Figure 5.

Potent TGF-β MSC Immunosuppressive Ability Is Dependent on Cell-to-Cell Contact-Mediated Smad2/3 Signaling Requiring Successful PGE2/EP4 Binding

(A) Untreated or TGF-β MSCs with or without Smad2/3 inhibitor were co-cultured with T lymphocytes at a ratio of 1:10 (MSCs/lymphocytes) for 96 h in the presence of anti-CD3/CD28 polyclonal-stimulating beads. CTV was used to determine lymphocyte proliferation. Bar charts show CD4⁺ and CD8⁺ T lymphocyte proliferation for more than three generations and frequency (%) of CD4⁺Foxp3⁺ cells in co-culture wells. (B) Untreated or TGF-β MSCs were seeded in the top compartment of a 0.8-μm transwell, and polyclonally stimulated lymphocytes were placed in the bottom compartment in a 24-well flat-bottom plate and co-cultured for 96 h. Bar charts show CD4⁺ and CD8⁺ T lymphocyte proliferation for more than three generations and frequency (%) of CD4⁺Foxp3⁺ cells in co-culture wells. (C) Untreated or TGF-β MSCs with or without AMP-CP were co-cultured with polyclonally stimulated T lymphocytes for 96 h. Bar charts show CD4⁺ and CD8⁺ T lymphocyte proliferation for more than three generations and frequency (%) of CD4⁺Foxp3⁺ cells in co-culture wells. (D) TGF-β MSCs with or without EP4 receptor antagonist were co-cultured with polyclonally stimulated T lymphocytes for 96 h. Bar charts show CD4⁺ and CD8⁺ T lymphocyte proliferation for more than three generations and frequency (%) of CD4⁺Foxp3⁺ cells in co-culture wells. Error bars show mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, by one-way ANOVA, Tukey’s post hoc test (n = 3).