Purified anti mouse cd28

ID8 cells were provided by Gordon Freeman (DFCI). Cells were cultured in DMEM with 10% FBS (Heat inactivated). The rest of the cell lines were received from ATCC and cultured in their recommended media. Splenocytes were cultivated in SILAC RPMI (Thermo # 89984) media supplemented with: 10% Heat Inactivated FBS, Sodium pyruvate 1 mM, Glutamine 1 mM, Penicillin (50 U/ML) Strptomycin (50μg/ml), 2-Mercaptoethanol; 0.00005 M, (Sigma M7522), IL7: 5 ng/ml (PeproTech, #217–17 murine IL7), IL15: 100 ng/ml (PeproTech, #210–15 murine IL15), N-acetyl L Cysteine: 0.5 M (Sigma A9165), Lysine 218 uM and Arginine: 75 uM (or as indicated). Recombinant arginase was purchased from (R&D Biosystems, 5868AR010). Purified anti-mouse CD3 (cat# 100202) and purified anti-mouse CD28 (cat# 102102) antibodies were purchased from BioLegend. IFNg ELISA was performed using Biolegend (Mouse IFN-γ ELISA MAX).

Anti–CD3ε-PerCP (100326), anti–CD8a-AF700 (100730), anti–CD8a-APC (100712), anti–PD-1-PE/DZ594 (109116), anti–Ki67-FITC (652410), anti–TCF1-PE (655208), anti–IFN-γ-BV421 (505830), anti–IFN-γ-PerCp/Cy5.5 (505822), anti–TNF-α-APC/Cy7 (506344), anti–TIM-3-PE/Cy7 (119716), anti–CD45-BV510 (103138), anti–Ly108 (Slamf6)-PE (134606), anti–PD-1-FITC (135214), anti–CD45.1-PE (110708), anti–CD45.2-AF488 (109816), purified anti-mouse CD3ε (100340), and purified anti-mouse CD28 (102116) were obtained from Biolegend. Anti–CD8-FITC (D271-4) and tetramer-SIINFEKL-APC (TS-5001-2c) were purchased from MBL. Cell Stimulation Cocktail Plus Protein Transport Inhibitors and FOXP3/TF Staining Buffer Set were bought from eBioscience. Naive CD8a⁺ T Cell Isolation and Tumor Dissociation Kits were obtained from Miltenyi Biotec.