Cells resident to precision templated 40-µm pore scaffolds generate small extracellular vesicles that affect CD4+ T cell phenotypes through regulatory TLR4 signaling.

Precision porous templated scaffolds (PTS) are a hydrogel construct of uniformly sized interconnected spherical pores that induce a pro-healing response (reducing the foreign body reaction, FBR) exclusively when the pores are 30-40µm in diameter. Our previous work demonstrated the necessity of Tregs in the maintenance of PTS pore size specific differences in CD4+ T cell phenotype. Work here characterizes the role of Tregs in the responses to implanted 40µm and 100µm PTS using WT and FoxP3+ cell (Treg) depleted mice. Proteomic analyses indicate that integrin signaling, monocytes/macrophages, cytoskeletal remodeling, inflammatory cues, and vesicule endocytosis may participate in Treg activation and the CD4+ T cell equilibrium modulated by PTS resident cell-derived small extracellular vesicles (sEVs). The role of MyD88-dependent and MyD88-independent TLR4 activation in PTS cell-derived sEV-to-T cell signaling is quantified by treating WT, TLR4ko, and MyD88ko splenic T cells with PTS cell-derived sEVs. STAT3 and mTOR are identified as mechanisms for further study for pore-size dependent PTS cell-derived sEV-to-T cell signaling. STATEMENT OF SIGNIFICANCE: Unique cell populations colonizing only within 40µm pore size PTS generate sEVs that resolve inflammation by modifying CD4+ T cell phenotypes through TLR4 signaling.