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⁺ tissue-resident 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.