Lymphocyte serum free medium

Alendronate sodium trihydrate, CaCl₂, ethanol, and NaOH were purchased from Sigma‐Aldrich. All chemical reagents were analytically pure and used without further purification. Fetal bovine serum (FBS), phosphate‐buffered saline, and lymphocyte serum‐free medium were purchased from Corning. McCoy's 5A medium, penicillin‐streptomycin (100×), and 0.25% Trypsin‐EDTA were purchased from Cytiva. Calcein‐AM and propidium iodide (PI) were purchased from Dojindo. Human TNF‐α and IFN‐γ ELISA kits were purchased from Abcam. Firefly luciferase substrate XenoLight D‐luciferin potassium salt was purchased from PerkinElmer. Female BALB/c mice (5 weeks), nude mice (5 weeks), and NOD/SCID mice (5 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All mice were maintained under specific pathogen‐free conditions. Animal care and experiments were carried out under institutional and National Institutes of Health protocol and guidelines. Animal work described in this manuscript has been approved and conducted under the oversight of the Animal Care and Use Committee of Sun Yat‐sen University (SYSU‐IACUC‐2021‐B0815).

Peripheral blood mononuclear cells (PBMCs) were prepared from the heparinized venous blood of healthy adult volunteers by Ficoll-Hypaque (Amersham Biosciences) density gradient centrifugation. Human CD4⁺CD45RA⁺ naïve T cells were sorted from PBMCs by using the CD4⁺ T cell isolation kit (Miltenyi) and CD45RA microbeads (Miltenyi) through auto-MACS (Miltenyi Biotec, Germany) in a two-step procedure of magnetic beads sorting (purity >95%). Naïve T cells were activated and induced to iTregs with anti-CD3/CD28 mAb-coated Dynabeads (1 bead to 5 cells, Gibco) in the presence of recombinant IL-2 (100 IU/ml, R&D) and TGF-β (1 ng/ml, R&D) in lymphocyte serum-free medium (Corning) with 10% fetal bovine serum (Sigma) for 72 h.