Figure 1.
Bortezomib and AG 490 induce apoptosis in BC3 PEL cell line.
PEL cells were exposed to Bortezomib, AG 490 or both for the indicated times, then resuspended in Annexin V binding buffer and stained with Annexin V (a). The effect of the drugs was also evaluated on nuclear fragmentation (sub-G0/G1 phase) that represent the apoptotic cells. Cells were fixed in ethanol/water (70/30, vol/vol), washed in PBS containing RNAse and then stained with Propidium Iodide (PI) (b). The analysis was performed by flow cytometry. The reduction of the percentage of Annexin V positive (c) or the sub-G0/G1 cells (d), obtained with Z-VAD-fmk pre-treatment at 100 µM, is also shown. The experiments were performed simultaneously with 1a and 1b at 24 hours. Mean plus SD is indicated.
Figure 2.
Effect of apoptotic BC3 and BCBL1 cells on DC.
CD83 and CD86 expression was verified by FACS analysis as indicator of DC maturation induced with BC3 cells (a) and BCBL1 (b) treated with Bortezomib, AG 490 or both for 24 hours and cocultured with iDC for 12 hours. Staining with isotype control (filled histograms) is also shown. Phagocytosis of Bortezomib and AG 490 treated BC3 cells (stained with CFMDA-green) by DC (stained with anti-CD1a/PE-red) in a four hour assay. The phagocytosis of apoptotic cells is indicated by the percentage of CFMDA-PE double positive cells by flow cytometric analysis (c). Results are representative of three independent experiments.
Figure 3.
Bortezomib and AG 490 induce immunogenic BC3 cell death.
Induction of HSP90, CRT and HSP70 traslocation on BC3 cell surface induced by Bortezomib, AG 490 or both after 12 hours (a) or 24 hours (b) treatments was evaluated by flow cytometric analysis. Mean of the percentage of positive cells plus SD of three independent experiments is indicated. Plasma membrane localization of CRT after Bortezomib, AG 490 or Bortezomib plus AG 490 treatments was also analyzed by immunofluorescence on BC3 fixed with 4% paraformaldehyde in PBS for 30 min and the arrows indicate the presence of CRT on the surface of the cells (c). One representative experiment out of three is shown. The effect of Z-VAD-fmk pre-treatment (100 µM) before exposure to Bortezomib and AG 490 on HSP90, CRT and HSP70 surface expression was evaluated by flow cytometric analysis. Mean of percentage of positive cells plus SD of three independent experiments is indicated (d). Western blot analysis showing the total HSP90 and 70 expression after 24 hours treatment of BC3 with Bortezomib, AG 490 or both (e–f). Results are representative of two independent experiments.
Figure 4.
Inhibition of immunogenicity of Bortezomib and AG 490 killed BC3 PEL cells.
DC activation obtained by coculture with BC3 cells untreated or killed by Bortezomib or AG 490 (a) or with killed BC3 coated with neutralizing antibody cocktail directed against HSP90, 70 and CRT before starting the coculture with iDC (b) or iDC coated with anti-CD91 mAb before starting the coculture with killed BC3 (c). Results are representative of three independent experiments. The staining with isotype control (filled histograms) is also shown. Reduction of CD91 expression by RNAi on DC was evaluated after 72 hours by Immunoblotting (d) and by FACS analysis (black empty hystogram in comparison with gray empty hystogram) (e). The effect of CD91 knockdown on the DC activation mediated by coculture with Bortezomib-killed BC3 cells (black empty histograms) in comparison with the control DC (gray empty histograms) (f). The staining with isotype antibody only (filled histograms) is also shown. Results are representative of three independent experiments.