Tumor vaccines have shown promise in early clinical trials. Among them, tumor cells genetically engineered to secrete biologically active granulocyte-macrophage colony-stimulating factor (GM-CSF) can generate a systemic antitumor immune response. Although the minimal required GM-CSF dose produced by modified tumor cells to achieve a measurable antitumor effect is well known, no data examined whether an upper therapeutic limit may exist for this vaccination strategy. Because recent data demonstrate an immunosuppressive effect of GM-CSF produced by growing tumors, we thus sought to determine whether high GM-CSF doses administered in a vaccine formulation could impair antitumor immunity. Using a vaccine strategy involving a GM-CSF-producing bystander cell line (B78H1-GM) admixed with autologous tumor, we assessed the impact of varying doses of GM-CSF while maintaining a constant antigen dose. Our results defined a threshold above which a GM-CSF-based vaccine not only lost its efficacy, but more importantly for its clinical implications resulted in substantial immunosuppression in vivo. Above this threshold, GM-CSF induced Gr1؉ /CD11b ؉ myeloid suppressor cells that substantially impaired antigen-specific T-cell responses and adversely affected antitumor immune responses in vivo. The dual effects of GM-CSF are mediated by the systemic and not local concentration of this cytokine. Myeloid suppressor cell-induced immunosuppression is mediated by nitric oxide production via inducible nitric oxide synthase (iNOS) because the specific iNOS inhibitor, L-NMMA, restored antigenspecific T-cell responsiveness in vitro. Taken together, our data demonstrated the negative impact of supra-therapeutic vaccine doses of GM-CSF and underscored the importance of identifying these critical variables in an effort to increase the therapeutic efficacy of tumor vaccines.

A major limitation of adoptive immunotherapy is the availability of T cells specific for both terminally differentiated tumor cells and their clonogenic precursors. We show here that marrow-infiltrating lymphocytes (MILs) recognize myeloma cells after activation with anti-CD3/CD28 beads with higher frequency than activated peripheral blood lymphocytes from the same patients. Furthermore, activated MILs target both the terminally differentiated CD138 + plasma cells and the myeloma precursor as shown by profound inhibition in a tumor clonogenic assay. The presence of antigen in the marrow microenvironment seems to be important for the maintenance of tumor specificity. Taken together, these results highlight the intrinsic tumor specificity of MILs and describe a novel approach for the generation of tumor-specific T-cell populations suitable for adoptive immunotherapy of multiple myeloma. (Cancer Res 2005; 65(5): 2026-34)

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