Clofoctol promotes systemic T-cell redistribution and intratumoral cytotoxicity to improve the response to PD-1 blockade in glioblastoma

Glioblastoma (GBM) is largely refractory to immune checkpoint blockade (ICB), which may be attributable in part to its immunosuppressive microenvironment and systemic T-cell dysfunction. Drug repurposing provides an efficient strategy for identifying potential immunomodulatory agents. The antibiotic clofoctol (Clo) has shown antitumor activity; however, its effects on the immune landscape of GBM remain poorly understood. In this study, we evaluated the therapeutic efficacy and potential immunomodulatory mechanisms of Clo using an orthotopic GL261 GBM model. Flow cytometric analysis showed that Clo treatment increased the intratumoral representation of CD8 + T cells and enhanced the expression of cytotoxic effector molecules, consistent with a more immunologically active tumor microenvironment. Analysis of bone marrow cell composition further suggested that Clo partially alleviated tumor-associated T-cell accumulation in the bone marrow and promoted their systemic redistribution. Notably, depletion of CD8 + T cells markedly attenuated the antitumor efficacy of Clo, indicating that CD8 + T cells contribute substantially to its therapeutic effects. In vitro co-culture experiments further showed that Clo pretreatment enhanced the cytotoxic activity of CD8 + T cells, accompanied by increased expression of granzyme B (GZMB), perforin (PRF1), and IFN- γ . Mechanistically, ITSA-MS identified BCL9 as a candidate Clo-responsive protein, while subsequent western blot analyses showed that Clo treatment was associated with increased phosphorylation of VAV1, AKT, and ERK. These findings suggest the potential involvement of BCL9-associated signaling in Clo-mediated T-cell activation, although the precise regulatory relationship requires further investigation. Moreover, combined treatment with Clo and anti-PD-1 antibody increased intratumoral CD8 + T cell abundance and effector activity, achieved greater tumor control, and prolonged survival compared with either treatment alone. Collectively, these findings suggest that Clo may exert complementary immunomodulatory effects by facilitating the systemic redistribution of CD8 + T cells and enhancing their cytotoxic function, supporting its further investigation as a potential combination strategy for improving the response of GBM to ICB.