Figure 1
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.

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.

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