Fig. 1: TLS-associated immune landscape in HNSCC.

a Workflow shows the collection and processing of fresh samples of primary oral cavity HNSCC tumors for scRNA-seq, scTCR/BCR-seq, and spatial transcriptomics repertoire analysis. The collection of fresh tumor samples from HNSCC patients (n = 14 independent samples) was used to identify the status of TLS by IHC staining and classified into nTLS (n = 4 independent samples), imTLS (n = 4 independent samples), and mTLS (n = 6 independent samples). Subsequently, the fresh tumor sample was divided into three parts and subjected to different processes, including: (1) scRNA-seq of whole tumor cells; (2) MACS targeting CD45⁺ cells in tumor, followed by paired scRNA-seq and scTCR/BCR-seq; and (3) spatial transcriptomic sequencing for the tumor sample. b UMAP plot of 248,336 cells profiled by all scRNA-seq, colored by cell types. The 11 broad cellular lineages contain ECs, lymphatic ECs, stromal cells (pericytes, fibroblasts), lymphoid cells (T/NK cells, B cells, plasma cells), myeloid cells, neutrophils, and cancer cells. Below the UMAP plot, the number of identified cells for each cell type is included. c UMAP plot colored by patients (n = 14 independent samples) from whole tumor cells or sorted CD45⁺ cells profiled by scRNA-seq. d UMAP plot colored by origin of the cells and the number of cells, either from whole tumor cells (n = 123,432) or sorted CD45⁺ cells (n = 124,904). e UMAP plot colored by nTLS (66,903), imTLS (61,415), and mTLS (120,018)—three different TLS statuses profiled by scRNA-seq. f UMAP plot colored by TCR/BCR information profiled by scTCR/BCR-seq. The cell types for scRNA-seq of whole tumor cells and the composition difference of CD45⁺ cells (g), H&E and IHC of tumor slides (h), and spatial transcriptomics of tumor tissue (i) in nTLS, imTLS, and mTLS statuses. The analysis was conducted using a generalized linear model (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 (indicating enrichment) to blue (signifying depletion), encodes the log₂-transformed odds ratios, while the sizes of the depicted points are governed by the Bonferroni-adjusted −log₁₀(P values), accentuating the statistical significance of observed variations. TLS tertiary lymphoid structures, OCT optimal cutting temperature, H&E hematoxylin and eosin, IHC immunohistochemistry, ECs endothelial cells.