Anti lag 3

Tumor tissues were first minced and passed through 70 μm filters to obtain single-cell suspension. Cells were then stained with Viability Ghost Dye 510 (Tonbo Biosciences, 13-0870) for 10 min at 4 °C and blocked by anti-CD16/32 (Tonbo Bioscience, 70-0161) (1 to 100 dilution) at 4 °C for 10 min. Cells were further stained with anti-CD45 (Invitrogen, 11-0451-82), anti-CD3 (Tonbo Biosciences, 65-0031-U100), anti-CD8 (BD Pharmingen, 557654), anti-CD44 (BioLegend, 103041), anti-CD62L (BioLegend, 104424), anti-PD1 (BD Biosciences, 563059), anti-TIM3 (BioLegend, 119704), and anti-LAG3 (Invitrogen, 25223182) in staining buffer (2% FBS in PBS). For nuclear transcription factor staining, cells were permeabilized using a FoxP3/transcription factor staining kit (eBioscience, 00-5523-00) and then stained with anti-Ki67 (Invitrogen, 48-5698-82). For cytokine staining, cells were stimulated by anti-CD3/CD28 (Thermo Fisher, 11452D) at 37 °C overnight and then treated with BD GolgiPlug (BD Biosciences, 550583) at 37 °C for 5 h. After surface staining, cells were permeabilized using a BD Cytofix/Cytoperm kit (BD Biosciences, 554714) and stained with anti-IFNγ (BioLegend, 505826) and anti-TNFα (BioLegend, 506314). All antibodies were used at 1:150 dilution. Cells were then fixed with 1% paraformaldehyde and analyzed by BD FACSCelesta. Data were analyzed using BD FACSDiva and FlowJo software.

Flow cytometry and tetramer were performed as previously described^{55 (link)}. Briefly, single cell suspended splenocytes were incubated with gp33-tetramer and np396-tetramer for 15 minutes and gp-66 tetramer for 30 minutes at 37 °C. After the incubation, surface antibodies were added, including anti-CD8 or anti-CD4 along with anti-IL7R, anti-PD-1, anti-CD62L, anti-CD44, anti-KLRG-1, anti-TIM3, anti-Lag-3, and anti-2B4 antibodies (eBioscience) for 30 minutes at 4 °C. For intracellular cytokine stain single suspended splenocytes were incubated with the LCMV specific peptides gp33 and np396 or the MHC-II epitope gp61. After 1 h, Brefeldin A (eBioscience) was added, followed by an additional 5 h incubation at 37 °C. After surface stain with anti-CD8 or anti-CD4 antibodies (eBioscience) cells were fixed with 2% formalin and permeabilized with 0.1% Saponin and stained with anti-IFNγ and anti-TNF antibodies (eBioscience) for 30 min at 4 °C.