LGALS9 blockade augments vaccine-induced immune responses against prostate cancer

Background: Prostate-specific membrane antigen (PSMA)-based vaccination represents a promising immunotherapeutic strategy for prostate cancer; however, its efficacy remains constrained by tumor-induced immune evasion and insufficient activation of antigen-presenting cells. Galectin-9 (LGALS9), an immunoregulatory lectin that contributes to immune suppression, is therefore an attractive target for overcoming tumor-induced immune tolerance. Methods: Recombinant adenoviral vaccines encoding PSMA and LGALS9 (Ad-PSMA and Ad-LGALS9) were generated and validated for antigen expression in vitro and in vivo. Therapeutic efficacy was evaluated in murine subcutaneous, bone metastatic, and humanized prostate cancer models. Vaccine-induced immune responses were characterized by flow cytometry, ELISA, enzyme-linked immunospot (ELISpot), immunohistochemistry, EdU proliferation assays, cytotoxic T lymphocyte assays, and cell depletion experiments. Results: Pre-immunization with Ad-LGALS9 significantly potentiated the therapeutic efficacy of the Ad-PSMA vaccine in subcutaneous, bone metastatic, and humanized prostate cancer models, resulting in pronounced tumor growth inhibition and prolonged survival. Mechanistically, LGALS9 targeting enhanced dendritic cell (DC) activation and maturation, upregulating CD80, CD86, major histocompatibility complex-II, and CD40 expression and promoting efficient antigen cross-presentation. This facilitated robust priming and expansion of multifunctional CD8+ T cells producing interferon-γ, interleukin-2, and tumor necrosis factor-α, which mediated potent cytotoxicity against PSMA-expressing tumor cells. Furthermore, LGALS9 immunization induced high-titer neutralizing antibodies that disrupted the LGALS9/TIM-3 inhibitory axis, alleviating T-cell exhaustion. Combined Ad-LGALS9/PSMA vaccination established durable memory CD8+ T-cell responses that conferred protection against tumor rechallenge. Conclusions: Targeting LGALS9 enhances DC-mediated CD8+ T-cell immunity and synergistically augments the therapeutic efficacy of tumor vaccines, representing a promising immunotherapeutic strategy for prostate cancer.