Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer. While the number of HNSCC cases is decreasing, the number of oropharyngeal SCC (OSCC) is rising. OSCC are increasingly (45-90%) caused by human papillomavirus type 16 (HPV16). Intriguingly, patients with HPV-induced OSCC (HPV16+) respond better to therapy than HPV-negative (HPV16-) OSCC. This is independent of nodal status, age, stage, tumor differentiation or gender. We hypothesize that tumors of HPV16+ OSCC patients display a different immune contexture that contributes to a better response of these tumors to standard therapy. To test our hypothesis, an exploratory in-depth analysis of immune infiltrates in the tumor microenvironment (TME) of 13 HPV16- and HPV16+ OSCC patients was performed using 36-parameter mass cytometry (CyTOF) analysis.
Tumor cell suspensions were prepared by mechanistic dissociation using Gentlemacs and cryopreserved until CyTOF analysis. HPV16 status of the tumors was determined by GP5+/6+ PCR and p16 immunohistochemistry staining. HPV16 E6/E7-specific T cell reactivity within HPV16+ tumors was determined by proliferation assay on directly ex vivo tumor samples and/or IL-2-expanded tumor infiltrating lymphocyte (TIL) batches. In-depth tumor immune infiltrate analysis was performed by CyTOF technology on ex vivo tumor samples from 13 OSCC patients, of which four were found to be HPV16-, four HPV16+ but HPV16 immune response-negative (HPV16+ IR-) and five HPV16+ immune response-positive (HPV16+ IR+).
Analysis of the TME through 36-parameter CyTOF revealed clear phenotypic differences between immune cells infiltrating the TME of HPV16 IR+, HPV16+ IR- and HPV16- tumors. Whereas HPV16+ IR- tumors were strongly infiltrated with B cells, HPV16+ IR+ tumors were strongly infiltrated with effector memory CD4+ and CD8+ T cells with a highly activated, i.e. CD38+, HLA-DR+ and/or PD-1+, phenotype. Interestingly, subsequent unsupervised hierarchical clustering through the CITRUS algorithm led to the identification of two distinctive populations of activated CD4+ T cells and one population of CD103-expressing tissue-resident effector memory CD8+ T cells that were present at significantly higher levels in HPV16+ IR+ patients.
In conclusion, our data revealed that distinct immune cell populations infiltrate the TME of HPV16+ IR+ tumors that may contribute to a better response of these tumors to standard therapy.