Anti lag 3 antibody

A T-cell costimulation assay was conducted as previously described^{15 (link)}. Briefly, splenocytes isolated from C57BL/6 J mice were treated with 2.5 μg/mL anti-CD3ε mAb (Biolegend, San Diego, USA), 1.25 μg/mL anti-CD28 mAb (Biolegend) and 5 ng/mL recombinant mouse IL-2 (PeproTech) for 12 h. Thereafter, the pretreated splenocytes were seeded into 96-well plates (NEST Biotechnology, Wuxi, China) at 5 × 10⁴/well and incubated with recombinant murine FGL1 (10 μg/mL), anti-LAG-3 antibody (BioXcell, Lebanon, USA) or control for 72 h. The supernatant was collected, and the IFN-γ levels were determined using a Mouse IFN-γ Valukine ELISA Kit (R&D, Minneapolis, USA) according to the manufacturer’s instructions.

YSK12-C4 was synthesized as previously described [14 (link)]. The commercially available reagents used are as follows: Cholesterol (Sigma-Aldrich, St. Louis, MO, USA); 1,2-Dimirystoyl-sn-glycerol methoxyethyleneglycol 2000 ether (DMG-PEG2k) (NOF Corporation, Tokyo, Japan); Cyclic di GMP (c-di-GMP) (Cayman chemical, Ann Arbor, MI, USA); anti-programmed cell death ligand 1 (anti-PD-L1) antibody (clone: 10F.9G2), anti-CTLA-4 antibody (clone: 9H10), and anti-lymphocyte activation gene 3 (anti-LAG-3) antibody (clone: C9B7W) (Bio X Cell, West Lebanon, NH, USA).
Renca cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and were cultured with RPMI1640 medium (high glucose) containing 10% fetal bovine serum (FBS), 100 units/mL of penicillin/streptomycin (P/S), and 1 mM of sodium pyruvate.
Female BALB/c mice (6–8 weeks old) (Japan SLC Inc., Shizuoka, Japan) were housed under specific pathogen-free (SPF) conditions. The animal experiments described herein were approved by the Animal Committee of Hokkaido University (approval number: 20-0176). All methods were conducted based on the guidelines set by Hokkaido University and the guidelines established for Animal Research: Reporting of In Vivo Experiments (ARRIVE).