Hypothesis\nUrolithin A (UA) enhances vaccine‑induced immunity in older adults by promoting a SIRT3‑dependent short form (SL‑SIRT3) that deacetylates LC3B at mitochondrial‑associated membranes (MAMs), thereby coupling mitophagy to improved antigen processing and CD8⁺ T‑cell memory formation.\n\n## Mechanistic Rationale\nUA‑induced SL‑SIRT3 deacetylates SOD2, reducing mitoROS as shown in hypertensive rats【https://pmc.ncbi.nlm.nih.gov/articles/PMC12885030/】. Recent work shows SIRT3 also resides at MAMs where it can deacetylate LC3B, a key autophagy protein required for phagophore elongation【https://doi.org/10.1016/j.molmed.2017.08.001】. Deacetylated LC3B exhibits higher affinity for phosphatidylethanolamine, accelerating mitophagosome formation and facilitating mitochondrial‑ER calcium flux that optimizes MHC‑I loading of viral peptides. Consequently, naïve‑like CD8⁺ T cells expanded by UA【https://www.news-medical.net/news/20251103/Urolithin-A-recharges-aging-immune-cells-and-boosts-mitochondrial-fitness-in-midlife-adults.aspx】 acquire a stem‑like memory phenotype (TCF7⁺/LEF1⁺) with superior proliferative capacity upon antigen re‑encounter.\n\n## Testable Predictions\n1. In human peripheral blood mononuclear cells (PBMCs) from UA‑supplemented older adults, SL‑SIRT3 levels will rise concomitantly with decreased LC3B acetylation (measured by immunoprecipitation‑Western blot).\n2. The magnitude of LC3B deacetylation will correlate with increased mitochondrial‑ER contacts (visualized by EM or split‑GFP MAM sensors) and with higher ex vivo OVA‑peptide MHC‑I complex stability.\n3. Individuals classified as high UA producers (based on fecal urolithin phenotyping) will exhibit greater influenza‑vaccine‑induced hemagglutination inhibition titers and a larger pool of CD8⁺ TCF7⁺/LEF1⁺ cells than low producers, independent of baseline microbiota diversity.\n\n## Experimental Design\nA double‑blind, placebo‑controlled trial (n=120, 65‑80 y) will receive 1 g UA or maltodextrin daily for 28 days before seasonal influenza vaccination. PBMCs will be collected at baseline, day 28 (pre‑vaccine), and day 56 (post‑vaccine). Outcomes:\n- SL‑SIRT3 and acetyl‑LC3B WB.\n- MAM frequency via confocal microscopy of MitoTracker‑ER‑GFP constructs.\n- Antigen presentation assay using HLA‑A*02:01 tetramers loaded with influenza M1 peptide.\n- Vaccine efficacy: HAI titers and CD8⁺ T‑cell cytokine polyfunctionality (IFN‑γ, TNF‑α, IL‑2).\n- Microbiome stratification: fecal metabonomics to quantify UA precursors (ellagitannins) and derived urolithins.\n\nFalsification: If UA supplementation fails to increase SL‑SIRT3 or decrease LC3B acetylation, or if these biochemical changes do not predict improved vaccine responses, the hypothesis is refuted.\n\n## Broader Impact\nLinking a gut‑derived postbiotic to organelle‑communication hubs provides a mechanistic bridge between microbiome variability and individualized immunotherapy, suggesting that UA‑based regimens could be tailored to improve vaccine efficacy in aging populations.