Bortezomib enhances dendritic cell (DC)-mediated induction of immunity to human myeloma via exposure of cell surface heat shock protein 90 on dying tumor cells: therapeutic implications

Abstract

Most anticancer chemotherapies are immunosuppressive and induce nonimmunogenic tumor cell death. Bortezomib, a specific inhibitor of 26S proteasome, has shown clinical activity in several human tumors, including myeloma. Here we show that the uptake of human myeloma cells by dendritic cells (DCs) after tumor cell death by bortezomib, but not gamma irradiation or steroids, leads to the induction of antitumor immunity, including against primary tumor cells, without the need for any additional adjuvants. The delivery of activating signal from bortezomib-killed tumor cells to DCs depends on cell-cell contact between DCs and dying tumor cells and is mediated by bortezomib-induced exposure of heat shock protein 90 (hsp90) on the surface of dying cells. The combination of bortezomib and geldanamycin (an hsp90 inhibitor) leads to greater apoptosis of tumor cells but abrogates their immunogenicity. These data identify drug-induced exposure of endogenous heat shock proteins on the surface of dying cells as a mechanism of immunogenic death of human tumors. Specific targeting of bortezomib to tumors may enhance their immunogenicity and the induction of antitumor immunity.

Figure 1

Bortezomib induces immunogenic cell death in human myeloma. (A) Expansion of myeloma-reactive T cells by DCs loaded with U266 tumor cells killed by bortezomib or γ irradiation. Monocyte-derived DCs alone or loaded with U266 tumor cells were matured using LPS as a maturation stimulus. The tumor-loaded and unpulsed DCs were each used to stimulate autologous T cells for 2 weeks. IFN-γ producers against unpulsed DCs, U266, or control cag cells as control were analyzed by ELISPOT assay. Data shown are mean/SD of 1 representative experiment of 3. (B) Same set up as in panel A. In addition to using DCs loaded with killed tumor cells, DCs also were pulsed with U266 cell killed by bortezomib and γ irradiation and opsonized with anti-CD138 monoclonal antibody. IFN-γ producers against U266 cells were analyzed by an overnight ELISPOT assay. Data shown are summary of 3 independent experiments with different blood donors. (C) Immature monocyte-derived DCs from HLA-A2⁺ donors were loaded with U266 myeloma cells coated with isotype control or anti-CD138 antibody. DCs were then used to stimulate autologous T cells. On day 7, T cells were restimulated with same DCs. After 14 days of culture, T cells were stimulated overnight in ELISPOT plates with autologous DCs pulsed with 10 μM HLA-A2 restricted peptides derived from MAGE-A3, NY-ESO-1, or 2.5 μM of an overlapping 15-mer peptide library derived from survivin. IFN-γ producers were quantified by an ELISPOT assay. Data shown are mean/SD of 3 independent experiments on 3 blood donors. *P value for comparison with γ-irradiated tumor cell–loaded DCs, P < .05. (D) Cytotoxic activity of myeloma-reactive T cells expanded by DCs loaded with U266 tumor cells killed by bortezomib, dexamethasone, or γ irradiation. CFSE-labeled target cells were incubated with T cells at the ratio of 1:20 for 6 hours and percentage of dead cells determined by TO-PRO3 staining. Data shown are representative of 3 experiments. (E) Kinetics of apoptosis after bortezomib or dexamethasone treatment. Either annexin V–positive or annexin V/TO-PRO3 double-positive cells was considered to be apoptotic. Live cells are defined as annexin V–negativeTO-PRO3 negative. Data are representative of 3 separate experiments. (F) Proportion of early (annexin V positive/TO-PRO3 negative) or late (annexin V/TO-PRO3 double-positive) tumor cells in the course of apoptosis induced by bortezomib or dexamethasone. Representative result of 3 experiments. (G) Representative FACS plot showing the stage of apoptosis of tumor cells used for pulsing of DCs, 24 hours after the induction of apoptosis.

Figure 2

Enhanced immunogenicity of bortezomib-killed myeloma cells in patients with multiple myeloma. (A) DCs from patients with multiple myeloma (MM) were pulsed with autologous CD138-positive tumor cells killed by γ irradiation, dexamethasone, or bortezomib, matured with LPS, and used for the stimulation of autologous T cells for 2 weeks. IFN-γ producers against unpulsed DCs or DCs pulsed with autologous tumor cells were analyzed by ELISPOT assay. Data shown mean/SD of representative patient of 3 patients tested. *P < .05. (B) Intracellular IFNγ production by autologous tumor cell–specific CD4 and CD8 T cells. T cells were expanded as in panel A, and IFNγ production after stimulation with autologous DCs loaded with killed tumor cells was analyzed by flow cytometry.

Figure 3

Mechanism of bortezomib-induced immunogenic cell death: role of cell surface hsp90 on the immunogenicity of bortezomib-induced cell death. (A) Phenotype of dendritic cells after interaction with bortezomib-killed U266 cells. Day 5 immature DCs were cultured for 24 hours with U266 myeloma cells killed by γ irradiation, dexamethasone, or bortezomib. Second set of cocultures was set up in transwells to prevent DCs/tumor cells contact. After 24 hours, expression of CD80 and CD86 on DCs was analyzed by flow cytometry. Mean fluorescence intensity is shown. *P value for comparison with γ-irradiated tumor cell–loaded DCs, P < .05. (B) Induction of inducible HSP70 (iHPS70) and HSP90 expression on U266 treated with bortezomib. Apoptosis of U266 myeloma cells was induced by γ irradiation or bortezomib and surface expression of HSP70 and HSP90 analyzed 24 hours later by flow cytometry. Preincubation of tumor cells with geldanamycin 1 hour prior to the addition of bortezomib inhibits the increase in HSP90 expression. (C) HSP90 expression 24 hours after bortezomib treatment on myeloma (U266 and cag), mantle cell lymphoma (NCEB1), and breast cancer (MCF-7) cell lines. (D) Kinetics of HSP90 expression on U266 cells after dexamethasone or bortezomib treatment analyzed by flow cytometry. (E) Phenotype of dendritic cells after interaction with dexamethasone or bortezomib-killed U266 cells at different stages of apoptosis. U266 cells were killed by bortezomib or dexamethasone, and cocultures with day 5 immature DCs were set up at 6, 12, and 24 hours after induction of apoptosis. After 24 hours, expression of CD86 on DCs was analyzed by flow cytometry. Mean fluorescence intensity is shown. *P value for comparison with immature DCs, P < .05. (F) Kinetics of U266 myeloma cell apoptosis after γ irradiation, bortezomib treatment, or combined bortezomib + geldanamycin (GDC) treatment. Percentage of live cells was followed for 24 hours by flow cytometry. Live cells were defined as annexin V negative/To-Pro3 negative. (G) Inhibition of increase in CD86 expression on DCs after interaction with bortezomib-killed myeloma cells by GDC or anti-HSP90 antibody. Immature DCs were cultured for 24 hours with U266 myeloma cells killed by bortezomib and expression of CD86 analyzed by flow cytometry. Preincubation of tumor cells with geldanamycin or anti-HSP90 mAb prior to the induction of apoptosis by bortezomib abrogates increase in CD86 expression. Staining with isotype control (gray line) and anti-HPS90 mAb (black line) is shown. Error bars represent mean ± SD.

Figure 4

Geldanamycin abrogates increased immunogenicity of bortezomib-killed myeloma cells. (A) DCs were pulsed with U266 myeloma cells killed by γ irradiation, bortezomib alone, or with myeloma cells preincubated with geldamamycin for 1 hour before treatment by bortezomib. Tumor-loaded DCs with or without additional maturation stimulus with LPS were then used as stimulators of autologous T cells for 2 weeks. On day 7, T cells were restimulated with fresh tumor cell–pulsed DCs. On day 14, IFNγ production by tumor-reactive T cells in response to U266 myeloma cells was quantified by an overnight ELISPOT. Response to an HLA-A2–negative cell line cag or unpulsed autologous DCs was used as a control. Data shown are mean/SD of 3 independent experiments on 3 blood donors. (B) DCs were fed with apoptotic U266 tumor cells killed by γ irradiation or bortezomib, with or without 1 hour preincubation of U266 cells with geldanamycin. Pulsed DCs then were activated with LPS or left untreated. DCs then were used to stimulate T cells as in panel 2A. On day 14 of culture, IFNγ production in response to U266 tumor cells was monitored by intracellular cytokine flow cytometry. Data are representative of similar experiments on 3 donors. Response to HLA-A2 negative cag cells was used as a negative control and did not exceed 0.1% (not shown).

Figure 5

Induction of antitumor immunity by bortezomib-killed primary myeloma cells without the need for exogenous DC maturation stimulus. Monocyte-derived DCs from patients with MM were pulsed with autologous CD138-positive tumor cells killed by γ irradiation, dexamethasone, or bortezomib in the absence of any DC maturation stimuli and used for the stimulation of autologous T cells for 2 weeks. IFN-γ producers against unpulsed DCs or DCs pulsed with autologous tumor cells were analyzed by an ELISPOT assay. Data shown are representative (mean/SD) of 3 tested patients.