Abstract
Immune checkpoint inhibitors (ICIs), particularly those targeting programmed cell death protein 1 (PD-1), have revolutionized oncology; however, variable clinical responsiveness underscores the necessity of identifying host-extrinsic modulatory factors. The gut microbiome has emerged as an essential orchestrator of systemic antitumor immunity, yet the exact chemical mediators translating microbial signals into durable immunological responses remain incompletely defined. In this study, we investigated the biochemical and immunological mechanisms through which gut microbiome-derived metabolites modulate anti-PD-1 efficacy using targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) and syngeneic murine melanoma models. Longitudinal profiling of fecal and serum metabolomes revealed marked elevations of short-chain fatty acids (SCFAs), notably butyrate, and tryptophan catabolites, specifically indole-3-propionic acid (IPA), in anti-PD-1-responsive mice compared to non-responders. Antibiotic-induced depletion of commensal microbiota ablated therapeutic responsiveness, which was substantially restored by oral administration of exogenous IPA or sodium butyrate. Mechanistic immunophenotyping demonstrated that IPA supplementation promoted the expansion of tumor-infiltrating CD8+ effector T cells, upregulating interferon-gamma (IFN-γ) and granzyme B expression while attenuating terminal exhaustion via aryl hydrocarbon receptor (AhR) activation. Similarly, butyrate enhanced CD8+ T-cell metabolic fitness and cellular cytotoxicity through histone deacetylase inhibition. These findings demonstrate that microbial small molecules act as distal biochemical switches that optimize immune checkpoint blockade, providing a rationale for metabolite-guided adjuvant strategies in clinical immunotherapy.
