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).