CD4⁺ naive cells from WT mice were isolated and cultured in Treg differentiation media with or without IL-1β (n = 6 per group). (A–C) Foxp3^eGFP expression by flow cytometry after 5 days of Treg differentiation with or without IL-1β (D and E). iTregs were sorted based on Foxp3^eGFP expression and cocultured with bone marrow cells in the presence of M-CSF (20 ng/mL) and RANKL (20 ng/mL) (n = 3 per group). (D and E) High-dimensional analyses of sorted Foxp3^eGFP+ iTregs differentiated with or without IL-1β by mass cytometry. (D) viSNE plots of iTregs differentiated with or without IL-1β. (E) Difference in protein expression between iTregs differentiated with or without IL-1β. (F–H) Sorted Foxp3^eGFP+ iTreg cells differentiated with or without IL-1β were cocultured with WT macrophage precursor cells. After 7 days of coculture, cells were stained for tartrate-resistant acid phosphatase (TRAP) and TRAP⁺ osteoclasts were measured (surface area) and counted. Scale bars: 1 mm. (I and J) RANKL-deficient Tregs were generated by crossing FoxP3-YFP-Cre mice with Rankl^fl/fl mice. Littermate mice that do not express FoxP3-YFP-Cre were utilized as WT controls. (I) Histogram depicting RANKL expression on WT and Rankl^–/– Tregs. (J) Osteoclastogenic activity of WT and Rankl^–/– Tregs differentiated in the presence or absence of IL-1β. Data are expressed as mean ± SEM. Statistical significance was determined using Mann-Whitney U test (B and C), unpaired t test (F), or 1-way ANOVA (G–I). *P < 0.01, **P < 0.001, ***P < 0.0001.