Fibroblast-Treg Interaction Assay

Murine dermal fibroblasts were prepared from WT and Fli1^+/− mice and maintained as described previously [13 (link)]. Splenic Tregs were isolated from WT mice with a CD4⁺CD25⁺ Treg cell isolation kit (130-091-041; Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured in RPMI 1640 medium supplemented with FCS. Murine dermal fibroblasts (1 × 10⁵ cells) and CD4⁺CD25⁺ T cells (3 × 10⁵ cells) were cocultured in 24-well plates for 2 days. Then, cells were analyzed on a FACSVerse flow cytometer. In some experiments, cocultured cells were treated with anti-mouse IL-33 antibody (M187-3; MBL, Nagoya, Japan) or antimouse IL-6 antibody (MAB406-SP; R&D Systems).