Nonpolarizing CD4+ T Cell Activation

For nonpolarizing CD4⁺ T cell (Th₀) activation, a protocol previously described elsewhere was used (18 (link)). Briefly, aseptically purified naïve CD4⁺ T cells were resuspended in complete media supplemented with 100 U/ml IL-2 (Peprotech; Rocky Hill, NJ, USA) and 10 µg/ml anti-TGF-β (Bioxcell; West Lebanon, NH, USA) and plated on sterile 24 well tissue culture plates (Corning/Life Sciences; Tewksbury, MA, USA) previously coated with 4 µg/ml anti-mouse CD3ε (Clone 145–2C11, Biolegend; San Diego, CA, USA) and 4 µg/ml anti-mouse CD28 (Clone 37.12, Biolegend). Cells were incubated for up to 96 h in a 5% CO₂ humidified tissue culture incubator at 37°C.