Monensin solution

For the detection of cytokine-secreting T cells, their secretion was inhibited by addition of 2 mM monensin solution (eBiosciences) for 10 hours prior to T cell harvesting. T cells were fixed and permeabilized using the FoxP3-staining buffer kit (eBiosciences, 00–5523) according to manufacturers´ instructions. Extracellular markers were stained then T cells (including unstained controls) were re-suspended in 100 µL of Fixation/Permeabilization reagent and were incubated for 20 min on ice. The master mix for intracellular staining was prepared with 2% mouse serum in permeabilization buffer and relevant antibodies (Supplementary Table 1). The cell pellets were re-suspended in 100 µL of Master Mix. Unstained controls were re-suspended in permeabilization buffer. T cells were incubated for 30 min on ice in the dark. Fluorescently labelled cells were acquired on a LSRII (BD Biosciences) using Diva Software. Compensation was set-up using single positive stained beads or cells and voltages were adjusted accordingly. Compensation was automatically calculated by the Diva Software. The events counted were dependent on the number of cells available and were generally between 20000 and 100000 acquired events per sample and identical numbers were acquired within one experiment. Analysis of flow cytometry data was performed using FCS Express (De Novo).

Spleen cells were further seeded in triplicates at 5 × 10⁶ cells/well in a 24-well plate and stimulated with WH121 (10 μg/mL) in the presence of 1 μg/mL anti-CD28/CD49d (eBioscience CA, USA) for 20 h at 37°C under 5% CO₂. For the last 4 h of the stimulation, 3 μg/mL brefeldin A and 2 μM monensin solution (eBioscience CA, USA) were added. The cells were washed in FACS buffer (1% FCS-PBS) and stained for 30 min at RT with anti-CD4-PE-Cy7 (clone GK1.5, eBioscience), anti-CD8α-PE (clone53-6.7, eBioscience), anti-CD62L-FITC (clone MEL-14, BD Pharmingen), and anti-CD44-APC-Cy7 (clone IM7, BD Pharmingen) mAbs. After the cells had been washed and permeabilized with the Cytofix/Cytoperm kit (BD Pharmingen CA, USA), the intracellular cytokines were stained with anti-IFN-PerCP-Cy5.5 (clone XMG1.2; eBioscience CA, USA) and anti-IL-2-allophycocyanin (clone JES6-5H4; eBioscience CA, USA) mAbs for 30 min at RT. The cells were washed twice, resuspended in FACS buffer and analyzed using an LSRII multicolor flow cytometer (BD Biosciences CA, USA). The absolute number of IFN-γand/or IL-2 positive CD4⁺ and CD8⁺ T cells, T_CM (central memory T cells, CD62L^hiCD44^hi) and T_EM (effector memory T cells, CD62L^loCD44^hi) cells were analyzed with FlowJo software (Tree Star Inc., OH, USA) as previously described [25 (link)], respectively. The data are represented as the mean ± SD per group (n = 6).

Mo/Ma were harvested and stained with anti-human CD14 AlexaFluor-647 or FITC, CD206 APC, Gal-9 PE, CD16 PE, CD40 PE-Cy5 (Biolegend), HLA-DR PE-Cy7, B7.H4 Biotin (eBioscience, San Diego, CA, USA) or isotype controls in PBS with 2% FSB on ice for 20 min. T cells were harvested and stained with mAb to human CD3 PerCP, CD27 APC-Alexa750, CD28 APC and Tim-3 PerCP-eFluor710 (eBioscience).
To detect CD40L expression, PE-labeled anti-human CD40L/CD154 antibodies were cultured with T cells in the presence of anti-CD3 mAb and Monensin solution (eBioscience) during 40 h. Intracellular staining for phosphotyrosine was performed an AlexaFluor-647-conjugated anti-human phosphotyrosine (PY20, Biolegend) antibody.
The production of ROS and NO was evaluated using the molecular probes: H₂DCF-DA (10 μM, Invitrogen Inc.) and DAF-FM DA (10 μM, Molecular Probes, Inc.), respectively.
The assessment of phagocytosis was performed using 1 μm-Fluoresbrite Yellow Green (YG) Carboxilate Microspheres (Polysciences, Inc., Warrington, PA, USA; 1 : 25 cell/microspheres ratio), which were added to CCs for 30 min. Afterwards, the uptake of YG-microspheres was evaluated in CD14+ cells.
All samples were acquired on a FACS Canto II (BD Biosciences, San Jose, CA, USA) and then analyzed with Flow Jo software (LLC, Ashland, OR, USA).

To evaluate cytokine production of CD4+ T cells, PBMCs and platelets were co-cultured in a ratio of 1:1, 1:10, 1:50, 1:100, and 1:500. PBMC were stimulated with 0.5 µg/mL Phytohaemagglutinin (PHA; Merck KGaA, Darmstadt, Germany) for 6 h in the presence of Monensin Solution (Thermo Fisher Scientific Inc., Rockford, IL, USA) to inhibit protein transport and improve cytokine staining. Cells were harvested and stained for CD4, CD41, PD-1, and PD-L1 as described below. Intracellular staining with INF-γ-APC or TNF-α-APC was performed after using the Fixation/Permeabilization Solution Kit (Becton Dickinson, Heidelberg, Germany) according to the manufacturer’s protocol.
Cytokine production of CD4+ T cells from HNSCC patients could not be investigated due to non-sufficient isolated cell numbers of PBMC and platelets from those patients.

Single-cell suspensions obtained from the blood, spleen, and lymphocyte node were used for restimulation in vitro to measure cytokine secretion ability. At indicated time points, single-cell suspensions were seeded into 96-well plate. The cells were pre-stimulated with 1 μM SIINFEKL (N4) peptide (synthetized by China Abcepta Biotech Ltd., Co.) for 30 min. Then, cells were restimulated for another 4 h, and meanwhile, the medium was added eBioscience Brefeldin A (Invitrogen, no. 00-4506-51) and eBioscience Monensin Solution (Invitrogen, no. 00-4505-51).