Multimodal single-cell and spatial profiling reveals altered T cell-mediated immunity and B-cell follicular architecture in non-metastatic lymph nodes of patients with aggressive non-small cell lung cancer

Regional lymph nodes (LNs) in the thoracic cavity serve as essential immunological hubs that coordinate humoral and cell-mediated responses against the development and progression of non-small cell lung cancer (NSCLC). To investigate immune dysregulation in the non-metastatic regional LNs of patients with aggressive NSCLC, we performed multimodal profiling on 36 LNs from 11 patients undergoing curative-intent resection including CITE-seq, scRNA-seq, and Imaging Mass Cytometry (IMC). Regional N1 LNs from patients with more aggressive disease (stage IB–IIIA) exhibited a significant enrichment of dysfunctional CD8⁺ T cells and regulatory T cells (Tregs) compared to N2 LNs and LNs from patients with less aggressive disease (stage IA). These immune subsets were spatially co-localized with mature regulatory dendritic cells (mregDCs; CD1c⁺, TIM3⁺, LAMP3⁺), forming an immunosuppressive niche uniquely enriched in the N1 LNs of higher-stage patients. Concurrently, higher-stage N1 LNs contained larger number of “decorticated” B-cell follicles characterized by decreased encapsulation of the mantle zone layer surrounding the germinal centers. This mantle zone disorganization was associated with increased spatial niches involving Tregs, CD68⁺ CD163⁺ TIM3⁺ Macrophages, CD163⁺ TIM3dim Monocytic-Myeloid Derived Suppressor Cells (M-MDSC), plasma B cells, and a decrease in spatial niches involving CD4⁺ T helper cells and fibroblastic reticular cells (FRCs). Together, our findings reveal parallel alterations in humoral and cell-mediated immunity within the regional LNs of patients with aggressive NSCLC.