To develop more effective immunotherapy strategies for patients with multiple myeloma (MM), it is important to identify novel tumor antigens. Heat shock protein gp96, a highly conserved glycoprotein, is a chaperon molecule that carries intracellularly processed peptides from proteins synthesized by the cells, including those of tumor-specific or -associated proteins. Recent studies in solid tumors have shown that tumor-derived gp96 is immunogenic and potent at stimulating the generation of tumor-specific cytotoxic T lymphocytes (CTLs). In this study, we examined whether myeloma-derived gp96 can be used as potent myeloma antigen. To generate myeloma-specific CTLs, immature dendritic cells (DCs), obtained from cultures of blood monocytes from HLA-A2+ healthy individuals or patients, were pulsed with gp96 protein purified from the myeloma cell line U266 (A2+), and induced maturation using the cytokine cocktail. Autologous T cells were then stimulated every two weeks with these DCs, and cytotoxicity was examined against gp96-pusled DCs, myeloma cell lines, and primary myeloma cells isolated from patients. After several cycles of in vitro stimulation, specific CTL lines were obtained, which consisted of both CD4+ and CD8+ T cells. These CTLs demonstrated not only specific cytotoxicity against gp96-pusled DCs and the cell line U266, but also significantly killed A2+ primary myeloma cells. No killing was observed against A2+ normal lymphocytes including B cells or A2 myeloma cell lines and primary myeloma cells from patients. Using the cold target inhibition assay, we showed that the same CTLs mediated the killing of both gp96-pulsed DCs and myeloma tumor cells. The cytotoxicity was MHC class I- and, to a lesser extent, class II-restricted, indicating that the gp96 contained both class I- and II-restricted tumor-derived peptides and that myeloma cells naturally present these peptides in the context of their surface MHC molecules. Upon antigen stimulation, these CTLs secreted predominantly interferon-g, detected by the ELISPOT assay and intracellular staining, indicating that they belong to the type-1 T-cell subsets. Furthermore, the CTLs lysed the target cells mainly through the perforin-mediated pathway because Concanamycin A but not Brefeldin-A, the selective inhibitors for perforin- or Fas-, respectively, mediated pathways, abrogated the cytolytic activity of the cells. These results therefore show that myeloma-derived gp96-specific CTLs are able to lyse myeloma tumor cells including primary myeloma plasma cells from patients and, thus, provide a rationale for gp96-based immunotherapy in MM.

Present address: MD Anderson Cancer Center, Department of Lymphoma and Myeloma, 1515 Holcombe Boulevard, Unit 0903, Houston, Texas 77030.

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