Reduced HLA class I on the myeloma cell surface results in NK cell–mediated recognition and lysis. (A) Cells were incubated with increasing concentrations of HLA-ABC blocking or isotype control antibody. PI-negative cells were gated for analysis of HLA class I expression. The percentages indicate the amount of HLA class I blocked. (B) A chromium release cytotoxicity assay demonstrated increased recognition of targets with HLA blocked. Data were reported as mean (± SD). (C) Bortezomib treatment (10 nM for 24 hours) induced a 60% decrease in cell-surface HLA class I. PI–negative cells were analyzed for HLA class I expression. The percentage of HLA class I reduction was determined by: 100 × (MFI of control − MFI of HLA-blocked or bortezomib-treated cells)/MFI of control. (D) Class I reduction by bortezomib treatment resulted in increasing killing of JJN3. Interestingly, the level of lysis was comparable between the untreated targets with 65% blocking of HLA class I and the bortezomib-treated targets, which had a 60% decrease in cell-surface HLA class I. The HLA class I devoid line K562 and bortezomib-treated JJN3 cells with 100% blocking of HLA class I were used as positive controls for maximum lysis. The “0% blocked” targets were incubated with an isotype control antibody. Panels B and D pertain to the same experiment. Data were reported as mean (± SD).