Fig. 2

Immunomodulatory capacity of MSCs stimulated with TLR3 and TLR4 ligands in vitro. (a) To study the immunosuppressive capacity of untreated MSCs and MSCs pretreated for 1 hour with poly(I:C) or LPS on T-cell response, we performed an in-vitro T-cell stimulation assay at different ratios of MSCs:splenocytes, as described in Methods. MSCs were either unstimulated or were stimulated with poly(I:C) (10 μg/ml) or LPS (500 ng/ml) for 1 hour before being cocultured with T cells in complete RPMI medium. Previously, splenocytes were labeled with CTV and stimulated with Con A and finally cultured with MSCs at 1:5, 1:10, or 1:20 ratios for 3 days. T-cell proliferation was evaluated by flow cytometry, gating on CD3⁺ cells. (b) Secretion of nitric oxide (NO) by MSCs in coculture with splenocytes at a 1:10 ratio was measured using a modified Griess reagent. (c) IL-6 mRNA expression evaluated by RT-qPCR. (d) IL-6 secretion, measured by ELISA. Data expressed as the mean ± SED. A Mann–Whitney test was performed, *p < 0.05, **p < 0.01,***p < 0.001. MSCsPoly MSCs pretreated with poly(I:C) for 1 hour, MSCsLPS MSCs pretreated with LPS for 1 hour