Phenotypical and functional analysis of natural killer cells from primary human head and neck squamous cell carcinomas

Abstract

Abstract The purpose of this study was to determine if natural killer (NK) cells from primary human head and neck squamous cell carcinomas (HNSCC) expressed altered phenotypical and functional capabilities compared to matched NK cells from the periphery. NK cells are a critical part of the host defense against cancer through direct cytotoxicity of transformed cells and the release of pro-inflammatory cytokines. To facilitate the targeting of NK cells for immunotherapy we phenotyped human HNSCC NK cells using flow cytometry and RNA-SEQ analysis of matched flow-sorted PBMC and tumor infiltrating NK cells. We found that tumor infiltrating NK cells of HNSCC patients, significantly downregulated activating receptors NKG2D, DNAM-1, NKp30, CD16 and 2B4 compared to matched blood NK cells (n=13). Surprisingly, the majority of inhibitory KIR receptors on tumor-infiltrating NK cells were decreased. However, the inhibitory receptors NKG2A and PD-1 were found to be highly overexpressed in tumor-associated NK cells. In addition, RNA-SEQ analysis of flow sorted NK cell populations from matched blood and TIL of HNSCC patients (n=9) demonstrated significant differential expression of multiple immune pathways. Functional assays confirmed that tumor infiltrating NK cells have reduced cytotoxicity and produce significantly less IFNγ after ex-vivo stimulation (n=10). We believe that this study provides the first in-depth look at NK cells from primary HNSCC tumors. We found that tumor associated NK cells express a profoundly different phenotype than NK cells from the blood of the same patients, with multiple activating receptors downregulated and the inhibitory receptors NKG2A and PD-1 overexpressed.