Rapamycin(RAPA) is a macrolide antibiotic and is widely used in clinical practice(Choudhury et al., 2024; Kraaijeveld et al., 2019). Emerging evidence suggests that RAPA modulates cellular proliferation and differentiation via the mTOR signaling pathway, thereby extending lifespan and mitigating age-related pathologies(Chen et al., 2009; Weichhart, 2018). In elderly populations, short-term RAPA administration enhances immune competence, remodels the microbiota, and exerts sustained anti-aging effects persisting for at least three months post-treatment(Bitto et al., 2016). Intriguingly, even in young individuals, transient RAPA exposure has demonstrated anti-senescence benefits(Breed et al., 2018). Despite these findings, the dynamic alterations in thymic architecture and concomitant T-cell immunity after short-term application of RAPA in adolescence are still lacking in exploration.

Thymus, the first organ to show age-related changes in human body, regresses with age after puberty reaches its peak(Boehm et al., 2013). The thymic output of memory T cells in aging thymus decreases, the diversity of TCR library shrinks, and immune dysfunction and immunosenescence occur(Thomas et al., 2020). As an important central immune organ, it is mainly composed of thymic stromal cells and thymocytes. Thymic epithelial cells (TEC) is the most important type of thymic stromal cell. And the thymus function is mainly coordinated by TEC, which is usually divided into capsule, subcapsule area, cortical area, cortex-medullary junction area and medullary area according to tissue characteristics(Nitta et al., 2020). This structural compartmentalization underpins the thymic microenvironment essential for T-cell development and selection.

Thymic involution can be triggered by oxidative stress, infection, or radiation, yet regenerative processes often ensue upon cessation of these stressors(Ashby et al., 2023; Cosway et al., 2023; Miller, 2020). Notably, thymic regeneration therapies have shown promise in attenuating aging phenotypes and age-associated diseases. Intriguingly, the anti-aging efficacy of RAPA may partially arise from thymic rejuvenation following transient treatment(Fahy et al., 2019; Abbott, 2024). To systematically delineate the temporal dynamics of RAPA-induced thymic remodeling, we employed a short-term low-dose RAPA regimen (1 mg/kg/d, i.p. for 3 days) in 6–8 weeks female mice. This way allowed characterization of acute thymic involution and subsequent natural regeneration kinetics at 3 and 14 days post-withdrawal. In fact, endogenous thymic regeneration is regulated by TEC, thymocyte proliferation and apoptosis(Duah et al., 2021; Liang et al., 2022). Interleukin-7 (IL-7) and metformin (MET), pivotal regulators of thymic homeostasis, exhibit therapeutic potential in preserving thymic function and counteracting immunosenescence. IL-7 deficiency leads to profound thymic hypoplasia, while exogenous IL-7 supplementation partially reverses age-related thymic involution(Barata et al., 2019; Chaudhry et al., 2016). Similarly, Xu's research shows that MET could increase thymus volume, and another study shows that MET ameliorates D-galactose-induced thymic involution via mitochondrial function modulation(Liu et al., 2023; Xu et al., 2024). Building on these insights, we further investigated whether IL-7 or MET administration could augment endogenous thymic regeneration post-RAPA withdrawal, aiming to elucidate their mechanistic roles in this process.

In summary, this study provides a comprehensive analysis of thymic dynamics following short-term, low-dose RAPA exposure, integrating multimodal assessments of regenerative kinetics and therapeutic interventions(e.g. IL-7/MET). Our findings advance the understanding of thymus-centric mechanisms underlying RAPA-mediated anti-aging effects, offering a foundation for thymic regeneration therapies to promote immune reconstitution.