Cooperative IFN-γ and TLR9 signaling reprogram tumor-associated macrophages to enhance antitumor immunity in melanoma.

Tumor-associated macrophages (TAMs) often adopt immunosuppressive phenotypes that promote tumor progression and limit the efficacy of immunotherapy. Although Toll-like receptor 9 (TLR9) agonists and interferon-γ (IFN-γ) possess immunostimulatory properties, their antitumor efficacy as monotherapy remains limited, particularly in poorly immunogenic tumors. Here, we investigated whether coordinated activation of innate immune pathways could reprogram macrophages and enhance antitumor immunity in melanoma. Using genome-wide cap analysis of gene expression (CAGE), we found that combined CpG DNA and IFN-γ stimulation induced extensive transcriptional reprogramming in macrophages, characterized by activation of M1-associated genes, enhancer remodeling, alternative transcription start site usage, and induction of transcription factors associated with inflammatory and cytotoxic responses. Functionally, this combination markedly enhanced macrophage-mediated phagocytosis of live tumor cells and induced sustained antitumor activity that persisted for at least 10 days after stimulation. In the poorly immunogenic B16F10 melanoma model, intratumoral administration of CpG DNA in combination with IFN-γ significantly suppressed tumor growth and prolonged survival compared with single-agent treatment. This antitumor effect was associated with increased infiltration and activation of macrophages and CD8+ T cells, without a corresponding expansion of regulatory T cells. Overall, these findings suggest that coordinated IFN-γ and TLR9 signaling promotes TAM reprogramming and is associated with enhanced antitumor immunity and the conversion of an immunologically cold tumor microenvironment into a more immune-responsive tumor microenvironment, highlighting the potential of an innate immune-targeted therapeutic strategy.