Monensin

Human peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy donors with informed consent at the Blood Centre of Anhui Province using Ficoll-Hypaque (Solarbio, Beijing, China) density gradient centrifugation. For analysis of NK cell and CD8⁺ T cell activation, PBMCs were cultured in RPMI medium supplemented with 10% FBS in the absence or presence of 50 IU/mL rhIL-2, 10 ng/mL rhIL-15 or 35.7 ng/mL IL-15/SuIL-15Rα-IgG4 Fc complexes (a molar equivalent dose of rhIL-15) for 24 h. For analysis of NK cell and CD8⁺ T cell proliferation, PBMCs were labeled with 5 μM CFSE (Biolegend) and then incubated for 7 days in the indicated conditions mentioned above. After incubation, cells were harvested, washed with PBS, and then resuspended in PBS containing 10% mouse serum (Future, Guangzhou, China) at 4 °C for 30 min prior to staining with fluorescently conjugated antibodies at 4 °C for 30 min in the dark. For intracellular staining, cells were incubated with 10 ng/mL monensin (Abcam) for 4 h at 37 °C in a 5% CO₂ incubator followed by staining for extracellular markers. Cells were then fixed, permeabilized (Invitrogen) and stained for intracellular molecules. The stained cells were subsequently washed, detected by a CytoFLEX Flow Cytometer and analyzed by CytExpert software. The antibodies used in this study were listed in Additional file 1: Table S2.

Single-cell suspensions were prepared from tumors as previously described [52 (link)] and stimulated prior to staining with panels of antibodies for flow cytometry analysis of the T cells. In total, 2.5 × 10⁶ cells were cultivated for 3 hours in 2 mL of Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich, Merck KGaA) supplemented with 10% FCS (Biosera), 100 IU/mL penicillin, 100 μg/mL streptomycin (Biosera), and 50 µM 2-mercaptoethanol and containing 81 nM phorbol 12-myristate 13-acetate, 1.34 µM ionomycin, 2 µM monensin, and 10.6 µM brefeldin A (Abcam, Cambridge, UK).