Abstract
Background: Radiotherapy (RT) remodels the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are key mediators of TME, yet how RT reprograms TAMs toward a programmed death ligand- 1(PD-L1)⁺ immunosuppressive phenotype remains unclear. Materials and Methods: Esophageal squamous cell carcinoma (ESCC) subcutaneous xenografts in immunodeficient mice received localized RT or sham treatment. Tumor-infiltrating PD-L1⁺ TAMs were quantified via multiplex immunofluorescence and flow cytometry. Extracellular vesicles (EVs) derived from irradiated ESCC cells (IR-EVs) were isolated and characterized by nanoparticle tracking analysis and transmission electron microscopy. Functional assays included co-culture of IR-EVs-educated macrophages with autologous CD8⁺ T cells. RNA sequencing identified DYNLL1-AS1 as the most upregulated lncRNA in IR-EVs. Mechanistic studies employed RNA pull-down, mass spectrometry, RNA immunoprecipitation, and dual-luciferase reporter assays. Clinical validation utilized ESCC specimens for RNA in situ hybridization. Prognostic significance was assessed via Kaplan-Meier and Cox regression analyses. Results: RT triggered ESCC cells to secrete DYNLL1-AS1-enriched EVs, which reprogrammed macrophages into PD-L1⁺ immunosuppressive TAMs. IR-EVs-educated macrophages suppressed CD8⁺ T cell proliferation and IFN-γ/ Granzyme B secretion. Mechanistically, DYNLL1-AS1 bound SEC22B, enabling its interaction with FOXP1 to activate PD-L1 transcription via promoter binding. In vivo, EVs carrying DYNLL1-AS1 counteract anti-PD-L1 therapy by suppressing CD8+ T cell function and promoting tumor growth. In ESCC patients, high DYNLL1-AS1 expression correlated with PD-L1⁺ TAM density, poor immunotherapy response, and reduced survival. Multivariate analysis confirmed DYNLL1-AS1 as an independent prognostic factor. Conclusions: Radiation-induced DYNLL1-AS1 in ESCC EVs drives PD-L1⁺ TAMs immunosuppression via SEC22B/ FOXP1 signaling. Combining DYNLL1-AS1 inhibition with PD-L1 blockade may reverse RT-induced immunosuppression, offering a transformative strategy for ESCC radio-immunotherapy.