The use of natural killer (NK) cells is a promising approach in the field of cancer immunotherapy; however, combination treatments are required to enhance the effects of NK cell immunotherapy. In this study, we assessed the potential of irradiation and cisplatin as a chemoradiotherapy (CRT) regimen to augment the effects of NK cell immunotherapy in head and neck squamous cell carcinoma (HNSCC). NK cells were expanded using our recently established K562-OX40 ligand and membrane-bound interleukin (IL)-18 and IL-21 feeder cells in the presence of IL-2/IL-15 from peripheral blood of healthy donors. The results showed an increase in the purity of NK cells and expression of activation markers such as NKG2D and lymphocyte function–associated antigen 1 during the expansion process, which is positively correlated to the NK cell infiltration and overall survival in patients with HNSCC. CRT induced NK cell activation ligand (ULBP2) and adhesion molecules (ICAM-1, -2 and -3) on HNSCC, leading to enhanced cytotoxicity of NK cells against HNSCC. Our findings suggest that the NK cells have a potent anti-tumor effect in combination with CRT against HNSCC. We hypothesized that NK cells expanded by our recently established K562-OX40L-mbIL-18/21 feeder cells can increase the expression of NKG2D and LFA-1, thereby increasing the recognition and adhesion, respectively. In contrast, the treatment of cancer cells with irradiation (RT) and cisplatin not only directly killed HNSCC but also upregulated the expression levels of MICA and MICB, ULBP 1 and ULBP 2, and ICAM-1, 2 and 3 on HNSCC. The upregulation of these markers will enhance NK cells–HNSCC interaction, thereby leading to enhanced NK cell killing activity against HNSCC cells. [Display omitted] [ABSTRACT FROM AUTHOR]