Fig. 4: TLS-associated heterogeneity of B cell states in HNSCC.

a UMAP plot of B cell subclusters identified from scRNA-seq. Subclusters are numbered and colored by identity: B cells (clusters 1–6) and plasma cells (clusters 7–9). b Left, GLM-based dot plot showing TLS status-specific enrichment of B cell subclusters. The analysis was conducted using a GLM with a binomial distribution and a logit link function. Estimated marginal means and contrasts were computed with P values indicating the statistical significance of the observed differences. The P values were adjusted using the Bonferroni correction method. A color gradient, transitioning from red (representing enrichment) to blue (representing depletion), encodes the log₂-transformed odds ratios, while the sizes of the depicted points are governed by the Bonferroni-adjusted −log₁₀(P values), highlighting the statistical significance of observed variations. Middle, heatmap of signaling pathway activity scores across B cell and plasma cell subclusters. Right, heatmap of normalized expression of genes associated with immunoglobulin heavy chain constant region, which correlate with types of Ig across B cell subclusters. c Pairwise comparisons of kernel density estimates in UMAP space. The color gradient from red to blue indicates decreasing enrichment of B cells and plasma cells in different TLS statuses. d UMAP plots showing normalized expression profiles of cell-type-specific markers in B cell subclusters. e Ranking of GO Biological Process terms in subclusters of B cells. A color gradient, transitioning from red (representing enrichment) to blue (representing depletion), encodes the Benjamini–Hochberg-adjusted −log₁₀(P values), highlighting the statistical significance of observed variations. Benjamini–Hochberg-adjusted P values were obtained by a two-tailed Wilcoxon rank-sum test. f PAGA analysis of B cells and plasma cells. Each color represents a subcluster of B cells or plasma cells. g Left, Schematic illustrates that as B cells differentiate from naive B cells to GC B cells, the maturation progressively increased, and the GC reaction time was extended. Right, scaled module scores within these subclusters of B cells with respect to two pseudotime trajectories and genes associated with activation, class switch recombination machinery and class switch recombination interactors. h Volcano plot showing differentially expressed genes (DEGs) of total B cells between non-mTLS (imTLS/nTLS) and mTLS statuses (red dots: Benjamini–Hochberg-adjusted P < 0.05 and log₂(fold change) > 10, gray dots: adjusted P > 0.05, blue dots: adjusted P < 0.05 and log₂(fold change) < −10). Benjamini–Hochberg-adjusted P values were obtained by a two-tailed Wilcoxon rank-sum test. i UMAP plot embedding for subclusters of B cells and plasma cells, colored by clone size (upper left) and UMAP embedding for subclusters of B cells and plasma cells in different TLS status, colored by subcluster, indicating the different clone size by dot size. A color gradient, transitioning from red (representing enrichment) to blue (representing depletion), encodes the Z-score normalized enrichment score, while the sizes of the depicted points are governed by the Benjamini–Hochberg-adjusted −log₁₀(P values), highlighting the statistical significance of observed variations. Benjamini–Hochberg-adjusted P values were obtained by a two-tailed Wilcoxon rank-sum test. j Heatmap and pie chart showing the number of clonotypes shared between neighboring functional subclusters of B cells and plasma cells in different statuses of TLS. For the heatmap, the color represents the number of shared clonotypes. For the pie chart, the color represents the status of TLS, and the size represents the number of shared clonotypes. k–n Boxplot showing number of clonotypes of total B cells, plasmablasts, plasma cells, and FCRL4⁺ B cells in different TLS status (n = 4 independent samples nTLS, n = 4 independent samples imTLS, n = 5 independent samples mTLS, one-tailed Mann–Whitney U-test; for box plots: box center line, median; box limits, upper and lower quartiles; box whiskers, maximum and minimum values). Ig immunoglobulin, MHC major histocompatibility complex, Th2 T helper 2 cell, GC germinal center.