Il 12

T-cell differentiation experiments were performed as described previously.^E8 Briefly, FoxP3- CD4+ T cells were purified and activated using anti-CD3/CD28 beads (Dynabeads, Invitrogen). For T_H1 differentiation, 1 × 10⁵ T cells were stimulated in the presence of IL-2 (20 U/mL; eBioscience), IL-12 (20 ng/mL; Tonbo), and anti–IL-4 (10 μg/mL; Tonbo). For Treg-cell differentiation, cells were stimulated in the presence of IL-2 (100 U/mL; eBioscience), TGF-β (5 ng/mL; Tonbo), and anti–IFN-γ (10 μg/mL; Tonbo). After 4 days, T cells were stimulated with ionomycin (500 ng/mL) and phorbol 12-myristate 13-acetate (50 ng/mL) in the presence of monensin (eBioscience) for 4 hours before intracellular staining. FoxP3 staining was performed using FoxP3/transcription factor staining kit (Tonbo).

Single cell suspensions of splenocytes were prepared by cutting the spleen into small pieces and forcing them through 70 μm filters. CD4⁺-TLs were enriched by mouse CD4 MicroBeads (Miltenyi) as per manufacturer’s protocol. The anti-CD3-coated 96-well plate was prepared by an overnight incubation with 100 μl anti-CD3ε antibodies (5 μg·ml⁻¹ in PBS; eBioscience) at 4°C. Next, 2× 10⁵ naïve CD4⁺-TLs cells were cultured in 100 μl/well of CD4⁺-TL activation culture medium (RPMI-1640 supplemented with 5% heat inactivated FBS, 100 IU·ml⁻¹ penicillin/streptomycin, and 25 ng·ml⁻¹ amphotericin B, 55 μmol·L⁻¹ β-Mercaptoethanol (Sigma), 10 ng·ml⁻¹ IL-2 (R&D Systems)). For Th1-skewed stimulation, 10 μg·ml⁻¹ anti-IL4 antibody (clone 11B11) and 20 ng·ml⁻¹ IL-12 (both from Tonbo)) were added to the medium. The cells were incubated at 37°C with 5% CO₂ for 96 hours.
If cell tracking was required, the CD4⁺-TLs were labeled with CFSE (Thermo Fisher) using a mouse lymphocyte optimized method^{24 (link)}. Mice received intravenous injection of 10⁶ CD4⁺-TLs unless otherwise noted.