations, fresh PBMCs were incubated with 20 μg/ml antibody overnight before harvest and FACS staining. THP-1 cells were cultured in RPMI-based media at a density ∼2.5 × 10⁵/ml before 5 ng/ml PMA stimulation where indicated. After 48 h, cells were detached with enzyme-free dissociation buffer before the FACS staining protocol. Gating of cells was performed with the following strategy: CD4 T cells (CD4 TN; CD3⁺CD4⁺CD45RO⁻), antigen-experienced CD4 T cells (CD3⁺CD4⁺CD45RO⁺), CD8 TN cells (CD3⁺CD8⁺CD45RO⁻), antigen-experienced CD8 T cells (CD3⁺CD8⁺CD45RO⁺), B cells (CD3⁻CD14⁻CD19⁺), monocytes (high side scatter; CD3⁻CD19⁻CD14⁺), or NK cells (CD3⁻CD19⁻CD14⁻CD56⁺) in human whole blood.

PBMCs were isolated from peripheral blood (diluted 1.6 times with 10% phosphate buffered saline) by density gradient centrifugation (800 × g for 25 min at room temperature) over Isolymph (specific gravity 1.077) (CT Scientific Supply Corp., Deer Park, NY, USA) in Leucosep tubes (Greiner Bio-One, Tokyo Japan) for use in the NK cell activity assay. The supernatant (isolated PBMCs) was washed twice with saline solution and the efficiency of cell separation was confirmed by flow-cytometry (Accuri C6, Becton Dickinson and -Company, Franklin Lakes, NJ, USA).

Buffycoats containing plasma, red blood cells and Peripheral Blood Mononuclear Cells (PBMCs) of six different healthy donors were obtained from the Dutch bloodbank Sanquin (collected in March and June in 2019). To obtain the plasma of the same time point as the PBMCs, the buffycoats were centrifuged 10 min at 2000 rpm, the plasma was taken and centrifuged again for 10 min at 11,000 rpm to eliminate residual cell debris. PBMCs were isolated by density gradient centrifugation separation using LeucoSep tubes (Greiner, Alphen aan de Rijn, The Netherlands) for the separation of different cell layers, using Lymphoprep density gradient medium (StemCell technologies, Dieren, The Netherlands) according to manufacturer’s protocol. The cells were cryopreserved in 10% dimethyl sulfoxide/Fetal Calf Serum (DMSO/FCS) until further analysis.

Human PBMCs were isolated from heparinized blood of healthy donors or buffycoats by means of a standard Ficoll density centrifugation protocol (LymphoprepTM, Axis Shield, Oslo, Norway, cat. no. 1114544). The PBMCs of HNSCC patients and healthy donors were isolated from heparinized blood using Leucosep tubes (Greiner Bio-One, Kremsmünster, Austria, cat. no. 227290). Human NK cells were isolated from PBMCs using an NK cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany, cat. no. 130-092-657) according to the manufacturer’s protocol. The purity of the NK cells was at least 95%. PBMCs and NK cells were cultured in RPMI 1640 supplemented with 10% FBS and 2 mM L-glutamine. Cultures were maintained at 37 °C in a humidified atmosphere with 5% CO₂. Tubes with freshly drawn heparinized blood of healthy donors or buffycoats were purchased from Sanquin (Amsterdam, The Netherlands). The Institutional Review Board of Amsterdam UMC, location VUmc, approved the collection of blood and the use of immune cells from HNSCC patients, who provided informed consent (2008.071|A2016.035). Clinical details are provided in Supplementary Table S1. Pathological TNM staging was used for patients treated with surgery, while clinical TNM staging was used for patients treated otherwise.

Porcine PBMCs were isolated from blood samples collected in Vacutainer tubes EDTA-K2, diluted 1:1 in PBS and then used to obtain PBMC by density-gradient centrifugation with Histopaque 1077 (Sigma) and Leucosep tubes (Greiner Bio-One) as described (27 (link)). The number of live PMBCs was calculated using a Neubauer chamber (Immune Systems) and cell staining with 0.4% Trypan Blue (Sigma) in PBS. In general, fresh cells were used in the experiments and those remaining were cryo-preserved in 90% FBS and 10% DMSO in liquid nitrogen. The minimum number of cells used for freezing was 2x10⁷/vial.