Transcriptomic differences in cytotoxic T cells between the inflamed vs. non-inflamed tumor microenvironment of head and neck squamous cell carcinoma (HNSCC)

Introduction Stroma, cancer and immune cells in the HNSCC tumor microenvironment (TME) employ mechanisms of resistance to immunotherapy, leading to a low response rate of 15-20 %. We aim to use single-cell RNA sequencing (ScRNAseq) to explore the differences between inflamed (infiltrated) and non-inflamed (non-infiltrated) HNSCC tumors.

Methods 19 HNSCC tumor specimen were dissociated to produce single cell suspensions for scRNAseq and sorted into CD45+ and CD45- samples. ScRNAseq was performed using the 10x Genomics 3’ single cell kits. Immune infiltration status was determined by H&E staining. Data aggregation and normalization (CellRanger) as well as visualization (scanpy) was performed.

Results We identified 31 different cell clusters, with 22 clusters formed by immune cells (PBL and TIL) while non-immune cells formed 9 clusters. Canonical immune cells (B cells, cytotoxic T cells, helper T cells, etc.) as well as sub-states (activation, senescence and exhaustion) were delineated. Inflamed tumors (‘hot’ TME, n = 9) showed a higher number of relative CD8⁺ T cells versus non-inflamed (‘cold’) tumors. Gene set enrichment analysis (GSEA) showed IFN? and IFNa responses as well as allograft rejection-associated gene sets were enriched in CD8⁺ T cells from inflamed tumors, while in cells from non-inflamed tumors TNF, apoptosis and hypoxia signaling were upregulated. Further, cytotoxic T cells from the infiltrated TME showed an enrichment of effector genes such as NKG7 and CCL5, GZMH.

Conclusions Using scRNAseq of HNSCC tumors, we can show that that CD8⁺ cytotoxic T cells are enriched in inflamed tumor and show a more effector-like transcriptome. This underlines the importance of qualitative rather than only quantitative differences in evaluating tumor immune infiltration.

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Publication History

Article published online:
10 June 2020