Bortezomib down-regulates the cell-surface expression of HLA class I and enhances natural killer cell-mediated lysis of myeloma

Abstract

Human leukocyte antigen class I molecules expressed by tumor cells play a central role in the regulation of natural killer (NK) cell-mediated immune responses. The proteasome inhibitor bortezomib has demonstrated significant activity in multiple myeloma (MM). We hypothesized that treatment of MM with bortezomib results in the reduction of cell-surface expression of class I and thereby sensitizes MM to NK cell-mediated lysis. Here we report that bortezomib down-regulates class I in a time- and dose-dependent fashion on all MM cell lines and patient MM cells tested. Downregulation of class I can also be induced in vivo after a single dose of 1.0 mg/m(2) bortezomib. Bortezomib significantly enhances the sensitivity of patient myeloma to allogeneic and autologous NK cell-mediated lysis. Further, the level of decrease in class I expression correlates with increased susceptibility to lysis by NK cells. Clinically relevant bortezomib concentrations do not affect NK-cell function. Our findings have clear therapeutic implications for MM and other NK cell-sensitive malignancies in the context of both allogeneic and autologous adoptively transferred NK cells.

Figure 1

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).

Figure 2

Bortezomib reduces HLA class I expression on JJN3 and primary MM in a dose- and time-dependent fashion. MM cells were incubated with various doses of bortezomib for indicated times. PI-negative cells were gated and analyzed for class I expression. (A) Dose response: JJN3 was exposed to increasing doses of bortezomib for 24 hours. (B) Time course: JJN3 cells were incubated at 10 nM bortezomib for increasing durations of time. (C) Dose response: Patient MM was exposed to increasing doses of bortezomib for 24 hours. (D) Time course: Patient MM was incubated at 10 nM bortezomib for increasing durations of time. Class I expression was also tested on JJN3 after bortezomib by spinning-disc confocal microscopy. (E) Dose response: JJN3 was exposed to increasing doses of bortezomib for 24 hours. (F) Time course: JJN3 cells were incubated at 10 nM bortezomib for increasing durations of time. Images were acquired using a Zeiss Axiovert 200M microscope (Carl Zeiss) fitted with a BD Bioimaging CARV II spinning-disc confocal accessory. Mid-cell confocal images were presented (original magnification, ×250). The fluorescence intensity was quantified using IPLab version 3.9.5 software and corrected by substracting the blank field intensity. A total of 30 cells per group from 3 independent experiments were considered and the mean intensity (± SD) was plotted. (G) The kinetics of apoptosis on JJN3 after drug treatment. JJN3 was treated with 10 nM bortezomib for indicated times. Cells were stained with annexin V and PI and analyzed by FACS. The data represent 1 of 3 individual experiments. 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).

Figure 3

Provision of exogenous HLA-C binding peptides rescues expression of HLA-C during bortezomib treatment and also prevents the bortezomib–mediated increase in NK cell–mediated lysis. (A) JJN3 cells were cocultured in the presence or absence of the HLA-C binding peptides during 16 hours treatment with bortezomib. Human β₂M was added in the medium. Cells were gated on PI–negative cell population and analyzed by flow cytometry for HLA-C expression. The percentage HLA-C expression was shown. Data were reported as means (± SD) of 3 individual experiments. (B) JJN3 cells were treated with 50 nM bortezomib for 18 hours in the presence or absence of the HLA-C binding peptides or irrelevant peptides. Treated and untreated cells were then used as targets in a standard 4-hour ⁵¹Cr release assay. K562 was used as a positive control for maximum lysis. Data were reported as means (± SD).

Figure 4

Bortezomib down-regulates HLA class I in vivo on patient MM cells. Comparison of surface expression of HLA class I on the pretreatment and posttreatment of primary MM cells from 6 patients. The paired samples were cryopreserved, simultaneously thawed, and analyzed in the same experiment. PI-negative cells were analyzed for HLA class I expression. Open curves were before bortezomib. The black curves were 48 hours after a single dose of bortezomib 1.0 mg/m². The percentage of class I expression decrease was determined by 100 × (MFI of pretreatment sample − MFI of posttreatment sample)/MFI of pretreatment sample. Statistically significant difference of class I expression between the pre- and post- bortezomib treatment samples was present (P = .002, Student t test). The viability of pretherapy (▭) and after therapy () samples after thawing was similar.

Figure 5

Bortezomib treatment enhances the sensitivity of patient MM to allogeneic and autologous NK cell–mediated lysis. The patient MM cells were treated with 10 to 50 nM bortezomib for 16 to 24 hours. Treated and untreated cells were then used as targets in a standard 4-hour ⁵¹Cr release assay. UPN indicates unique patient number.

Figure 6

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).

Figure 7

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.