Anti tigit

Sorted Teff and Tregs from healthy donors were activated with anti-CD3/CD28 beads (1:5 ratio) (Life Technologies) and 1000 UI IL-2 for 5 days a 37°C. Then, 100 uL of OSCC and control secretomes were added to 2x10⁵ Teff or 2x10⁵ Tregs (in 100uL) in XVIVO-15 serum-free medium 48h a 37°C. After the incubation, the supernatants were stored for further cytokine production measurement using the Cytokine Bead Array Th1/2/17 Kit (BD) and the cells were counted (CountBright Absolute Counting Beads), stained with Live/Dead dye (Life Technologies), anti-CXCR3, anti-CCR4, anti-CCR6, anti-CCR8, anti-PD-1 and anti-TIGIT (all BioLegend) and analyzed by flow cytometry. For the analysis of cells after secretome co-culture, cells were washed after co-culture with secretome and cultured in new media X-VIVO15 (LONZA) serum-free medium for 48 h at 37°C with anti-CD3/CD28 beads (1:5 ratio) (Life Technologies) and 1000 UI IL-2. After the incubation, the supernatants were stored for further cytokine production measurement and the cells were stained with Live/Dead dye (Life Technologies), anti-CCR6, anti-CCR8, anti-PD-1 and anti-TIGIT (all BioLegend).

Cryopreserved PBMC were isolated and used for immunophenotypic staining as previously described (108 (link)). Cells were stained with Zombie NIR fixable viability stain (BioLegend, San Diego, USA) and the following anti-human monoclonal fluorochrome-conjugated antibodies: anti-CD45RA, anti-CD4, anti-TCR-γ/δ (BD Biosciences, Heidelberg, Germany), anti-TCR-Vδ2 (Beckman Coulter Life Sciences, Indianapolis, USA), anti–HLA-DR, anti-CD27, anti-CD279 (PD-1), anti-TIGIT, anti-CD8, anti-CD28, anti-CD39, anti-CD38, anti-CD19, anti-CD3, anti-CD73 and anti-CD14 (all BioLegend) (Supplementary Table 2). Cells were incubated for 30 minutes at room temperature with the respective antibodies. After washing, cells were fixated with 4% paraformaldehyde. All samples were run on a Becton Dickinson LSR Fortessa flow cytometer with FACS Diva version 8 (BD Biosciences).

CD4+ T cells were enriched by magnetic activated cell sorting (MACS) using 20 µl of anti-CD4 MicroBeads (anti-CD4 MicroBeads, human, Miltenyi Biotec, Germany) and 80 µl MACS buffer (PBS with 0.5% BSA and 2 mM ethylenediaminetetraacetic acid) per 10⁷ cells. CD4+ T cells were stained for surface expression in MACS buffer at a concentration of 2 × 10⁸ cells/ml. Cells were washed and stained with 4′,6-diamidino-2-phenylindole (1:250) and then sorted using the flow cytometer BD FACSAria II (BD Biosciences, Germany) into the following CD4+ T-cell subpopulations: Treg (CD25highCD127low) and non-Treg: TN (CD45RA+ CCR7+), TCM (CD45RA-CCR7+), TEM (CD45RA− CCR7−), and TEMRA (CD45RA+ CCR7−).
Antibodies used for surface staining were anti-CCR7 (GO43H7, 1:50), anti-CD25 (M-A251, 1:100), anti-CD45RA (HI100, 1:50), anti-CD127 (A019D5, 1:100), anti-CD3 (UCHT1, 1:200), anti-CD4 (OKT4, 1:200), anti-PD-1 (EH12.2H7, 1:20), anti-TIGIT (A15153G, 1:20), anti-TIM-3 (F38–2E2, 1:20), and LAG-3 (11C3C65, 1:20) from BioLegend, and anti-KLRB1 (191B8, 1:10), anti-KLRF1 (4A4.D10, 1:50), and anti-KLRG1 (REA261, 1:10, 1:50 since 2018/06 due to more concentrated formulation) from Miltenyi Biotec. See also Supplementary Fig. 5 for the pre-gating strategy.