Eliminating PD‐L1 on Dendritic Cell Extracellular Vesicles for Immunotherapy Potentiates Immune‐Mediated Tumour Rejection in Mice

ABSTRACT Extracellular vesicles (EVs) are emerging as promising vehicles for cancer immunotherapy, yet the molecular determinants of their immunogenicity remain poorly defined. While PD‐L1 expression on cancer‐derived EVs has been shown to suppress immune responses, its role on immunotherapeutic EVs remains unexplored. In this study, we investigated how eliminating PD‐L1 from antigen‐loaded bone marrow‐derived dendritic cell (BMDC) EVs affects the efficacy of EV‐based cancer vaccines. We generated and characterized ovalbumin (OVA)‐loaded BMDC EVs from wild‐type (WT) and PD‐L1 −/− C57BL/6 mice. EVs were administered intravenously into WT mice in immunization experiments and in therapeutic and prophylactic B16 OVA‐secreting melanoma models. Immune responses were assessed by flow cytometry, ELISpot, and ELISA, and tumour growth was monitored. Proteomic analysis confirmed high EV purity and similar protein profiles between WT and PD‐L1 −/− EVs, with PD‐L1 being the major difference. Functionally, PD‐L1 −/− EVs induced significantly stronger anti‐tumour responses in vivo, particularly in the prophylactic setting. Mice treated with PD‐L1 −/− EVs showed increased CD8 + T cell tumour infiltration, enhanced IFNγ secretion, and higher tumour rejection rates compared to WT EVs (72.7% vs. 37.5%). Additionally, the frequency of tumour‐infiltrating, antigen‐specific CD8 + T cells was significantly higher in PD‐L1 −/− EV‐treated mice. In summary, BMDC‐derived EVs loaded with antigen are potent immune stimulators, and removal of immune checkpoint molecules such as PD‐L1 further enhances their immunogenicity. These findings support the development of engineered EVs as improved platforms for cancer immunotherapy.