Pb009 3 c

Tregs (PB009-4F-2, Allcells, Shanghai, China) at a density of 1 × 10⁶ cells/mL were seeded in InstantOne ELISA kit (85-86112-11, Invitrogen, Carlsbad, CA, USA) microplates at 20 μL/well. The plates were then incubated for 2 h at 37 °C. IL-2WT, IL-2K35C, and IL-2K35C-moFA were added to each well at the concentration of 0.1 μg/mL and 0.01 μg/mL. Ten microliters of cell lysis mix (5× was added to each of the InstantOne ELISA assay wells after shaking (~300 rpm) for 10 min. The absorbance of the plates was measured at 450 nm using a Spectramax M5.
CD8⁺ T cells (PB009-3-C, Allcells, Shanghai, China) at a density of 1 × 10⁶ cells/mL were seeded into an InstantOne phospho-STAT5 ELISA kit microplate at 20 μL/well and incubated for 2 h at 37 °C. IL-2WT, IL-2K35C, and IL-2K35C-moFA were added to each well at a concentration of 2 μg/mL. For other steps, please refer to the Tregs method.

The microporous 2-compartment co-culture system was applied for co-culture. Transfected NSCLC cells were inoculated in the upper chamber, while CD8⁺ T (PB009-3-C, ALLCELLS, Shanghai) cells were inoculated in the lower chamber, allowing direct contact between NSCLC cells and immune cells. CD8⁺ T cells were sorted using EasySep™ direct human CD8⁺ T cell isolation kit (STEMCELL, Vancouver, BC, Canada). Annexin V-FITC apoptosis kit (Becton Dickinson, Franklin Lakes, NJ, USA) was used for flow cytometry to determine the percentage of CD8⁺ T cells or apoptotic CD8⁺ T cells according to the manufacturer's instructions.