The immunomodulatory capacity of aggregated MSCs is enhanced through synergy with glucocorticoid steroids

Background \u0026 Aim Upon local delivery, MSCs aggregate into spheroids leading to alterations in phenotype. While MSCs immunomodulatory potential in 2D cultures has been extensively studied, the effect of aggregation on this potential is less understood. Using a series of in vitro potency assays, we sought to evaluate changes to human MSCs ability to suppress activated human T cells and explore the interactions of these cells with locally delivered steroids. Methods, Results \u0026 Conclusion We used a T-cell suppression assay to compare the ability of MSCs, either in 2D or aggregated into spheroids, to suppress T-cell proliferation. 6 human MSC donors, 3 from bone marrow and 3 from umbilical cord, were co-cultured with PBMCs from 3 independent donors creating 18 distinct MSC:PBMC donor pairings. After 6 days of co-culture, PBMCs were analyzed by flow cytometry and the percent PBMC proliferation was calculated for each donor pairing. For adherent MSCs, all 18 pairings resulted in strong suppression of PBMC proliferation, while none of the spheroid MSCs showed any suppression (A). The addition of budesonide, a glucocorticoid steroid, to the co-culture resulted in a significant improvement in spheroid MSC suppression (A). This observation led us to examine the interaction between budesonide and spheroid MSCs mechanistically. While neither budesonide nor aggregated MSCs alone were capable of affecting PBMC proliferation, when combined they displayed an apparent synergy and suppressed a significant degree of PBMC proliferation. Profiling activity of COX-2 and IDO revealed distinct changes in PGE2 (B) and kynurenine (C) production by MSC spheroids with PGE2 over a 100-fold higher in MSC spheroid cultures compared to adherent MSC controls. To determine if the interaction between budesonide and MSC spheroids was mediated by elevated PGE2 levels, a series of inhibition and add-back experiments were performed using PBMC co-culture assays as a read-out. Blockade of EP2/EP4 receptors with small molecule inhibitors completely blocked the synergy between spheroid MSCs and budesonide (D) while replacing spheroid MSCs with synthetic PGE2 replicated the observed synergy (E). Conclusion We report a novel mechanism by which PGE2 from spheroid MSCs works in synergy with the glucocorticoid steroid, budesonide, to inhibit T cells. These findings shed light both on a distinct immunomodulatory phenotype of aggregated MSCs as well as their unique interactions with immunosuppressive drugs they may encounter in clinical settings.