The Proteasome Inhibitor Bortezomib Simultaneously Enhances NK Cell Tumor Cytotoxicity While Paradoxically Reducing Antigen Specific T-Cell Tumor Cytotoxicity.

The proteasome inhibitor bortezomib was recently found to render tumor cells susceptible to natural killer (NK) cell-mediated apoptosis in vitro and in vivo. This sensitization appears to occur as a consequence of this agent up-regulating surface expression of tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) on human malignant cells rendering them susceptible to TRAIL-mediated NK cell cytotoxicity. We hypothesized that bortezomib would likewise sensitize tumors to the cytotoxic effects of antigen specific T-cells through similar apoptotic pathways, thereby providing an incentive to use bortezomib as a universal immune-sensitizing agent. The HLA-A2+, gp100+, MART-1+ melanoma cell lines 526 and 624 were treated with 10nM bortezomib for 18 hrs then were analyzed by

1. FACS for expression the cell surface markers (HLA-ABC, MIC-A/B, TRAIL-R1/2 and Fas) and

2. Cr51 cytotoxicity assay for susceptibility to CD8+/HLA-A2+ restricted gp100 and MART-1 specific CTL-mediated lysis.

As observed previously, NK cell-mediated apoptosis was significantly higher in tumor cells treated with bortezomib compared to untreated tumor cells. In contrast, an unanticipated and significant reduction in CTL-mediated cytotoxicity was observed in tumors treated with bortezomib compared to untreated tumors; at an effector:target ratio of 3:1, NK cell cytotoxicity increased from 43±2% to 70±2% (p<0.01) while gp100 CTL cytotoxicity decreased from 34±4% to 18±2% (p<0.01) in 624 melanoma cells after exposure to bortezomib (figure). This inhibition in T-cell killing was not due to changes in tumor surface expression of MHC class I, MIC-A/B, TRAIL receptors or Fas. Remarkably, CTL-mediated cytotoxicity was restored to baseline in tumor cells that were pulsed with gp100 antigen following bortezomib treatment, suggesting proteasome inhibition by bortezomib altered or impaired the processing and presentation of the gp100 tumor antigen.

Conclusions: Exposure of malignant cells to bortezomib results in simultaneous divergent effects on innate NK cell and adaptive T-cell anti-tumor immunity. While tumors exposed to bortezomib have enhanced susceptibility to NK-cell cytotoxicity, proteasome inhibition appears to disrupt antigen presentation potentially reducing tumor specific CTL effector responses. These findings suggest antigen specific T-cell responses such as graft-vs-host disease, and T-cell mediated graft-vs-tumor effects might be altered when bortezomib is administered following allogeneic hematopoietic cell transplantation.

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Figure. Melanoma cell line (624) was treated with bortezomib [10 nM] and analyzed for susceptibility to NK cell (left) and gp100-specific CD8+ CTL (middle) - mediated cytotoxicity in a 5h Cr51 cytotoxicity assay. Right - bortezomib-treated and untreated gp100:209 peptide pulsed 624 melanoma cells analyzed for susceptibility to gp100-specific CD8+ CTL-mediated cytotoxicity at a E:T ratio of 4:1

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Figure. Melanoma cell line (624) was treated with bortezomib [10 nM] and analyzed for susceptibility to NK cell (left) and gp100-specific CD8+ CTL (middle) - mediated cytotoxicity in a 5h Cr51 cytotoxicity assay. Right - bortezomib-treated and untreated gp100:209 peptide pulsed 624 melanoma cells analyzed for susceptibility to gp100-specific CD8+ CTL-mediated cytotoxicity at a E:T ratio of 4:1

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