Size-dependent immunomodulation by MSC secretome: soluble factors target innate pathways, components larger than 100 kDa regulate T cell proliferation.

BACKGROUND: Mesenchymal stromal cells exert their immunoregulatory effects through a complex secretome constituted of soluble factors and extracellular vesicles (EVs). While the immunomodulatory activity of the secretome has been demonstrated, the contribution of each fraction remains poorly defined. In particular, there is little knowledge about which bioactive molecules are responsible for the effect. METHODS: Human peripheral blood mononuclear cells (PBMCs) were treated with resiquimod in presence of clarified or concentrated secretome by tangential flow filtration (TFF), or fractions derived from ultracentrifugation. Supernatant was collected and used to treat THP-1 dual cells, a reporter cell line, to evaluate NF-κB and IRF pathway immunomodulation. T cell proliferation was measured via dye dilution and flow cytometry. Human PBMCs were treated with PHA/IL-2 in presence of clarified or concentrated secretome. RESULTS: Clarified secretome and the soluble factors fraction from ultracentrifugation inhibited NF-κB and IRF activation in a dose dependent manner. This effect is reduced in presence of concentrated secretome and lost when treated with pelleted EVs. Soluble factors below 5 kDa were responsible for this effect, which was partially mediated by Prostaglandin E2. However, T-cell proliferation was inhibited in the same dose-dependent manner by all TFF-concentrated secretome, regardless of cutoff size, while clarified samples had little effect. CONCLUSIONS: These findings indicate a complex mechanism of action where soluble factors and components larger than 100 kDa modulate immunity but through different pathways. This mechanistic distinction highlights the importance of considering secretome composition when designing cell-free MSC-based therapies.