Regulatory T Cells Isolation and Suppression Assay

Tregs were isolated from PBMCs and gingival tissues using a CD4⁺CD25⁺ Regulatory T Cells Isolation Kit (Miltenyi Biotec, Bergisch Gladbac, Germany), as described.^{(16 (link))} Prior to the isolation procedures, gingival tissues were submitted to enzymatic digestion protocol as described in the Isolation of inflammatory cells from periodontal tissues and flow cytometric analysis section. Briefly, CD4⁺ T cells were pre-enriched by depletion of non-CD4⁺ T cells using a cocktail of biotin-labeled antibodies, anti-biotin magnetic beads, and an magnetic bead column (Miltenyi Biotec, Bergisch Gladbac, Germany); to isolate CD4⁺CD25⁺ T cells, enriched CD4+ T cells were incubated with PE-labeled anti-CD25 antibody and anti-PE magnetic beads. Then CD4⁺CD25⁺ T cells were positively selected using a MS magnetic bead column. The purity of CD4⁺CD25⁺ T cells was more than 95%, as confirmed by flow cytometry. To determine the isolated Treg cells’ suppressive activity, 5 × 10⁴ CD4⁺CD25⁻ cells were treated with 2μg/mL anti-CD3 and anti-CD28 (eBioscience) for 12 hours as effector cells, then incubated with or without isolated Tregs at 2:1 ratios of for 72 hours in complete medium containing RPMI 1640 (Sigma, St. Louis, MO, USA) supplemented with 5% FCS, as described.^{(17 (link))} Cell proliferation was assessed by [3H]thymidine incorporation as measured by scintillation counting.^{(18 (link))}