MM cells exposed to bortezomib have decreased HLA class I expression on the cell surface. (A) Bortezomib induced identical down-regulation of HLA class I on MM cell lines after gating on PI negative or PI and annexin V double negative cells. MM cell lines were exposed to 10 nM bortezomib for 24 hours, and then stained with annexin V FITC/HLA-ABC allophycocyanin/PI. Class I expression on the cell surface was analyzed by flow cytometry; 8226/R5 was a bortezomib–resistant MM cell line. The data represent one of 3 individual experiments. The percentage of HLA class I decrease was determined by: 100 × (MFI of control − MFI of treated cells)/MFI of control. (B) Reduction of HLA class I expression was tested on patient MM after bortezomib treatment. The patient MM cells were treated with 10 to 50 nM bortezomib for 16 to 24 hours. UPN indicates unique patient number. (C) Viability of MM cell lines after exposure to bortezomib. MM cells were incubated with 10 nM bortezomib for 24 hours. Cells were stained with annexin V and PI and analyzed by flow cytometry. One of 3 representative experiments was shown. The percentages are shown of cells that are annexin V and PI double negative (lower left quadrant), double positive (upper right quadrant), solely annexin V positive (lower right quadrant), and solely PI positive (upper left quadrant).