Anti mouse il 4

The plates were coated with anti-mouse CD3 (5 μg/mL) (BioLegend) overnight at 4 °C, and the concentration of the purified naïve CD4⁺T cells was adjusted to 2 × 10⁶/mL. Anti-mouse CD28 (5 μg/mL) (BioLegend), IL-6 (100 ng/mL) (Peprotech), TGF-β1 (1 ng/mL) (Peprotech), IL-23 (5 ng/mL) (Novoprotein), anti-mouse IL-4 (10 μg/mL) (BioLegend), and anti-mouse IFN-γ (10 μg/mL) (BioLegend) were added, mixed with QU (0, 1, 3, 10 μM) (sigma) and incubated for 3 days.

All CD4⁺ T cells or naïve (CD4⁺CD44^lowCD62L^highCD25⁻) T cells were isolated from the spleen and LN of 8- to 12-week-old mice, as described in the previous section. For CD4⁺ CD25⁻ T_conv cell isolation, CD4⁺ CD25⁺ T_reg cells were removed from the population of all CD4⁺ cells by using a CD25-positive selection kit (catalog no. 130-091-072, Miltenyi Biotec; or catalog no. 18783, STEMCELL Technologies). For polarization, isolated cells were stimulated on plate-bound anti-CD3 and anti-CD28 in flat-bottom 96-well plate in absence (T_H0) or in the presence of specific T_H subtype–polarizing cytokines for 3 to 4 days. For T_H1 cells, rmIL-12 (25 ng/ml; BioLegend, San Diego, CA) and anti-mouse IL4 (10 μg/ml; BioLegend). For T_H17 cells, rmTGFβ1 (2.5 ng/ml; Tonbo Biosciences, San Diego, CA), rmIL-6 (50 ng/ml; Tonbo Biosciences), rmIL-23 (25 ng/ml; BioLegend), rmIL-1β (25 ng/ml; BioLegend), and anti-mouse IFNγ (10 μg/ml). For T_reg cells, rmTGFβ1 (10 ng/ml) and rmIL-2 (100 U/ml; BioLegend). To activate TCR signaling in the T lymphocyte subpopulation of cells isolated from the spleen and LN, soluble anti-CD3 (100 ng/ml; 145-2C11) antibodies were added to the whole cell suspension in round-bottom 96-well plates under specific T_H subtype-polarizing conditions as stated above for T_H1, T_H17 and Treg cells.