Hypothesis\nIntermittent fasting (IF) restores thymic epithelial cell function, thereby increasing naïve T‑cell output and reversing immune‑driven aging.\n\n## Rationale\nAged immune cells drive systemic aging through SASP secretion, mitochondrial dysfunction, and poor senescent‑cell clearance [1][2]. Thymic involution limits the supply of naïve T cells that could replace exhausted or senescent lymphocytes [5]. IF activates autophagy and inhibits mTOR, pathways known to preserve cellular homeostasis and promote mitophagy [7]. In thymic epithelial cells, autophagy improves mitochondrial quality, reduces ROS, and sustains FOXN1 expression, a transcription factor essential for thymopoiesis.\n\n## Predictions\nIf IF rejuvenates the thymus, then in aged mice subjected to a 24‑hour fast twice weekly for 8 weeks we expect:\n- Increased thymic cellularity and FOXN1+ epithelial cells relative to ad libitum controls.\n- Elevated recent thymic emigrants (RTEs) measured by sjT‑CRC circles in peripheral blood.\n- Reduced frequency of senescent CD4⁺/CD8⁺ T cells (p16^INK4a^+ or SA‑β‑gal^+) in spleen, liver, lung and bone marrow.\n- Lower circulating SASP factors (IL‑6, TNF‑α, MCP‑1) and decreased inflammaging markers.\n- Improved healthspan readouts such as grip strength, glucose tolerance and delayed onset of age‑related pathology.\n\n## Experimental Design\n1. Animals: 20‑month‑old C57BL/6J mice, n=15 per group (IF vs control).\n2. IF regimen: 24‑hour fast followed by 24‑hour refeed, twice weekly; controls receive uninterrupted access to food.\n3. Readouts (after 8 weeks):\n - Thymic flow cytometry for epithelial subsets (EpCAM^+Ly51^−, Foxn1^+).\n - qPCR for Foxn1, autophagy genes (LC3‑II, p62).\n - Mitochondrial ROS measurement with MitoSOX.\n - Peripheral blood sjT‑CRC quantification.\n - Senescent T‑cell staining (p16, SA‑β‑gal).\n - Serum cytokine panel (IL‑6, TNF‑α, MCP‑1).\n - Functional assays: grip strength, intraperitoneal glucose tolerance test.\n4. Statistical analysis: Two‑tailed t‑tests or Mann‑Whitney where appropriate; significance set at p<0.05.\n\n## Potential Outcomes\n- Supportive outcome: IF group shows significant thymic FOXN1 increase, higher RTEs, fewer senescent T cells, lower SASP, and better healthspan. This would falsify the null hypothesis that IF does not affect thymic output and would support the idea that restoring immune competence can retard aging.\n- Refutative outcome: No measurable differences between IF and control groups across all readouts. This would refute the hypothesis and suggest that thymic rejuvenation via IF is insufficient to alter immune‑driven aging, prompting investigation of alternative routes (e.g., targeted senolytes or cytokine neutralization).\n\n## Implications\nA positive result would reposition intermittent fasting not merely as a metabolic intervention but as a strategy to reset the immune system’s upstream role in aging. It would provide a mechanistic link between nutrient‑sensing pathways, thymic regeneration, and systemic inflammaging, offering a testable, low‑cost avenue to delay multiple age‑associated phenotypes.
