B3GNT3 defines a spatially restricted dysfunctional T-cell niche and predicts immunotherapeutic outcomes in lung adenocarcinoma

Abstract Background Immunotherapy response in lung adenocarcinoma (LUAD) remains highly heterogeneous, underscoring the need for biologically informative markers of tumor immune dysregulation. B3GNT3, a glycosyltransferase implicated in several malignancies, is poorly characterized in LUAD with respect to the tumor immune microenvironment. Methods We integrated bulk, single-cell, and spatial transcriptomic data with weighted gene co-expression network analysis (WGCNA) to delineate immune networks associated with B3GNT3 in LUAD. Prioritized candidates were validated through molecular and functional assays in tumor cell lines and tumor-T-cell cocultures. A B3GNT3-based prognostic nomogram was constructed and evaluated in clinical cohorts. Results Elevated B3GNT3 expression correlated with advanced tumor stage and inferior overall survival. B3GNT3 high tumors exhibited exclusion and exhaustion features, and single-cell analysis confirmed enrichment of exhausted T cells (PD-1 + , TIM-3 + ). Network modeling identified the CD70 – CD27 axis as the key mediator. Spatial transcriptomics and multiplex immunofluorescence demonstrated that B3GNT3 and CD70 colocalize within exhausted T-cell-enriched tumor regions, delineating a spatially restricted dysfunctional niche. Mechanistically, B3GNT3 interacts with CD70 and regulates its N-glycosylation and protein stability, thereby prolonging its half-life. In tumor-T-cell cocultures, B3GNT3-dependent CD70 promoted T-cell activation and exhaustion, both reversed upon B3GNT3 knockdown, which also induced G1-phase arrest and suppressed tumor growth in vitro and in vivo. The nomogram accurately predicted 1-, 3-, and 5-year overall survival. Conclusions B3GNT3 defines a biologically distinct LUAD subtype characterized by a spatially restricted dysfunctional immune niche and adverse clinical outcome, supporting its evaluation in prognostic stratification and mechanism-guided therapeutic exploration.