Uncovering immune checkpoint heterogeneity in oral squamous cell carcinoma using single cell RNA-sequencing data highlights three subgroups of patients with distinct immune phenotypes

Objectives: In head and neck squamous cell carcinoma (HNSCC), PD-1/PD-L1 inhibitors remain inefficient in most patients, which points to the need for better characterization of immune checkpoint (ICP) molecule expression. Material and methods: We evaluated the expression of 22 ICP ligands (ICPL) in 2,176 malignant cells from 10 patients in a public single-cell RNA-sequencing dataset and in two cohorts of HNSCC patients for which gene expression data are available. Results: Based on ICPL expression, malignant cells formed three distinct clusters characterized either by a strong expression of ICPL together with an immune phenotype linked to IFN-gamma response (cluster 1) or by a weak ICPL expression and little response to IFN-gamma (clusters 2 and 3). Malignant cells from cluster 3 showed a high PD-L1 expression associated with NRF2 signature. The relevance of 3 groups of patients, i.e "high ICPL/high IFN-gamma", "low ICPL/low IFN-gamma" or "low ICPL/low IFN-gamma/high PD-L1" was confirmed in a cohort of 259 OSCC whole tumor samples from TCGA and in the CLB-IHN cohort including patients treated with PD1/PD-L1 inhibitors. The heterogeneous expression of ICPL among patients' malignant cells was associated with immunologically distinct microenvironments, evaluated with the "hot/cold" and the Tumor microenvironment (TME) classification. Finally, the "low ICPL/low IFN-gamma/high PD-L1" group 3 displayed a poor prognosis in the TCGA cohort. Conclusion: Hence, the global picture of ICPL gene expression in malignant cells from HNSCC patients may contribute to the broader issue of improving immunotherapy strategies though a better stratification of patients and the design of new treatment combinations.