Tumor-associated CD207 + cDC2 drives TLS formation via TNF family cytokines in non-small cell lung cancer

Background: Dendritic cells (DCs) are specialized antigen-presenting cells that orchestrate innate and adaptive immunity, and within the tumor microenvironment (TME) they extend their function to modulate anti-tumor immune responses. The heterogeneity of DCs underlies their diverse immune functions, with conventional type 2 DCs (cDC2s) showing particularly high phenotypic and functional complexity, which enables context-dependent immune modulation. However, the characteristics and functions of tumor-associated DCs remain incompletely defined. Elucidating how these subsets arise and function could broaden our understanding of tumor immunity and contribute to the development of effective cancer immunotherapies. Methods: Tumor tissues (n=35) and adjacent normal tissues (n=24) from treatment-naïve, EGFR-wildtype NSCLC patients were enzymatically dissociated and subjected to single-cell RNA sequencing using the 5′ Chromium platform (10x Genomics). Trajectory and SCENIC analyses were performed to delineate cDC2 lineage programs. Functional validation included differentiation of CD207 + DCs from human monocytes, co-culture with HUVECs, and multiplex IHC (mIHC) on FFPE tumor slides from NSCLC patients. Deconvolution analyses of TCGA-LUAD and an in-house immunotherapy bulk RNA-seq cohort were conducted to assess clinical relevance. Results: We identified 10 transcriptionally distinct DC subtypes and found that CD207 + cDC2s were markedly enriched in tumors. Trajectory analysis defined CD207 + cDC2s as a distinct TME-enriched lineage characterized by reduced NF-κB signaling, chemotaxis, and T cell-regulatory programs, while enhanced TNF superfamily cytokine production and retinoic acid metabolism. SCENIC analysis implicated RARA/RXRA as key regulators of CD207 + cDC2 differentiation. CD207 + cDC2 frequency correlated with higher immune cell infiltration, especially CXCL13 + T cells. Consistently, LTB expression in CD207 + cDC2 correlated with high endothelial venule (HEV) and tertiary lymphoid structure (TLS) gene signatures, suggesting a role in lymphoid organization. Functionally, monocyte-derived CD207 + DCs induced adhesion molecules in endothelial cells in vitro , and mIHC confirmed that their co-localization with HEVs and increased TLS density. Higher CD207 + cDC2 abundance was associated with improved survival and immunotherapy response, highlighting their specialized role within TME in supporting lymphoid structuring and favorable outcomes in EGFR-wildtype NSCLC. Conclusion: CD207 + cDC2s constitute a distinct TME-enriched DC subtype that mediates endothelial activation and drives HEV and TLS formation via LTB. Their enrichment in tumors correlates with improved survival and enhanced immunotherapy response, indicating that CD207 + cDC2s promote lymphoid organization and favorable immune outcomes in EGFR-wildtype NSCLC.