Figure 2.

TGF-β MSCs Inhibit T Cell Proliferation while Inducing/Expanding Regulatory T Cells in Syngeneic In Vitro Co-culture Assays

Untreated or TGF-β MSCs 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. (A) Flow cytometry gating strategy used to assess CD4⁺ or CD8⁺ T cell proliferation. (B) Bar charts and histograms showing CD4⁺ and CD8⁺ T cell proliferation for more than three generations and representative flow cytometry plot overlays. (C) Flow cytometry gating strategy used to select CD4⁺Foxp3⁺ Tregs. (D) Frequency (%) of CD4⁺Foxp3⁺ Tregs after co-culture with untreated or TGF-β MSCs. (E) Total number of Tregs per 1 × 10⁵ lymphocytes after co-culture with untreated or TGF-β MSCs. (F and G) Median fluorescence intensity (MFI) of CD73 (F) and PD-L1 expression on Tregs (G) following 96 h of co-culture. Error bars show mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, by multiple unpaired, two-tailed Student’s t test and one-way ANOVA, Tukey’s post hoc test (n = 3–7).