TILs

A total of 1.0 × 10⁶ TILs were stained with Live/Dead Fixable Yellow (Thermo Fisher Scientific {"type":"entrez-nucleotide","attrs":{"text":"L34968","term_id":"522211","term_text":"L34968"}}L34968) at a 1:1,000 concentration for 30 minutes on ice, rinsed with staining buffer and then blocked with CD16/32 FCR blocker at a 1:100 concentration (BioLegend, 101319) for 30 minutes on ice. Cells were then stained with antibodies specific for cell surface markers at a previously validated concentration for 30 minutes on ice. Two panels of antibodies were used to immunophenotype TILs and they were as follows; Panel 1–Cd11c Pacific Blue (BioLegend, 117322), MHCII BV711 (BioLegend, 107643), CD86 FITC (BioLegend, 105006), CD11b PerCP CY 5.5 (BioLegend, 101227), Ly-6C PeCy7 (BioLegend, 128017), F4/80 APC (BioLegend, 123115), CD45 Alexa Fluor 700 (BioLegend, 103128), and Ly6-G APC/Fire 750 (BioLegend, 127651). Panel 2–CD45 Pacific Blue (BioLegend, 103126), CD3ϵ PerCP Cy 5.5 (BioLegend, 100218), CD45R (B220) PE-CY7 (BioLegend, 103221), CD8α Alexa Fluor 700 (BioLegend, 100730), CD49b (pan-NK cells) PE/Dazzle 594 (BioLegend, 108924), and CD4 APC/Fire 750 (BioLegend, 100460). After surface staining, cells were rinsed with PBS and fixed overnight at 4°C using a Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, 00–5523–00). After fixation cells were blocked in permeabilization buffer (Thermo Fisher Scientific, 00–5523–00) with 2% rat serum (Sigma-Aldrich, R9759) for 30 minutes at room temperature and then stained with antibodies specific for intracellular markers. Panel 1 contained antibodies specific for Arginase PE (Thermo Fisher Scientific, 12–3697–82), CD206 PE/Dazzle 594 (BioLegend, 141732) for T12 TILs and Panel 2 antibodies specific for contained Ifnγ FITC (BioLegend, 505806), Foxp3 PE (BioLegend, 126403), and Granzyme B APC (BioLegend, 372203). After staining, cells were washed twice with permeabilization buffer, resuspended in PBS and data were collected using the Attune NxT flow cytometer. Flow samples were properly compensated and analyzed using FlowJo v10 software (FlowJo, RRID:SCR_008520).