Fig. 3: TLS-associated heterogeneity of CD4⁺ T cell states in HNSCC.

a PAGA analysis of CD4⁺ T cells. Each color represents a subcluster of CD4⁺ T cells. b UMAP plots showing normalized expression profiles of cell-type-specific markers in CD4⁺ T cell subclusters. c Left, UMAP plot showing pseudotime trajectories of CD4⁺ T cell clusters, revealing two potential differentiation trajectories: exhaustion and Treg. Right, scaled module scores within these subclusters of CD4⁺ T cells with respect to two pseudotime trajectories and stem-like, functional, exhausted, and immunosuppressive markers. d Dot plot showing the analysis of enrichment for the four most significant Gene Ontology (GO) Biological Process terms across CD4⁺ T cell subclusters. 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. e UMAP plot embedding for CD4⁺ T cell subclusters, colored by clone size (upper left) and UMAP embedding for CD4⁺ T cell subclusters in different TLS status, colored by subcluster, indicating the different clone size by dot size. f Images of a mIHC-stained different subcluster of CD4⁺ T cells in HNSCC tumor with TLS status. Regions with a high density of central memory (CXCR5⁺CD4⁺) CD4⁺ T cells and progenitor exhausted (TCF1⁺PD-1⁺CD4⁺) CD4⁺ T cells. nTLS and imTLS were repeated four times independently with similar results, mTLS were repeated six times independently with similar results. Scale bars = 100 μm. g Heatmap and pie chart showing the number of clonotypes shared between neighboring functional clusters of CD4⁺ T 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. h–k Box plots showing the number of clonotypes of total CD4⁺ T cell, CD4⁺ Tconv cell, naive/central memory CD4⁺ T cell, and progenitor exhausted CD4⁺ T cell 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). l–o Boxplot showing fraction clonotypes of naive/central memory CD4⁺ T cell, progenitor exhausted CD4⁺ T cell, CD4⁺ Tconv cell (clusters 9–13) and Treg (clusters 15–20) in total CD4⁺ T cell clonotypes across 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). Tconv conventional T cell, Treg regulatory T cell, Tfh T follicular helper cell.