IL-12 pretreatments enhance IFN-α-induced Janus kinase-STAT signaling and potentiate the antitumor effects of IFN-α in a murine model of malignant melanoma

IFN-α 2b (IFN-α) has been used to treat patients with metastatic malignant melanoma and patients rendered disease-free via surgery but at high risk for recurrence. We hypothesized that IL-12 pretreatments would result in endogenous IFN-γ production, and that this, in turn, would up-regulate levels of Janus kinase-STAT signaling intermediates and lead to increased expression of genes regulated by IFN-α. Treatment of PBMCs with IL-12 stimulated a significant and dose-dependent production of IFN-γ. Pretreatment of PBMCs and tumor cells with IFN-γ-containing supernatants from IL-12-stimulated PBMCs led to up-regulation of STAT1, STAT2, and IFN regulatory factor 9 (IRF9) and potentiated IFN-α-induced STAT signaling within PBMCs and tumor cells. These effects were abrogated by neutralization of IFN-γ in the PBMC supernatants with an anti-IFN-γ Ab. Pretreatment of HT144 melanoma cells and PBMCs with IFN-γ or IFN-γ-containing supernatants enhanced the actions of IFN-α at the transcriptional level, as measured by real-time RT PCR analysis of the IFN-stimulated gene 15. Experiments in wild-type C57BL/6 and IFN-γ receptor knockout (B6.129S7-Ifngr^tm1Agt mice demonstrated that a regimen of IL-12 pretreatment, followed by IFN-α, could cure mice of i.p. B16F1 melanoma tumors (p < 0.007), whereas mice treated with either agent alone or PBS succumbed to fatal tumor burden. However, this treatment regimen did not significantly prolong the survival of IFN-γ-deficient (B6.129S7-Ifng ^tm1Ts) mice compared with mice treated with IFN-α alone. These results suggest that the response to IFN-α immunotherapy can be significantly enhanced by IL-12 pretreatment, and this effect is dependent upon endogenous IFN-γ production and its actions on melanoma cells.