Clone 8d4 8

Naïve CD4 T cells were cultured in precoated plates with anti‐CD3 (5 μg/mL, Clone OKT‐3, BioLegend, USA) + anti‐CD28 (1 μg/mL, Clone CD28.2，BioLegend, USA) antibodies and maintained in media supplemented with IL‐2 (10 ng/mL, PeproTech, USA) (Group Th0). Recombinant IL‐6 (50 ng/mL, PeproTech, USA), recombinant TGF‐β1 (10 ng/mL, PeproTech, USA), anti‐IFN‐γ mAb (10 μg/mL, Clone B27, BioLegend, USA), and anti‐IL‐4 mAb (10 μg/mL, Clone 8D4‐8, BioLegend, USA) were added to generate Th17 cells (Group Th17). recombinant TGF‐β1 (50 ng/mL, PeproTech, USA), anti‐IL‐6 mAb (10 μg/mL, Clone MQ2‐13A5, BioLegend, USA), anti‐IFN‐γ mAb (10 μg/mL, Clone B27, BioLegend, USA), and anti‐IL‐4 mAb (10 μg/mL, Clone 8D4‐8, BioLegend, USA) were added to generate Treg cells (Group Treg). Media were changed every 48 hours. This protocol was based on previous publications.17, 18 For intracellular cytokine analysis, brefeldin A (10 mg/mL, BioLegend, USA), phorbol 12‐myrstate 13‐acetate (PMA)(50 ng/mL, BioLegend, USA), and ionomycin (1 μg/mL, BioLegend, USA) were added 4 hours before the end of the culture. The cells were then harvested, stained, and analyzed through flow cytometry. After 5 days of culture, 8.3 ± 1.7% of CD4⁺IL‐17A⁺ cells and 13.2 ± 1.7% of CD4⁺Foxp3⁺ cells were obtained.