Fig 2: TLS formation in MIA tumors(A) The schema of TLS formation. The first stage is B & T cell aggregation. The second stage is follicle formation with T-zone and B-zone. The third stage is ectopic germinal center formation with formation with follicular dendritic cell in light-zone.(B) Hierarchical clustering of Z score normalized gene expression of B cell infiltration markers (CD19 and CD20), follicle phenotype markers (CXCR5 and CXCL13), and lymphocyte homing signal markers (CCR7 and CCL19). Color of the top bar represents tumor (red) or adjacent normal (blue) tissue. Correlations between expression levels from these six TLS markers and immune cell abundance corrected by tumor purity are shown in the right bar. p Values are calculated using Spearman correlation; ***p < 0.001; **p < 0.01; *p < 0.05; ns p > 0.05.(C) Typical observation field of MIA six-plex TLS panel staining. The merged-channel multispectral image was captured after 5-cycle TSA staining and DAPI counterstain, revealing a classic lymphoid structure residing in MIA stromal tissue. Single channels are shown for the area bounded by the white rectangle. Fake colors are assigned as DAPI: blue, CD8: yellow, CXCR5: pink, CD4: green, CD20: red, CD35: cyan. Tfh cells are highlighted with white arrows (lower right corner images).(D–G) Paired comparisons of CD20+ B cell density, CD4+ T cell density (E), CD35 + follicle dendritic cell density (F), and CD8+ T cell density (G) between non-follicle (Non-Fol) and TLS areas

Fig 3: High-dimensional flow cytometry and immunohistochemistry reveal distinct TIL-B phenotypes and increased tertiary lymphoid structures in HPV+ HNSCC.a viSNE plots of B cells collected from non-inflamed and inflamed tonsils, HPV+ and HPV− HNSCC TIL and paired PBL (Supplementary Fig. 7) were analyzed using Cytobank. Non-inflamed tonsil (n = 4), inflamed tonsil (n = 6), HPV + HNSCC (n = 3), HPV− HNSCC (n = 2). Bar plot displaying frequencies of GC B cells and plasma cells in non-inflamed tonsil (n = 9), inflamed tonsil (n = 16), HPV+ HNSCC (n = 9), HPV− HNSCC (n = 9). *P = 0.02 Student‘s 2-sided t test. b Bar plot for frequency of B cell subpopulations. Non-inflamed tonsil (n = 9), inflamed tonsil (n = 16), HPV+ HNSCC (n = 12), HPV− HNSCC (n = 13). **P = 0.004, ***P = 0.0009, *P = 0.03, One way ANOVA followed by Tukey’s multiple comparisons test. c Frequencies of T follicular helper (TFH), regulatory T follicular helper (TFHreg), regulatory T cell (Treg), T helper type 1 (TH1) and CD8 T cells in non-inflamed tonsil (n = 6), inflamed tonsils (n = 10), HPV+ TIL (n = 7), HPV− TIL (n = 8). *P = 0.01,**P = 0.009,****P < 0.0001. *P = 0.04, *P = 0.03, ****P < 0.0001. One-way ANOVA followed by Tukey’s multiple comparisons test. d Representative CD20+ IHC on HPV+ and HPV− HNSCC tumors (×4 magnification). e B cell infiltrate counted within tumor bed compared to TLS. Total numbers from n = 50, 25 HPV+, 25 HPV− were graphed. ****P < 0.0001, Student‘s 2-sided t test. f Tumor TLS by site within the oropharynx (tonsil vs. tongue). Total numbers from n = 50, 25 HPV+, 25 HPV− were graphed. **P = 0.0096, Student‘s 2-sided. Data are presented as mean values ± SEM. g Total number of tumor TLS and non-tumor TLS numbers in HPV+ and HPV− disease. Total numbers from n = 50, 25 HPV+, 25 HPV− were graphed. *P = 0.0249, Student‘s 2-sided t test. Data are presented as mean values ± SEM. h Correlation of CD20+ tumor TLS with tumor area. Total tumor area (mm2) for each patient tumor was calculated by a pathologist. *P < 0.05, non-parametric Spearman correlation. i Total tumor TLS independently counted for CD20+ and CD4+ (n = 50, 25 HPV+, 25 HPV−). ****P < 0.0001, ***P < 0.001, non-parametric Spearman correlation. j Total CXCR5 was scored for all cell types (n = 50, 25 HPV+, 25 HPV−).**P = 0.0012, Student‘s 2 sided t test. Source data are provided as a Source Data file.

Fig 4: TFH–B–TRM lymphocyte cooperation and their co-localization in lung tumors.a The dot plot summarizing the cell–cell interactions among CD8-C2 (TRM-like), B, and CD4-C9 (TFH-like) cells by CellPhoneDB analysis. Only significant ligand–receptor interactions are presented here. The size of the circle indicates the statistical significance, and the color gradient indicates the mean expression value of gene pairs. b Representative multiplexed immunofluorescence (IF) of TLS-like lesion in EGFR-WT (n = 14) (upper) (scale bar 100 µm: left, scale bars 50 µm: right magnified) and EGFR-MT (n = 14) (lower) (scale bar 100 µm: left, scale bars 50 µm: right magnified) tumors. Summarized graph for the area of TLS-like lesions in EGFR-WT (n = 5) and EGFR-MT (n = 4) tumors for single-cell analysis (**p = 0.005, two-sided test) (upper panel) and in EGFR-WT (n = 9) and EGFR-MT (n = 10) tumors from independent patients for validation (*p = 0.05, two-sided test) (lower panel). c Representative multiplexed IF of CD4, CXCL13, CD103, CD8, CXCR5, and CD20 in tumor specimens from EGFR-WT (n = 14) (left) (scale bar 100 µm) and EGFR-MT (n = 14) (right) (scale bar 100 µm) patients. d Summarized graph for the numbers of each cell type in total TLS-like lesions in EGFR-WT (n = 5) and EGFR-MT (n = 4) patients. The number of CD4+, CXCL13+CD4+ T cells, CD8+ T cells, CD103+ CD8+ T cells are significantly higher in EGFR-WT than EGFR-MT (*p = 0.05, **p = 0.005, two-sided test). e Summarized graph for the numbers of each cell type in total TLS-like lesions in EGFR-WT (n = 9) and EGFR-MT (n = 10) tumors from independent patients for validation. Consistently, the number of CD4+, CXCL13+CD4+ T cells, CD8+ T cells, CD103+ CD8+ T cells were significantly higher in EGFR-WT than EGFR-MT (**p = 0.005, ***p = 0.0005, two-sided test). f An illustrative summary of the proposed model for TFH–B–TRM lymphocyte cooperation in TLS for antitumor immunity in lung cancer. Data are represented as mean ± SEM. Source data are provided as a Source Data file.