Blockade of PD-L1/PD-1 signaling promotes osteo-/odontogenic differentiation through Ras activation

Abstract

The programmed cell death ligand 1 (PD-L1) and its receptor programmed cell death 1 (PD-1) deliver inhibitory signals to regulate immunological tolerance during immune-mediated diseases. However, the role of PD-1 signaling and its blockade effect on human dental pulp stem cells (hDPSCs) differentiation into the osteo-/odontogenic lineage remain unknown. We show here that PD-L1 expression, but not PD-1, is downregulated during osteo-/odontogenic differentiation of hDPSCs. Importantly, PD-L1/PD-1 signaling has been shown to negatively regulate the osteo-/odontogenic differentiation of hDPSCs. Mechanistically, depletion of either PD-L1 or PD-1 expression increased ERK and AKT phosphorylation levels through the upregulation of Ras enzyme activity, which plays a pivotal role during hDPSCs osteo-/odontogenic differentiation. Treatment with nivolumab (a human anti-PD-1 monoclonal antibody), which targets PD-1 to prevent PD-L1 binding, successfully enhanced osteo-/odontogenic differentiation of hDPSCs through enhanced Ras activity-mediated phosphorylation of ERK and AKT. Our findings underscore that downregulation of PD-L1 expression accompanies during osteo-/odontogenic differentiation, and hDPSCs-intrinsic PD-1 signaling inhibits osteo-/odontogenic differentiation. These findings provide a significant basis that PD-1 blockade could be effective immunotherapeutic strategies in hDPSCs-mediated dental pulp regeneration.

Fig. 1

PD-L1 expression is downregulated during hDPSCs differentiation into the osteo-/odontogenic lineage. a hDPSCs were harvested for the isolation of membrane and cytosolic fractions. Immunoblotting analyses were carried out and representative band intensity was quantified. b and c hDPSCs were cultured with or without ODM for the indicated days. Immunoblotting analyses were carried out and representative band intensity was quantified (b). Real-time PCR data for CD274, PDCD1, RUNX2, and DSPP (c). *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test. d and e hDPSCs were treated with or without A.A (l-ascorbic acid 2-phosphate), β-gly (β-glycerol phosphate), Dex (dexamethasone), or ODM for one day. Immunoblotting analyses were carried out and representative band intensity was quantified (d). Real-time PCR data for CD274 and PDCD1 (e). *P < 0.05; ***P < 0.001, Student’s t test.

Fig. 2

PD-L1 inhibits hDPSCs differentiation into the osteo-/odontogenic lineage. a and b The control siRNA or PD-L1 siRNA-transfected hDPSCs were cultured with or without ODM for 3 days (for PD-L1 and RUNX2) or 6 days (for DSPP). Immunoblotting analyses were carried out and representative band intensity was quantified (a). Real-time PCR data for RUNX2 and DSPP (b). *P < 0.05, two-way ANOVA test. c and d. The control siRNA or PD-L1 siRNA-transfected hDPSCs were cultured with or without ODM for 6 days (for ALP staining and activity assay) or 21 days (for Alizarin red S staining and quantification). ALP staining and activity assay were performed (c). Alizarin red S staining was performed and quantified (d). *P < 0.05; **P < 0.01, two-way ANOVA test. e The control siRNA or PD-L1 siRNA-transfected hDPSCs were cultured with or without ODM for one day. Immunoblotting analyses were carried out and representative band intensity was quantified. f The control siRNA or PD-L1 siRNA-transfected hDPSCs were cultured with ODM or the indicated inhibitors (U0126, 10 μmol·L⁻¹; PD98059, 10 μmol·L⁻¹; or MK-2206, 5 μmol·L⁻¹) for three days (for RUNX2) or six days (for DSPP). Immunoblotting analyses were carried out and representative band intensity was quantified.

Fig. 3

PD-1 inhibits hDPSCs differentiation into the osteo-/odontogenic lineage. a and b The control siRNA or PD-1 siRNA-transfected hDPSCs were cultured with or without ODM for 3 days (for RUNX2) or 6 days (for DSPP). Immunoblotting analyses were carried out and representative band intensity was quantified (a). Real-time PCR data for RUNX2 and DSPP (b). **P < 0.01, two-way ANOVA test. c and d The control siRNA or PD-1 siRNA-transfected hDPSCs were cultured with or without ODM for 6 days (for ALP staining and activity assay) or 21 days (for Alizarin red S staining and quantification). ALP staining and activity assay were performed (c). Alizarin red S staining was performed and quantified (d). *P < 0.05; **P < 0.01, two-way ANOVA test. e The control siRNA or PD-1 siRNA-transfected hDPSCs were cultured with or without ODM for one day. Immunoblotting analyses were carried out and representative band intensity was quantified. f The control siRNA or PD-1 siRNA-transfected hDPSCs were cultured with ODM or the indicated inhibitors (U0126, 10 μmol·L⁻¹; PD98059, 10 μmol·L⁻¹; or MK-2206, 5 μmol·L⁻¹) for 3 days (for RUNX2) or 6 days (for DSPP). Immunoblotting analyses were carried out and representative band intensity was quantified.

Fig. 4

Silencing of PD-L1/PD-1 promotes hDPSCs differentiation into the osteo-/odontogenic lineage via Ras activation. a hDPSCs were cultured with or without ODM for the indicated days. RBD pull-down assay and immunoblotting analyses were carried out, and representative band intensity was quantified. b hDPSCs were cultured with or without ODM for the indicated days. Immunoblotting analyses were carried out and representative band intensity was quantified. c hDPSCs were cultured with or without ODM or Abd-7 (20 μmol·L⁻¹) for one day. Immunoblotting analyses were carried out and representative band intensity was quantified. d and e hDPSCs were cultured with or without ODM or Abd-7 (20 μmol·L⁻¹) for 3 days (for RUNX2) or 6 days (for DSPP). Real-time PCR data for RUNX2 and DSPP (d). Immunoblotting analyses were carried out and representative band intensity was quantified (e). *P < 0.05; ***P < 0.001, two-way ANOVA test. f hDPSCs were cultured with or without ODM or Abd-7 (20 μmol·L⁻¹) for 2 days. Immunoblotting analyses were carried out and representative band intensity was quantified. g and h hDPSCs were cultured with or without ODM or Abd-7 (20 μmol·L⁻¹) for 6 days (for ALP staining and activity assay) or 21 days (for Alizarin red S staining and quantification). ALP staining and activity assay were performed (g). Alizarin red S staining was performed and quantified (h). **P < 0.01, two-way ANOVA test. i The control siRNA or PD-L1 siRNA-transfected hDPSCs were cultured with or without ODM for one day. RBD pull-down assay and immunoblotting analyses were carried out, and representative band intensity was quantified. j The control siRNA or PD-1 siRNA-transfected hDPSCs were cultured with or without ODM for one day. RBD pull-down assay and immunoblotting analyses were carried out, and representative band intensity was quantified. k The control siRNA, PD-L1 siRNA, or PD-1 siRNA-transfected hDPSCs were cultured with or without ODM or Abd-7 (20 μmol·L⁻¹) for 3 days (for RUNX2) or 6 days (for DSPP). Immunoblotting analyses were carried out and representative band intensity was quantified.

Fig. 5

PD-L1/PD-1 signaling blockade nivolumab promotes hDPSCs differentiation into the osteo-/odontogenic lineage. a hDPSCs were cultured with or without ODM for one day in the presence of IgG4 or Nivolumab (10 μg/mL). RBD pull-down assay and immunoblotting analyses were carried out, and representative band intensity was quantified. b hDPSCs were cultured with or without ODM for one day in the presence of IgG4 or Nivolumab (10 μg·mL⁻¹). Immunoblotting analyses were carried out and representative band intensity was quantified. c and d hDPSCs were cultured with or without ODM for 3 days (for RUNX2) or 6 days (for DSPP) in the presence of IgG4 or Nivolumab (1 or 10 μg·mL⁻¹). Immunoblotting analyses were carried out and representative band intensity was quantified (c). Real-time PCR data for RUNX2 and DSPP (d). **P < 0.01, Student’s t test. e and f hDPSCs were cultured with or without ODM for 6 days (for ALP staining and activity assay) or 21 days (for Alizarin red S staining and quantification) in the presence of IgG4 or Nivolumab (10 μg·mL⁻¹). ALP staining and activity assay were performed (e). Alizarin red S staining was performed and quantified (f). ***P < 0.001, Student’s t test. g Schematic diagram of the proposed mechanism.