Fig. 6

Immunoregulatory function of DPSCs. A Western blot analysis of PD-L1 and PCNA. A statistically significant reduction of PD-L1 expression was shown in DPSCs after aPBMCs co-culture + PD-L1 inib, similarly to DPSCs alone; ^§§P < 0.01 vs. DPSCs after aPBMCs co-culture. B Evaluation of cell proliferation by Ki-67 immunofluorescence analysis. Histograms report the mean percentage of Ki-67⁺ DPSCs. Nuclei were counterstained with DAPI (red square). Scale bar: 10 µm. C Evaluation of FasL expression by Real Time PCR analyses revealed that after aPBMCs co-culture with and without PD-L1 inhibitor, DPSCs statistically significant up-regulated mRNA levels of FasL in a time-dependent manner; *P < 0.05, **P < 0.01 vs. DPSCs alone. At 72 h, a statistically significant up-regulation of FasL was detected in DPSCs after aPBMCs co-culture additioned with PD-L1 inhibitor versus DPSCs after aPBMCs co-culture; ^§P < 0.05. D FasL expression was also evaluated by Western blot analysis in different experimental groups. Histograms reveal a statistically significant increase of FasL in DPSCs after aPBMCs co-culture with and without PD-L1 inib; *P < 0.05, **P < 0.01 vs. DPSCs alone. Data are represented as mean ± SD and one-way ANOVA followed by Newman-Keuls post-hoc test was carried out. E FasL expression (red) is confirmed by immunofluorescence analysis on Nestin⁺ DPSCs. Nuclei were counterstained with DAPI. Scale bar: 10 µm