Integrated single-cell and functional analyses define strategies to enhance dendritic cell immunostimulatory capacity.

Dendritic cells (DCs) are essential for the initiation and regulation of antitumor immune responses. Although DC vaccines hold great promise in personalized cancer immunotherapy, their efficacy remains limited by tumor-induced immunosuppression and insufficient DC maturation and activation. Recent studies suggest that DC maturation is accompanied by dynamic changes in immune checkpoint signaling, highlighting the need to better define functional DC states. To delineate these regulatory features, we performed single-cell RNA sequencing to characterize DC heterogeneity and transcriptional programs, identifying enrichment of immune checkpoint associated signatures, including PD-L1, within mature DC subsets. Subsequently, we evaluated the effects of PD-L1 blockade and CD40 stimulation on DC activation and function in vitro. We found that combined PD-L1 inhibition and CD40 engagement enhanced DC maturation and antigen-presenting capacity. We operationally define these functionally optimized DCs as “Enhanced DCs”, characterized by elevated IL-12 secretion, and improved T-cell priming efficiency. When loaded with tumor neoantigen peptides, Enhanced DCs effectively activated CD8⁺ T cells and promoted memory T-cell differentiation in co-culture assays. Together, this study integrates single-cell transcriptomic profiling with functional validation to demonstrate that targeted modulation of DC immune checkpoints and co-stimulatory pathways can improve the immunogenicity of DC, particularly when loaded with neoantigens. These findings provide a rational framework for optimizing DC-based cancer vaccines and advancing personalized immunotherapy approaches.