Interferon-alpha immunotherapy of melanoma: Signal transduction, gene transcription, and the role of suppressor of cytokine signaling proteins in immune cells

High dose interferon-alpha-2b (IFN-alpha-2b) is employed as an adjuvant therapy in melanoma patients who have undergone surgical resection of high-risk lesions. The precise molecular targets of IFN-alpha therapy are unknown, but likely involve signal transducer and activator of transcription 1 (STAT1) signal transduction within host immune effector cells. We hypothesized that STAT1-mediated signaling induces molecular targets important for mediating the anti-tumor effect of exogenously administered IFN-alpha. To identify the STAT1-dependent genes, microarray technology was utilized to characterize the gene expression profile of splenocytes from wild type (WT) and STAT1 -/- mice stimulated with IFN-alpha. Analysis showed that 25 genes required STAT1 signal transduction for optimal expression in response to IFN-alpha (i.e. Gzmb, Isg20, Ly6c; p < 0.001). Interestingly, human immune cells are also capable of inducing the homologues of these genes in response to IFN-alpha. Human PBMCs, CD3+ T cells, CD56 + natural killer (NK) cells, and CD14+ monocytes each exhibited a distinct and reproducible transcriptional profile following stimulation with IFN-alpha by microarray analysis. Analysis of gene expression within PBMCs from melanoma patients (n = 7) receiving high-dose IFN-alpha-2b (20 MU/m2 i.v.) also revealed significant upregulation of 23 genes (including IRF7, TAP1, TNFRSF6, USP18; p < 0.001). A comparison of the gene expression profile of in vitro IFN-alpha-stimulated PBMCs from normal donor cells with that of PBMCs obtained from melanoma patients receiving intravenous IFN-alpha revealed little overlap. However, within individual patients, the gene expression profile of in vitro IFN-alpha-stimulated PBMCs was largely predictive of the expression profile of gene expression following IFN-alpha administration. This indicated that an individual patient response to IFN-alpha immunotherapy could be predicted prior to treatment.; The phosphorylation of STAT1 (P-STAT1) within effector cells is highly variable among melanoma patients following IFN-alpha immunotherapy and at high doses of IFN-alpha P-STAT1 is down-regulated. We hypothesized that high doses of IFN-alpha administration would yield suboptimal levels of signal transduction and gene transcription and the induction of inhibitors by IFN-alpha may be the cause. PBMCs collected from metastatic melanoma receiving escalating doses of IFN-alpha-2b (5 MU/m2 and then 10 MU/m2) exhibited statistically equivalent levels of P-STAT1, P-STAT2 and the induction of interferon stimulated gene (ISG) transcripts. In addition, suppressors of cytokine signaling (SOCS) protein 1 and SOCS3 were induced to a greater degree with the higher dose of IFN-alpha. This suggests that a negative feedback loop is activated, thus inhibiting the effect of the higher dose. In fact, SOCS1 and SOCS3 are involved in the negative regulation of IFN-alpha activity including STAT1 activation and ISG transcription in vitro. The loss of function of SOCS1 and SOCS3 reveals that the anti-tumor effects of IFN-alpha can be enhanced in the mouse model of malignant melanoma. Impressively, IFN-alpha treatment eliminated lethal inoculums of melanoma in 70% of SOCS1-deficient mice, whereas all IFN-treated SOCS1-competent mice died. The anti-tumor effects of IFN-alpha in tumor-bearing SOCS1-deficient mice were markedly inhibited following depletion of CD8+ T cells. These results indicate that the anti-tumor response of immune effector cells to exogenous IFN-alpha is regulated by SOCS proteins. Additionally, we show that SOCS1 was an important regulator of immunosurveillance of developing cancer as SOCS1-deficient mice were protected from tumor formation.; These reports are the first to characterize the dependence of STAT1 in mediating the transcriptional response of immune cells to IFN-alpha. In addition, the studies remain the first to define the transcriptional response of immune subsets to IFN-alpha and to characterize t...