Pe cd4 gk1

For analysis of surface markers, the single‐cell suspensions of PBMCs or TILs were labelled with fluorochrome‐conjugated monoclonal antibodies specific to the relevant targets for 30 min. The primary antibodies include: anti‐human PE/cy7‐CD3 (OKT3, eBioscience), FITC‐CD4 (OKT4, eBioscience), Alexa Fluor 700‐CD8 (HIT8a, BD), Brilliant Violet 421‐TIGIT (MBSA43, eBioscience), APC‐CD96 (NK92.39, eBioscience), PE‐CD226 (c11A8, BioLegend) and FITC‐CD69 (FN50, BioLegend). For the detection of intracellular markers, cells were cultured with PMA/ionomycin/brefeldin A (eBioscience) for 4 h and then were permeabilised using a fixation/permeabilization kit (eBioscience). The primary antibodies include: anti‐human APC/CY7‐IFN‐γ (4S.B3, eBioscience), Alexa Fluor 647‐TNF‐α (MAB11, eBioscience), Pacific Blue‐granzyme B (QA16A02, BioLegend), and PE‐Ki‐67 (Ki‐67, BioLegend). For mouse cell staining, the primary antibodies include: anti‐mouse APC/cy7‐CD45 (30‐F11, BioLegend), FITC‐CD3 (17A2, BioLegend), PE‐CD4 (GK1.5, BioLegend), Alexa Fluor 700‐CD8 (Mp6‐XT22, BioLegend), PE/cy7‐CD69 (H1.2F3, eBioscience), PE‐IFN‐γ (XMG1.2, eBioscience), Brilliant Violet 421‐TNF‐α (Mp6‐XT22, BioLegend), APC‐IL‐2(JES6‐5H4, BioLegend), APC‐NK1.1 (S17016D, BioLegend) and BV421‐Foxp3 (MF‐14, eBioscience).

Extracted tumors were minced completely (1–3 mm cubes) by using scalpels in two drops of dissociation solution (1 mg/ml collagenase D (11088858001 Roche, Indianapolis, IN) and 1 mg/ml DNase I (10104159001 Roche) in RPMI 1640) on ice. Tumor pieces were further digested for 45 min with dissociation solution (about 1 g tumor in 10 ml dissociation solution) at 37 °C water bath with manually periodical shaking. The digested tissue suspension was aspirated into a 20 ml syringe with 14 g cannula attached and clumps were triturated 15 times and filtered through 40 um strainer to get the single cell suspension  followed by staining with fluorescent antibodies from Biolegend: PE CD4 (GK1.5, 100407), Brilliant Violet 605 CD8a (53–6.7, 100744), PE CD11b (M1/70, 101207), Alexa Fluor 700 F4/80 (BM8, 123130), Alexa Fluor 488 CD3 (17A2, 100210), APC MHC II (M5/114.15.2, 107613). Stained cells were analyzed using BD LSRFortessa X-20 flow cytometer. Data were collected with BD FACSDiva v 8.0.1 software and were analyzed using FlowJo v 10 software.