Overexpression of PD-L1 in mesenchymal stem/stromal cells and their derived extracellular vesicles enhances immunomodulatory properties and therapeutic efficacy in nonobese diabetic mice

Abstract

Background & Aim

Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of pancreatic β cells, necessitating lifelong insulin therapy. Programmed death ligand-1 (PD-L1) is critical in maintaining peripheral tolerance and immunological homeostasis. This study investigates the protective effects of mesenchymal stem/stromal cells (MSCs) overexpressing PD-L1 and their derived extracellular vesicles (PEVs) in murine models of T1D.

Methodology

PD-L1-engineered MSCs (PD-L1-MSCs) were generated by infecting human bone marrow-derived MSCs with a lentiviral vector encoding the human PD-L1 gene. The immunosuppressive properties and impact on islet cell death of PD-L1 MSCs and PEVs were evaluated in vitro by coculturing them with peripheral blood mononuclear cells (PBMCs) and murine islets. For the in vivo study, 8-week-old female NOD mice were given a single IV infusion of MSCs (n=17), PD-L1-MSCs (n=18), MSC-EVs (n=20), PEVs (n=20), or PBS (control, n=20), respectively. Blood glucose levels were monitored weekly for 25 weeks. In separated groups of mice, the pancreas, pancreatic lymph nodes (PLN), and spleen were collected 3 weeks post-treatment to assess immune cell infiltration, T cell profiling and function via H&E staining and flow cytometry. Statistical differences were analyzed using one-way ANOVA with post-hoc correction.

Results

In vitro, PD-L1-MSCs and PEVs effectively suppressed T cell proliferation and increased T regulatory cells (Tregs, p<0.01 vs. control), highlighting their immunomodulatory potential. Islet viability was significantly improved when cocultured with PD-L1-MSCs (viability: 75.3±5.3%) or PEVs (91.3±1.9%), vs. controls (59.9±6.0%, p<0.05 vs control in each group). In vivo, PD-L1-MSCs or PEVs significantly reduced blood glucose and delayed T1D onset in NOD mice (CTR vs. PD-L1-MSC, p<0.05; CTR vs. PEV, p<0.01, Logrank test). H&E staining showed a significant decrease in immune cell infiltration in the pancreatic islets. In PLN, PD-L1 MSCs or PEVs decreased CD8⁺ T cell number (p< 0.03 vs control), and increased CD8⁺ T cell exhaustion and Tregs.

Conclusion

These findings suggest that PD-L1 overexpression in MSCs enhances their immunosuppressive and protective effects on pancreatic islets, partly by enhancing Tregs and CD8⁺ T cell exhaustion. This underscores the therapeutic potential of PD-L1-MSCs and PEVs in modulating immune responses in T1D and reveals the underlying cellular mechanisms.