Targeting Periodontitis with Treg-Derived Extracellular Vesicles: Modulation of Macrophages and CD8⁺ T-Cell Responses

Periodontitis is a chronic inflammatory disease characterized by alveolar bone loss driven by dysregulated immune responses. We previously showed that extracellular vesicles derived from retinoic acid-induced regulatory T lymphocytes (RA-Treg EVs) suppress pathogenic CD4⁺ T-lymphocyte responses and reduce alveolar bone loss during periodontitis. Herein, we investigated whether RA-Treg EVs also modulate macrophage and CD8⁺ T-lymphocyte responses during experimental periodontitis. Ligature-induced periodontitis was generated in mice, followed by local administration of RA-Treg EVs. Alveolar bone loss was analyzed by micro-computed tomography, and periodontal tissues and cervical lymph nodes were analyzed by flow cytometry to quantify antigen-presenting cells, macrophages, macrophage subsets, and CD8⁺ T lymphocytes. The direct effects of RA-Treg EVs on macrophage phenotype and CD8⁺ T-cell proliferation and activation were assessed in vitro. RA-Treg EV treatment attenuated alveolar bone loss and preserved trabecular microarchitecture. This effect was associated with reduced macrophage infiltration into periodontal tissues, modulation of macrophage polarization, and restoration of CD8⁺ T-cell abundance in periodontal tissues and draining cervical lymph nodes, without major changes in CD8⁺IFN-γ⁺ or CD8⁺RANKL⁺ cells. In vitro, RA-Treg EVs induced heterogeneous macrophage phenotypes distinct from the classical M1/M2 polarization states while markedly enhancing CD8⁺ T-cell proliferation and activation. These findings indicate that RA-Treg EVs preserve alveolar bone during experimental periodontitis while selectively modulating macrophage and CD8⁺ T-lymphocyte responses.