Figure 6. Cytotoxicity of NKG2A^null NK cells against tumor cells with endogenous HLA-E expression.

(A) Four-hour cytotoxicity of NK cells transduced with anti-NKG2A PEBL or GFP only (Control) against cell lines expressing endogenous HLA-E (see Supplemental Figure 10). EW8 and PLC/PRF/5 were transduced with luciferase. BrightGlo was added after 4 hours of coculture, and luminescence was measured using a Flx 800 plate reader. Cytotoxicity of U937 and OP-1 was measured by flow cytometry. Box (25th–75th percentile, median) and whiskers (minimum-maximum) plots from 3 experiments with cells from 3 donors (EW8, PLC/PRF/5), and 6 experiments with cells from 2 donors (U937, OP-1) in triplicate, at 2:1, 1:1, or 1:2 E/T. (B) Spheroid tumors of U2OS-mCherry were cocultured with NK cells at 1:2 E/T in triplicate and analyzed with IncuCyte Zoom System. Data are shown as mean (± SD) red calibrated unit (RCU)/μM². Representative images at end of culture are shown. Scale bars: 300 μm. (C) Four-hour cytotoxicity against cell lines exposed to IFN-γ (300 ng/ml; 12 hours). Plots are from 3 experiments with NK cells from 3 donors (EW8, PLC/PRF/5), and 6 with NK cells from 2 donors (U937) in triplicate at 2:1, 1:1, or 1:2 E/T. (D) Similar experiments targeting cells exposed for 12 hours to conditioned medium (C.M.) from 24-hour cocultures of NK cells with the respective cell lines. Four-hour cytotoxicity was compared with that against cells not exposed to conditioned medium. (E) Four-hour cytotoxicity against primary AML cells from 4 patients, exposed to IFN-γ (300 ng/ml; 12 hours). Data are from 4 experiments with NK cells from 2 donors in triplicate at 2:1 and 1:1 E/T. (F) NKG2A-negative NK cells from 3 donors were stimulated with K562-mb15-41BBL for 7 days, transduced with anti-NKG2A PEBL or GFP alone, and then exposed to IL-12 (20 ng/ml) for 5 days. Percentage of NKG2A⁺ cells at each stage is shown. (G) PEBL-transduced and control NK cells were exposed to IL-12 and tested in 4-hour cytotoxicity assays against K562-GpHLA-E cells. Data are shown as mean (± SD) of triplicate measurements at each E/T. **P < 0.01; ***P < 0.001; ****P < 0.0001, t test.