Abstract
Immune-checkpoint inhibitors improve the survival of head and neck squamous cell carcinoma (HNSCC) patients. Although recent studies have demonstrated that the tumor immune microenvironment (TIME) has critical roles in immunotherapy, the precise mechanisms involved are unclear. Therefore, further investigations of TIME are required for the improvement of immunotherapy. The frequency of effector regulatory T-cells (eTregs) and the expression of immune-checkpoint molecules (ICM) on eTregs and conventional T-cells (Tconvs) both in peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) from HNSCC patients were analyzed by flow cytometry and their distributions were evaluated by multi-color immunofluorescence microscopy. High frequency eTreg infiltration into HNSCC tissues was observed and high expressions of CD25, FOXP3, stimulatory-ICM (4-1BB, ICOS, OX40 and GITR) and inhibitory-ICM (programmed cell death-1 [PD-1] and cytotoxic T-lymphocyte-associated protein-4 [CTLA-4]) were found on invasive eTregs. In contrast, the expression of stimulatory-ICM on Tconvs was low and the expression of inhibitory-ICM was high. In addition, ICM-ligands (programmed cell death-1 [PD-L1], galectin-9 and CEACAM-1) were frequently expressed on cancer cells. PD-L1 and galectin-9 were also expressed on macrophages. PD-1+ T-cells interacted with PD-L1+ cancer cells or PD-L1+ macrophages. This suggested that in TIL, eTregs are highly activated, but Tconvs are exhausted or inactivated by eTregs and immune-checkpoint systems, and ICM and eTregs are strongly involved in the creation of an immunosuppressive environment in HNSCC tissues. These suggested eTreg targeting drugs are expected to be a combination partner with immune-checkpoint inhibitors that will improve immunotherapy of HNSCC.