Enhanced NK-cell cytotoxicity against bortezomib-treated JJN3 correlates with down-regulation of class I, rather than interactions between MM cells and NK-cell receptors not belonging to the KIR family. (A) After exposure to bortezomib (10 nM) for 14 hours, HLA class I was down-regulated, and DR4 and DR5 were up-regulated. (B) Increased NK-cell killing was not abolished by blocking TRAIL on NK cells. JJN3 was exposed to 10 nM bortezomib for 14 hours. ⁵¹Cr labeled JJN3 was incubated at 37°C with NK cells at different ratios. The supernatants were collected after 4 hours and analyzed by gamma count. (C) Augmented NK-cell cytotoxicity correlated with decreased class I. JJN3 exposed to 10 nM bortezomib for 12 hours and 14 hours were used as targets in a 4-hour ⁵¹Cr release assay. (D) Bortezomib did not affect expression of NKG2D ligands, such as MICA/B, ULBP-1, -2, and -3 on JJN3. (E) Blocking of NKG2D on effectors did not have a major effect on the increased NK-cell killing of bortezomib-treated JJN3. JJN3 cells were treated with 10 nM bortezomib for 20 hours. (F) There was no significant up-regulated expression of NCR ligands on JJN3 after treatment. (G) Blocking of NCRs on NK cells did not have a major effect on the enhanced NK-cell lysis of bortezomib-treated JJN3. JJN3 was exposed to 10 nM bortezomib for 20 hours. Data were reported as mean (± SD) for panels B,C,E, and G.