Fig. 5

Modulation of PD1/PD-L1 pathway in differentiated DPSCs and BM-MSCs. A After 3 weeks of induction, osteogenic differentiation of DPSCs was demonstrated by deposition of mineralized extracellular matrix as revealed by Alizarin Red stain and confirmed by immunofluorescence analyses of OCN and RUNX2. Undifferentiated DPSCs were used as negative control. Nuclei were counterstained with DAPI (yellow square). DIC image of undifferentiated DPSCs was reported on the right. B Western blot analysis of OPN in undifferentiated DPSCs and in differentiated DPSCs. Histograms show a statistically significant increased expression of OPN in differentiated DPSCs. **P < 0.01 versus DPSCs undiff. C Immunofluorescence and Western blot analyses demonstrating the modulation of PD-L1 expression in DPSCs. Representative images showed undifferentiated DPSCs labeled strongly positive only after co-culture with aPBMCs, whereas, after reaching osteogenic commitment no PD-L1 expression was detected. Scale bar: 10 µm. Western blot analysis of PD1 and PD-L1 showed that PD-L1 expression is statistically significant decreased in differentiated DPSCs after co-culture with aPBMCs in comparison to undifferentiated DPSCs co-cultured with aPBMCs; ***P < 0.001 vs. DPSCs undiff after aPBMCs co-culture. On the right side Western blot analysis showing caspase 3 expression in aPBMCs after co-culture with undifferentiated DPSCs (uDPSCs) and differentiated DPSCs (dDPSCs); *P < 0.05 versus aPBMCs after dDPSCs co-culture. D Down-regulation of PD-L1 in osteogenic differentiated BM-MSCs after aPBMCs co-culture was revealed by Immunofluorescence and Western blot analyses. Histograms show that PD-L1 expression was statistically significant reduced in BM-MSCs; ***P < 0.001 vs. BM-MSCs undiff after aPBMCs co-culture. On the right side Western blot analysis showing caspase 3 expression in aPBMCs after co-culture with undifferentiated BM-MSCs (uBM-MSCs) and differentiated BM-MSCs (dBM-MSCs); *P < 0.05 versus aPBMCs after dBM-MSCs co-culture. For each statistical analysis, data were expressed as mean ± SD