Flow cytometry
Expanded TIL was stained with fluorescent antibodies for human CD3 (BD, 564713), CD4 (BD, 557852), CD8 (BD, 564526), CD56 (BD, 555516), CD28 (BD, 555730), CD103 (Biolegend, 350216), CD39 (BD, 563679), TCF1 (Biolegend, 655208), PD-1 (BD, 564323), TIM-3 (BD, 565558), LAG-3 (BD, 565720), CD45RA (BD, 562885), CCR7 (BD, 565867). All cells were stained with a Live/Dead viability dye (eBioscience, 65-0865-14). For intracellular staining of TCF1, cells were fixed with Transcription Factor Buffer Set (BD, 562574), and then TCF1 was stained in accordance with the manufacturer’s protocol (Biolegend). Data were acquired on an MACSQuant Analyzer 10 flow cytometer (Miltenyi) and analyzed using FlowJo software (Treestar).