Anti cd8 53 6

Flow cytometry was used to analyze the tumor immune microenvironment. All tumor tissue samples per group were collected. Cells from the tumors were counted and cell surface markers were stained with the following fluorescently conjugated antibodies: anti-CD45 (30F11, BD Biosciences), anti-CD4 (RM4-5, BD Biosciences), anti-CD8 (53-6.7, Miltenyi Biotec), anti-CD3 (17A2, BD Biosciences), antiCD11b (M1/70, Invitrogen), anti-PD-1 (10F.9G2, BioLegend), anti-CD25 (PC61, Biolegend), anti-CD49b (DX5, BD Biosciences), anti-Granzyme b/Cryofix (GB11, Invitrogen), Viability UV Zombie. Flow cytometry data were acquired on the BD LSRFortessa X20 cytometer and FlowJo software (Ashland, OR, USA) was used for analyses.

Flow cytometry was used to analyze the tumor immune microenvironment. All tumor tissue samples per groups were collected, dilacerated, counted and cells surface were stained with the following fluorescently conjugated antibodies: anti-CD45 (30F11, BD Biosciences), anti-CD4 (RM4-5, Miltenyi Biotec), anti-CD8 (53-6.7, Miltenyi Biotec), anti-CD161 (PK136, Miltenyi Biotec), anti-CD3 (REA606, Miltenyi Biotec), anti-CD68 (FA-11, Miltenyi Biotec), anti-CD206 (C068C2, BioLegend) anti-CD11b (REA592, Miltenyi Biotec), anti-Ly-6C (1G7.G10, Miltenyi Biotec), anti-Ly6G (REA526, Miltenyi Biotec), anti-PD-1 (HA2-7B1, Miltenyi Biotec), anti-LAG3 (C9B7W, Miltenyi Biotec), anti-TIM3 (REA602, Miltenyi Biotec), anti-ICOS (REA192, Miltenyi Biotec), anti-PD-L1 (10F.9G2, BioLegend), anti-TIGIT (REA536, Miltenyi Biotec), anti-TNFα (REA636, Miltenyi Biotec), anti-FoxP3 (3G3, Miltenyi Biotec). After surface staining, cells were fixed and permeabilized using BD Cytofix/Cytoperm kit and then labeled with FoxP3 and TNFα. Flow cytometry data were acquired on the LSRII flow cytometer, FlowJo software was used for analyses and GraphPad Prism software was used for statistical analysis (Unpaired t test). Isotype controls were from Miltenyi Biotec. Gating schemes are shown on Figures S4, S5.