Fig. 3

MSCs stimulated with TLR3 and TLR4 ligands differentially modulate Th1 and Th17 differentiation and proliferation. T-helper cell differentiation (a, b, e, f) and proliferation (c, d, g, h) were assessed using naïve CD4⁺ T cells. Purified CD4⁺ cells were stimulated with a specific cocktail of cytokines, as described in Methods, to induce Th1 (a–d) and Th17 (e–h) differentiation in the absence or presence of MSCs pretreated with or without a TLR agonist. 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 CD4⁺ T cells in complete RPMI medium. MSCs were added at day 0 of the differentiation process in a 1:10 ratio (MSCs:T cells). Flow cytometry analysis, gating on CD4⁺ cells, and intracellular staining, using antibodies (mAb) for IFN-γ and IL17 to identify Th1 and Th17 lymphocytes, respectively, were performed. Representative density plots of six different experiments for Th1 and Th17 differentiation are shown. For proliferation analysis, CD4⁺ cells were previously labeled with CellTrace Violet (CTV) and analyzed (presented as histograms). Further analysis of the events of each cycle, described by the proliferation index (d, h). Th1 differentiation (b) and proliferation (d) with the MSCs pretreated with poly(I:C) and LPS. Th17 differentiation (f) and proliferation (h) with the MSCs pretreated with poly(I:C) and LPS. Bars represent the mean ± SEM, significant differences calculated using the Mann–Whitney test. *p < 0.05, **p < 0.001. MSCsPoly MSCs pretreated with poly(I:C) for 1 hour, MSCsLPS MSCs pretreated with LPS for 1 hour