Tissue-resident Limosilactobacillus reuteri modulates intratumoral arachidonic acid metabolism to enhance CD8+ T cell-mediated anti-PD1 response.

BACKGROUND: Tissue-resident bacteria have emerged as modulators of host responses to tumor immunotherapy. However, the immunological and metabolic mechanisms by which tissue-resident microbiota influence immune checkpoint blockade in colorectal cancer (CRC) remain incompletely defined. METHODS: We profiled the microbial composition of colorectal tissues from CRC patients and identified differentially enriched taxa between tumors and adjacent non-tumor tissues(NTs). By focusing on tissue-resident bacteria within the colorectal tumor microenvironment, immunocompetent mouse models were used to evaluate the impact of Limosilactobacillus reuteri (enriched in NTs) on tumor growth, survival, and anti-PD1 efficacy. Single-cell RNA sequencing combined with flow cytometric analysis was applied to characterize the phenotypic features of CD8⁺ T cells following anti-PD1 treatment with or without L.reuteri. Untargeted metabolomics combined with flow cytometry was conducted to investigate the association between tissue-resident L.reuteri and intratumoral lipid metabolic remodeling. RESULTS: L.reuteri administration significantly enhanced the efficacy of anti-PD1 therapy, reduced tumor burden (MC38: 95% CI −755.20 to −210.90 mm3, P < 0.0001; CT26: 95% CI −558.30 to −340.70 mm3, P < 0.0001), and prolonged survival (MC38: log-rank P < 0.01; CT26: log-rank P < 0.01) across multiple mouse models. Mechanistically, L.reuteri reduced CD8⁺ T-cell exhaustion and strengthened effector function. Untargeted metabolomic profiling revealed that L.reuteri remodeled intratumoral lipid metabolism, reflected by a moderate increase in arachidonic acid (AA) levels. These metabolic changes promoted CD8⁺ T-cell activation and cytotoxicity within the TME. CONCLUSION: Our study demonstrated that tissue-resident L.reuteri enhances anti-PD1 efficacy in CRC by alleviating CD8⁺ T cell exhaustion through reprogramming intratumoral lipid metabolism, highlighting the therapeutic potential of microbiota-based immunomodulation.