Smoking-Associated Enrichment of CXCL13 + Tfh Cells and VCAM1 + Fibroblasts Is Linked to Tertiary Lymphoid Structure Formation in Lung Adenocarcinoma

Background: Smoking history is associated with improved immune checkpoint inhibitor treatment responses.We investigated smoking-associated differences in the tumor microenvironment (TME) of lung adenocarcinoma (LUAD) and identified biomarkers associated with tertiary lymphoid structure (TLS) formation.Methods: Single-cell RNA-sequencing datasets from tumors and matched adjacent normal tissues from five current smokers and five never-smokers with LUAD were integrated, and the T-and NK-cell compartments were analyzed in detail.Key findings were further evaluated using public LUAD scRNA-seq datasets and validated by immunofluorescence in formalin-fixed paraffin-embedded LUAD tissues using QuPath-based image analysis.Results: In current smokers, follicular helper T (Tfh) cells were enriched in the TME, whereas CD4⁺ tissueresident memory T cells were reduced (RO/E, median [interquartile range (IQR)]: 2.1 [1.5-5.7] vs. 1.0 [0.5-1.5],p=0.040; and 0.7 [0.6-1.1] vs. 1.7 [1.6-2.4],p<0.001, respectively).The Tfh cluster showed high expression of CXCL13 and increased expression of PDCD1 and CTLA4.Immunofluorescence confirmed increased CXCL13⁺CD4⁺ cells in current smokers (83.0 [10.0-218.5]vs. 10.0 [2.0-60.5]cells/0.25 mm², p<0.001).Gene set analysis showed enrichment of a B-cell chemotaxis signature in the Tfh cluster (normalized enrichment score, 1.693; adjusted p=0.023), and CXCL13⁺CD4⁺ T-cell density correlated with CD20⁺ B-cell density (Spearman ρ=0.445, p<0.001).Current smokers also showed greater TLS burden, with higher TLS counts and larger TLS areas, and had increased VCAM1⁺ fibroblast abundance (283.0 [118.5-580.5]vs. 98.5 [32.5-191.0]cells/0.25 mm², p<0.001), which strongly correlated with CD20⁺ cell density (Spearman ρ=0.766, p<0.001).Conclusions: Smoking-associated enrichment of CXCL13⁺CD4⁺ Tfh cells was associated with B-cell accumulation and TLS formation in LUAD.These findings identify the CXCL13⁺ Tfh-B-cell axis, together with VCAM1⁺ fibroblast-rich stromal niches, as potential biomarkers and therapeutic targets in LUAD.