In vitro T-cell Killing Assay

In vitro cell-killing experiments were conducted using C57BL/6J spleen cells. CD3+ T cells were negatively selected by MojoSort Mouse CD3 T cell Isolation Kit (BioLegend, San Diego, CA, USA). CD3+ cells were incubated with various concentrations of S-CD3e-IT (1 pM to 10 nM) for 30 min in a complete RPMI medium (Hyclone, Logan, UT, USA) supplemented with 10% Fetal Bovine Serum (Hyclone, Logan, USA), 0.1% 2-mercaptoethanol (Gibco, Waltham, MA, USA), 1% L-Glutamine, and 1% antibiotic cocktail in 37 °C and 5% CO₂ incubator. After washing the cells twice, the cells were plated in flat-bottom 96-well plates (Falcon, Tewksbury, MA, USA) with plate-bound 5 μg/mL anti-CD3 (145-2C11, BioLegend) and soluble 2 μg/mL anti-CD28 (37.51, BioLegend, San Diego, CA, USA) in a complete RPMI medium supplemented as above. After 72 h, T subset viability was analyzed by Cytek Aurora flow cytometry (Cytek Biosciences, Fremont, CA, USA) using previously published protocol with modifications [59 (link),60 (link)]. The cell viability at different concentrations of S-CD3e-IT was normalized with that of the no-treatment (PBS) control.