T-cell Costimulation Assay for Evaluating Immune Responses

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.