T-Cell Culture

The spleens were retrieved from C57BL/6 WT mice. CD3⁺ T/CD8⁺ T cells were sorted using CD3 magnetic beads (BioLegend, 480024)/Dynabeads Untouched Mouse CD8 Cells Kit (Invitrogen, 11417D), and flow sorting was performed to ensure purity >95%. T cells were cultured for 72 h with recombinant IL-35 (rIL-35, 50–200 ng/mL; Adipogen; CHI-MF-11135-C025) or anti-EBI3 antibody/IL-35 (clone V1.4H6.25), or PBS, with a plate-bound anti-CD3 antibody (1 g/mL, eBioscience) and anti-CD28 (2 g/mL, eBioscience), and flow cytometry was used for assessment.

Spleens were retrieved from Foxp3-IRES-mRFP and Foxp3-IRES-mRFPx^Ebi3–/– mice. CD4⁺Foxp3⁺ T cells were sorted using CD4 magnetic beads (Invitrogen, 11415D) and flow sorting was performed to ensure purity >95%. The sorted cells were cultured for 18 to 48 h with recombinant IL-35, anti-EBI3 antibody or PBS, and with plate-bound anti-CD3 (5 g/mL) and anti-CD28 (2 g/mL). To block gp130 or STAT1 activity, the cells were cultured with SC144 (MedChemExpress, 895158-95-9) or fludarabine (NSC 118218, Selleck, S1491) for 24 h, and flow cytometry was used to assess the cells.

Mouse splenic CD4⁺Foxp3⁺ and CD8⁺T cells were sorted, as previously described. Treg were cocultured with CD8⁺ T cells (1:4, 1:8, 1:16, and 1:32) for 3 d and analyzed using flow cytometry.