Anti il 2

Splenic naïve CD4⁺ T cells (2.5 × 10⁴) from SFB young or middle-aged K/BxN mice were enriched by fluorescence-activated cell sorting (FACS Aria) and in vitro cultured in 96-well plates for 4 days in Th17 polarization conditions: anti-CD3ε (plate-coated, 2 μg/ml), anti-CD28 (2 μg/ml), anti-IL-2 (10 μg/ml, JES6-1A12, BioXCell), IL-6 (50 ng/ml, PeproTech), transforming growth factor (TGF)β1 (1 ng/ml, PeproTech), 6-formylindolo [3,2-b] carbazole (FICZ), (300 nM, Enzo Life Sciences).

An untreated, flat-bottom, 96-well plate was coated overnight at 4°C with 0.2 μg/mL anti-CD3 (clone 145-2C11, BD Biosciences) and 0.5 μg/mL anti-CD28 (clone 37.51, BD Biosciences) in PBS, washed with PBS, and blocked with coculture media (RPMI from Gibco containing 10% FBS from Atlanta Biologicals, 1% penicillin/streptomycin from Gibco, and 1× β-mercaptoethanol from Gibco) for at least 30 minutes at room temperature (RT). CD8⁺ T cells were isolated from the spleens of naive C57BL/6 mice using the untouched CD8⁺ T cell isolation kit (Miltenyi Biotec), following manufacturer’s instructions. Isolated CD8⁺ T cells were washed twice with PBS and stained with CTV dye (Life Technologies) following manufacturer’s instructions. Dye-labeled CD8⁺ T cells were then cultured on the anti-CD3/anti-CD28–coated plate at a density of 10⁵ cells per well in coculture media, and 2 μg/mL anti–IL-2 (using a mixture of both S4B6-1 and JES6-1A12 clones, Bio X Cell) was added at the beginning of coculture where indicated. The cells were cultured at 37°C and 5% CO₂ for 3 days. Following coculture, cells were stained and analyzed by flow cytometry.