Ab serum

TILs were suspended in AIM-V (Thermo Fisher Scientific, Tokyo, Japan) supplemented with 10% AB serum (Biowest, Nuaillé, France) and then treated with Golgi STOP (1:1000), PMA (50 ng/mL), and ionomycin (1 µM) for 4 h. Harvested cells were stained with Zombie Yellow Fixable Viability kit (BioLegend), anti-CD45-BV785, anti-CD45RA-APC-Cy7 [HI100], anti-PD-1-PE-Cy7, and anti-Tim3-PE. After fixing with Foxp3/transcription factor staining buffer set (Thermo Fisher Scientific), cells were stained with anti-CD3-BV421 [UCTH1], anti-CD4-APC [RPA-T4], CD8-BV711 [RPA-T8], anti-IL-2 [PerCP-eFluor710], anti-TNFα-BV510 [Mab11], and anti-IFNγ-FITC [4 S.B3].

NK cells were isolated from peripheral blood mononuclear cells (PBMCs) using the Human NK Cell Enrichment Cocktail-RosetteSep (StemCell Technologies Inc., 15065). Only populations displaying more than 95% of CD56⁺ CD3⁻ CD14⁻ NK cells were selected for the experiments. The isolated NK cells were cultured for 3 or 5 days in complete medium: RPMI 1640 (Lonza, 12-167F) 10% plus AB serum (Biowest, S4190-100), 1% penicillin/streptomycin (Lonza, 17-602E), 1% glutamine (Lonza, 17-605E) with 100U/ml IL-2 (Proleukin, Novartis), with 20ng/ml IL-15 (Miltenyi Biotech, 130-093-955), or with 20ng/ml IL-15 plus 0.1μg/ml IL-18 (MBL, B001-5) [53 (link)], in the presence or in the absence of BRAF-i (PLX4032, S1267) or MEK-i (PD0325901, S1036) (Selleckchem) dissolved in DMSO. All experiments were performed in accordance with approvals from the Liguria Regional Committee.