CD73-bearing Treg-derived extracellular vesicles modulate in vitro and in vivo immune response and prevent alveolar bone loss during periodontitis

Objectives: The CD73 ectoenzyme promotes immune regulation through the conversion of adenosine-mono phosphate (AMP) into adenosine, which after interaction with its receptors (A2aR and A2bR expressed on immune cells) upholds anti-inflammatory responses. T regulatory cells (Treg)-derived extracellular vesicles (TEVs) have been shown to be loaded with CD73, contributing to their immunomodulatory capacity in a cell-free manner. Periodontitis, a chronic inflammatory disease, develops a deregulated host immune response, where the formation of a inflammatory infiltrate and accumulation of bone pro-resorptive factors result in tooth-supporting tissue breakdown and alveolar bone loss. This study aimed to evaluate the effect of CD73-bearing TEVs on T-cell proliferation, activation, and phenotype in vitro and on periodontitis-induced immune response and alveolar bone resorption.
Methods: Magnetically isolated murine CD4⁺ T cells were differentiated into Tregs and characterized by flow cytometry (FC). From their recovered supernatant, TEVs were isolated by differential centrifugation then, quantified and characterized by Nanoparticle-Tracking-Analysis and Western Blot. The TEV immunosuppressive and modulatory capacities were evaluated in vitro on CD4⁺ and CD8⁺ T cells studying their effect on proliferation, activation, and phenotype. Besides, using a ligature-induced periodontitis mice model, the TEVs effect on periodontal immune response was assessed quantifying leukocyte infiltration by FC and the extent of alveolar bone loss by morphometric analysis.
Results: Tregs showed high expression of lineage markers Foxp3, CD25, and CD73. Isolated TEVs expressed CD73, displayed canonical EVs characteristics, downregulated the CD4⁺ and upregulated the CD8⁺ T cell proliferation and activation. During periodontitis, TEVs increased the frequency of CD73⁺ leukocytes, promoted anti-inflammatory response, and prevented alveolar bone loss.
Conclusions: CD73-bearing TEVs modulate CD4⁺ and CD8⁺ T cell response, impacts on local immune response and prevent peridontitis-induced alveolar bone loss.