FIGURE 2.

KIR is expressed uniquely in CD56^dim NK cells from patients with HNSCC and healthy individuals and cetuximab-activated NK cells-induced HLA-C upregulation on tumor cells in vitro. A, Frequency of NK cells (CD3^–CD56⁺ cells) from peripheral blood lymphocytes (PBLs) in patients with HNSCC. Top row shows gating strategy, second row shows CD56^dim and CD56^bnght NK cell populations, and bottom row shows KIR (CD158b) expression on CD56^dim NK cells. Representative gating dot plots shown for five patients with HNSCC. B, Percent of circulating KIR⁺ (CD158b/KIR2DL2/KIR2DL3) NK cells (CD3^-CD56⁺ cells) in PBL from patients with HNSCC, n = 20. C, Percent of circulating KIR+ NK cells (CD3^-CD56⁺ cells) in PBL from patients with head and neck cancer (HNSCC; n = 20) versus healthy individuals (n = 5) (Student’s t test ns = P > 0.05). D, NK cells were isolated from PBMC and cocultured with JHU029 tumor cells at 2 to 1 ratio for 24 hours in the absence of mAb or with IgG1 isotype control (10 μg/mL) or cetuximab (10 μg/mL). IFNγ concentration in culture supernatants was determined by ELISA and normalized to number of NK cells in each condition (data from triplicate experiments from three different donors, ANOVA, ***P < 0.001). E, NK cells from five healthy donors were cocultured with JHU029 or 93VU tumor targets for 24 hours in the absence of mAb, IgG1 control (10 ĝ/mL), or cetuximab (10 μg/mL), harvested, and HLA-C expression on tumor cells was determined by flow cytometry (ANOVA, *P < 0.05). Abbreviations: ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; HLA-C, human leukocyte antigen-C; HNSCC, head and neck squamous cell carcinoma; IFNγ, Interferon gamma; IgG1, immunoglobulin G-1; KIR, killer-cell immunoglobulin-like receptor; mAb, monoclonal antibodies; NK, natural killer [Color figure can be viewed at wileyonlinelibrary.com]