Introduction: The number of tumor-infiltrating lymphocytes (TIL) correlates with better survival and is increased in HPV^pos OPSCC. The expression of immune checkpoint molecules (ICM) affects anti-tumor T-cell function and immune response. To determine the extent to which HPVpos differs from HPV^neg OPSCC, single-cell RNA sequencing data from oropharyngeal carcinomas (OPSCC) were analyzed in addition to a transcription analysis of a TCGA and an Ulm/Southampton RNA sequencing data set.

Material and methods: The complete TCGA transcriptome data set, selected TCGA OPSCC cases (n = 66), and OPSCC cases from Ulm and Southampton (n = 51) were analyzed using the DeSeq2 pipeline in R. In addition, raw data from a published scRNA sequencing dataset from 26 OPSCC (18 HPV^neg and 8 HPV^pos) patients with blood samples (PBMC) and paired TIL samples (GSE139324) were bioinformatically processed and analyzed with the Seurat pipeline in R.

Results: In the transcriptome analysis, CD27 and TIM3 showed a statistically significant different expression depending on the HPV status. The scRNA analysis showed a statistically higher expression of VSIR in the PBMC and TILs of HPV^neg OPSCC. The LAG3 expression on PBMCs was reduced in HPV^neg OPSCC while there was no difference found on the TILs. Other differences were seen in the TILs: CD27, CD28, TIGIT, PD-1, BTLA, TNFRSF9 (CD137), and CD40 were elevated in HPV^pos OPSCC, while ICOS, TNFRSF4 (OX40), TNFRSF18 (GITR), and ENTPD1 (CD39) were increasingly expressed in HPV^neg OPSCC.

Conclusion: In this project, we describe the importance of ICM expression analysis for OPSCC depending on their HPV status. The identified expression profiles underpin the immunological differences of HPV^pos OPSCCs with enhanced T cell basal activation and point to a possible distinction in therapy and checkpoint inhibitor selection in the future.