Anti lag 3

CD8 + T cells from tumor-infiltrating lymphocytes were isolated using flow cytometry-assisted cell sorting. The purity of the sorted cells was >95 %. To detect cytokine production, lymphocytes were stimulated for 2 h in the presence of a cell-stimulating mixture (eBioscience, USA, 00-4975-93) and then stained with the surface markers anti-LAG-3 (BioLegend, USA, Cat. No. 125214) and anti-PD-1 (BioLegend, Cat. No. 135206), and CTLA4 (BioLegend, Cat. No. 106308) at a 1:100 dilution in PBS containing 1 % FBS. Following a 30 min incubation on ice, a cell fixation/permeabilization kit (BD Biosciences, USA, Cat. No. 554714) and a transcription factor fixation/permeabilization buffer (BioLegend, Cat. No. 424401) were employed for intracellular cytokine and nuclear transcription factor labeling, respectively. Anti-IFN-γ (BioLegend, USA, Cat. No. 505810), anti-TNF-α (BioLegend, Cat. No. 506306), and anti-TOX (Miltenyi Biotec, Germany, Cat. No. 130-120-337) were diluted 1:50 in permeabilization buffer and incubated overnight at 4 °C in the presence of Anti-IFN-γ and anti-TNF-α(BioLegend, USA). FlowJo software (Tree Star, USA) was used to analyze FACSCelesta (BD, USA) flow cytometry data. All analyses were performed using GraphPad Prism 8 (GraphPad Software, USA) and t-tests. P < 0.05 was deemed statistically significant.

Fresh tumor and spleen samples were minced, treated with 200 U/ml Collagenase I (Cat#ST2294, Beyotime Biotechnology), and filtered with a 100 μm filter to make a suspension of single cells. 2 × 10^{6 (link)} Cells were stained at 100 μl in PBS. Single-cell suspension is stained with the following antibodies: Live/Dead Fixable Violet Dead Cell (Cat# 2549272, Invitrogen), anti-CD3 (Cat# 100236, Biolegend), anti-CD4 (Cat# 100406; 100408, Biolegend), anti-CD8 (Cat# 100706, Biolegend), anti-Ki67 (Cat# 652425, Biolegend), anti-IFN-γ (Cat# 505860, Biolegend), anti-Perforin (Cat# 154306, Biolegend), anti-Foxp3 (Cat# 126404, Biolegend), anti-CD11b (Cat# 101212, Biolegend), anti-CD11c (Cat# 117352, Biolegend), anti-Gr-1(Cat# 108406, Biolegend), anti-TIM-3(Cat# 119727, Biolegend), anti-LAG-3 (Cat# 125241, Biolegend) and anti-PD-1 (Cat# 135227, Biolegend). Surface antigens were detected by NovoCyte Quanteon (Agilent) after staining with antibodies for 30 minutes in the dark. To detect intracellular cytokines, the samples were fixed with Fixation/Permeabilization Solution KiT (Cat# 554714, BD Biosciences) and incubated with intracellular antibodies for 30 minutes before running the machine. Datas were analyzed by NovoExpress.