ObjectiveOur goals were to evaluate the antitumour efficacy of Lactobacillus rhamnosus GG (LGG) in combination with immune checkpoint blockade (ICB) immunotherapies on tumour growth and to investigate the underlying mechanisms.DesignWe used murine models of colorectal cancer and melanoma to evaluate whether oral administration of LGG improves the efficacy of ICB therapies. We performed the whole genome shotgun metagenome sequencing of intestinal contents and RNA sequencing of dendritic cells (DCs). In a series of in vitro and in vivo experiments, we further defined the immunological and molecular mechanisms of LGG-mediated antitumour immunity.ResultsWe demonstrate that oral administration of live LGG augmented the antitumour activity of anti-programmed cell death 1 (PD-1) immunotherapy by increasing tumour-infiltrating DCs and T cells. Moreover, the combination treatment shifted the gut microbial community towards enrichment in Lactobacillus murinus and Bacteroides uniformis, that are known to increase DC activation and CD8+tumour recruitment. Mechanistically, treatment with live LGG alone or in combination with anti-PD-1 antibody triggered type I interferon (IFN) production in DCs, enhancing the cross-priming of antitumour CD8+ T cells. In DCs, cyclic GMP-AMP synthase (cGAS)/stimulator of IFN genes (STING) was required for IFN-β induction in response to LGG, as evidenced by the significant decrease in IFN-β levels in cGAS or STING-deficient DCs. LGG induces IFN-β production via the cGAS/STING/TANK binding kinase 1/interferon regulatory factor 7 axis in DCs.ConclusionOur findings have offered valuable insight into the molecular mechanisms of live LGG-mediated antitumour immunity and establish an empirical basis for developing oral administration of live LGG as a combination agent with ICB for cancer therapies.

Hypoxia inducible factor-1α (HIF-1α) up-regulates the expression of programmed death ligand-1 (PD-L1) in some extracranial malignancies. However, whether it could increase PD-L1 expression in intracranial tumor is still unknown. Here, we explored the relationship between HIF-1α and PD-L1 expression in glioma, and investigated their clinical significance. In glioma patients, HIF-1α and PD-L1 were overexpressed in high grade glioma tissues and were significantly associated with poor survival. In glioma cells, PD-L1 expression was induced under hypoxia condition, and the enhanced PD-L1 expression was abrogated by either HIF-1α knock-down or HIF-1α inhibitor treatment. Furthermore, ChIP-qPCR analysis showed the direct binding of HIF-1α to PD-L1 proximal promoter region, providing evidence that HIF-1α up-regulates PD-L1 in glioma. In glioma murine model, the combination treatment with HIF-1α inhibitor and anti-PD-L1 antibody caused a more pronounced suppressive effect on tumor growth compared to either monotherapy. Immunologically, the combination treatment improved both dendritic cell (DC) and CD8+ T cell activation. Overall, our results demonstrated that positive correlation between PD-L1 and HIF-1α in glioma, and provide an alternative strategy, inhibiting HIF-1α, as combination therapies with immunotherapies to advance glioma treatment.

The antitumor effects of ionizing radiation (IR) are mediated in part through activation of innate and adaptive immunity. Here we report that gut microbiota influences tumor control following IR. Vancomycin decreased the abundance of butyrate-producing gut bacteria and enhanced antitumor responses to IR. Oral administration of Lachnospiraceae, a family of vancomycin-sensitive bacteria, was associated with increased systemic and intratumoral butyric acid levels and impaired the efficacy of IR in germ-free (GF) mice. Local butyrate inhibited STING-activated type I IFN expression in dendritic cells (DCs) through blockade of TBK1 and IRF3 phosphorylation, which abrogated IR-induced tumor-specific cytotoxic T cell immune responses without directly protecting tumor cells from radiation. Our findings demonstrate that the selective targeting of butyrate-producing microbiota may provide a novel therapeutic option to enhance tumor radiation sensitivity.

Tumor-induced CD45−Ter119+CD71+ erythroid progenitor cells, termed “Ter cells,” promote tumor progression by secreting artemin (ARTN), a neurotrophic peptide that activates REarranged during Transfection (RET) signaling. We demonstrate that both local tumor ionizing radiation (IR) and anti–programmed death ligand 1 (PD-L1) treatment decreased tumor-induced Ter cell abundance in the mouse spleen and ARTN secretion outside the irradiation field in an interferon- and CD8+ T cell–dependent manner. Recombinant erythropoietin promoted resistance to radiotherapy or anti–PD-L1 therapies by restoring Ter cell numbers and serum ARTN concentration. Blockade of ARTN or potential ARTN signaling partners, or depletion of Ter cells augmented the antitumor effects of both IR and anti–PD-L1 therapies in mice. Analysis of samples from patients who received radioimmunotherapy demonstrated that IR-mediated reduction of Ter cells, ARTN, and GFRα3, an ARTN signaling partner, were each associated with tumor regression. Patients with melanoma who received immunotherapy exhibited favorable outcomes associated with decreased expression of GFRα3. These findings demonstrate an out-of-field, or “abscopal,” effect mediated by adaptive immunity, which is induced during local tumor irradiation. This effect, in turn, governs the therapeutic effects of radiation and immunotherapy. Therefore, our results identify multiple targets to potentially improve outcomes after radiotherapy and immunotherapy.

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