Cytotoxicity assays

Human sarcoma cell lines A673 (#CRL-1598, Ewing’s sarcoma) and SK-LMS (#HTB-88, leiomyosarcoma) were obtained from ATCC (Manassas, Virginia, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Nu-Serum and 1% penicillin-streptomycin. Cells were passaged every 3–5 days or sooner as needed. Adherent cells were removed using Tryp-LE (Thermo Fisher #12604021). Where indicated, cell lines were added to preincubated PBMCs or TILs at the indicated effector-to-target ratios in a 1:1 mixture of RPMI complete media to DMEM. Cytotoxicity was assessed by labeling target cells with CFSE (Invitrogen #{"type":"entrez-nucleotide","attrs":{"text":"C34554","term_id":"2370695","term_text":"C34554"}}C34554) for 5 min at room temperature. After 4 hours of effector/target contact, cells were stained with Fixable Viability Dye 780 and analyzed by flow cytometry for CFSE⁺FVD780⁺ proportion. For phenotypic analysis, PBMCs were preincubated with IL-15 as indicated and then exposed to target cells. On addition of target cells, 2.5 µL of CD107A-BV605 or CD107A-BV421 (clone H4A3, BioLegend) was added. After 60 min, Golgi Plug (brefeldin A) and Golgi Stop (monensin) were added per manufacturer’s instructions (BD Biosciences). Cells were incubated for another 3–5 hours then analyzed by flow cytometry.