Tehran University of Medical Sciences Iranian Journal of Allergy, Asthma and Immunology 1735-1502 0 0 2026 04 16 1 11 Yan Kang Department of Oncology, Liangxiang Hospital of Beijing Fangshan District, Capital Medical University, Fangshan, Beijing, China Baocen Xu Department of Thoracic Surgery, Xing’an League People’s Hospital, Ulanhot, Xing’an League, Inner Mongolia Autonomous Region, China Siyu Wang Department of Oncology, Liangxiang Hospital of Beijing Fangshan District, Capital Medical University, Fangshan, Beijing, China Shaozhi Xi Department of Comprehensive Surgery, The Second Medical Center & National Clinical Research Center for Geriatric Diseases, Chinese PLA General Hospital, Beijing, China 2025 10 16 2025 12 15 Programmed cell death protein-1/programmed cell death ligand-1 (PD-1/PD-L1) plays a pivotal role in tumor immune evasion. The efficacy of these treatments is limited by variable patient responses and adverse effects. It is necessary for a deeper understanding of the underlying biological mechanisms. This study used a 2-step Mendelian randomization (MR) approach to investigate causal relationships among gut microbiota, lipid and amino acid metabolic traits, and PD-1/PD-L1. The summary statistics for 412 traits of the gut microbiome (N=7738), 249 traits of serum metabolites (N=115 078), and 2 traits of PD-1/PD-L1 (N=3301) were derived from publicly genome-wide association studies. The primary method employed for MR was inverse-variance weighted regression. We conducted a series of sensitivity analyses to evaluate the reliability of the causal estimates. Subsequently, mediation analysis was undertaken to elucidate the pathway from gut microbiome to PD-L1, mediated by serum metabolic markers. Our analyses identified 28 gut microbial traits significantly affecting PD-L1 and 14 affecting PD-1, 8 of which remained consistently linked to PD-L1 after sensitivity analysis. Furthermore, 13 serum lipid and amino acid metabolic traits exhibited significant causal effects on PD-L1, with 6 remaining robust post analysis. Notably, Bacteroides dorei demonstrated a causal effect on PD-L1, mediated 9.6% by the metabolic biomarker phenylalanine. These findings highlight the intricate interplay among gut microbiome, metabolic biomarkers, and immune regulation. They suggest novel therapeutic targets for cancer treatment that emphasize the value of microbiome and metabolic biomarkers in improving immunotherapy outcomes and promoting personalized medicine. https://ijaai.tums.ac.ir/index.php/ijaai/article/view/4629 https://ijaai.tums.ac.ir/index.php/ijaai/article/download/4629/2322