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IL-27 exerts antitumor activity in murine orthotopic neuroblastoma, but only partial antitumor effect in disseminated disease. This study demonstrates that combined treatment with IL-2 and IL-27 induces potent antitumor activity in disseminated neuroblastoma metastasis. Complete durable tumor regression was achieved in 90% of mice bearing metastatic TBJ-IL-27 tumors treated with IL-2 compared with only 40% of mice bearing TBJ-IL-27 tumors alone and 0% of mice bearing TBJ-FLAG tumors with or without IL-2 treatment. Comparable antitumor effects were achieved by IL-27 protein produced upon hydrodynamic IL-27 plasmid DNA delivery when combined with IL-2. Although delivery of IL-27 alone, or in combination with IL-2, mediated pronounced regression of neuroblastoma metastases in the liver, combined delivery of IL-27 and IL-2 was far more effective than IL-27 alone against bone marrow metastases. Combined exposure to IL-27 produced by tumor and IL-2 synergistically enhances the generation of tumor-specific CTL reactivity. Potentiation of CTL reactivity by IL-27 occurs via mechanisms that appear to be engaged during both the initial sensitization and effector phase. Potent immunologic memory responses are generated in mice cured of their disseminated disease by combined delivery of IL-27 and IL-2, and depletion of CD8+ ablates the antitumor efficacy of this combination. Moreover, IL-27 delivery can inhibit the expansion of CD4+CD25+Foxp3+ regulatory and IL-17-expressing CD4+ cells that are otherwise observed among tumor-infiltrating lymphocytes from mice treated with IL-2. These studies demonstrate that IL-27 and IL-2 synergistically induce complete tumor regression and long-term survival in mice bearing widely metastatic neuroblastoma tumors.

The role of Fas in the regulation of solid tumor growth was investigated. Murine renal carcinoma (Renca) cells were constitutively resistant to Fas-mediated killing in vitro, but exhibited increased expression of Fas and sensitivity to Fas-mediated killing after exposure to IFN-γ and TNF. Transfected Renca cells overexpressing Fas were efficiently killed in vitro upon exposure to anti-Fas Ab (Jo2). When Fas-overexpressing Renca cells were injected into syngenic BALB/c mice, there was a consistent and significant delay in tumor progression, reduced metastasis, and prolonged survival that was not observed for Renca cells that overexpressed a truncated nonfunctional Fas receptor. The delay of in vivo tumor growth induced by Fas overexpression was not observed in IFN-γ−/− mice, indicating that IFN-γ is required for the delay of in vivo tumor growth. However, there was a significant increase of infiltrated T cells and in vivo apoptosis in Fas-overexpressing Renca tumors, and Fas-overexpressing Renca cells were also efficiently killed in vitro by T cells. In addition, a strong therapeutic effect was observed on Fas-overexpressing tumor cells by in vivo administration of anti-Fas Ab, confirming that overexpressed Fas provides a functional target in vivo for Fas-specific ligands. Therefore, our findings demonstrate that Fas overexpression on solid tumor cells can delay tumor growth and provides a rationale for therapeutic manipulation of Fas expression as a means of inducing tumor regression in vivo.

Mice bearing the experimental murine renal cancer Renca can be successfully treated with some forms of immunotherapy. In the present study, we have investigated the molecular pathways used by NK and T cells to lyse Renca cells. Renca cells normally express low levels of Fas that can be substantially enhanced by either IFN-γ or TNF-α, and the combination of IFN-γ + TNF-α synergistically enhances cell-surface Fas expression. In addition, cells pretreated with IFN-γ and TNF-α are sensitive to lysis mediated by Fas ligand (FasL)-expressing hybridomas (dllS), cross-linking of anti-Fas Abs or soluble Fas (FasL). Lysis via Fas occurs by apoptosis, since Renca shows all the typical characteristics of apoptosis. No changes in levels of bcl-2 were observed after cytokine treatments. We also examined cell-mediated cytotoxic effects using activated NK cells and T cells from gld FasL-deficient mice, and perforin-deficient mice, as well as wild-type C57BL/6 and BALB/c mice. Interestingly, the granule-mediated pathway predominated in killing of Renca by activated NK cells, while the Fas/FasL pathway contributed significantly to cell-mediated killing of Renca by activated T cells. These results suggest that killing of Renca tumor cells by immune effector cells can occur by both granule and Fas-mediated cytotoxicity. However, for the Fas-mediated pathway to function, cell surface levels of Fas need to be increased beyond a critical threshold level by proinflammatory cytokines such as IFN-γ and TNF-α.

The resistance of tumors to cell death poses a major clinical problem. Bortezomib (Velcade/PS-341), a specific and reversible proteasome inhibitor, sensitized renal (Renca) and mammary (4T1) mouse carcinomas to apoptosis induced by the TNF family ligand TRAIL or its receptor (DR5) agonist antibody. The molecular basis of the tumoritoxicity of the combination of bortezomib and TRAIL seemingly involved amplification of the extrinsic cell death pathway as increased expression of DR5, activation of caspase-8 and -3, and decreased apoptosis inhibitory proteins cFLIP, cIAP-1 and XIAP were observed in bortezomib-treated tumors. In an established tumor setting, anti-DR5 administration to mice pre-treated with bortezomib not only reduced lung metastases of Renca and 4T1 tumors but also significantly increased long-term survival in mice bearing Renca as compared to either agent alone. Encouragingly, no evidence of any overt tissue damage was noted in the treated mice. This provides the first experimental evidence that proteasome inhibition can be combined with DR5 agonist antibody for successful cancer therapy. Moreover, bortezomib administration 24 hours prior to adoptive transfer of T cells did not block their anti-tumor effector function. This suggests that tumor sensitization with bortezomib may also be combined with tumor-specific T cell immunotherapy of cancer. Funded in part by NCI contract N01-CO-12400.