Therapeutic Immune Reprogramming by Rapamycin Attenuates Plaque Inflammation and Lymphoid Immune Responses in Aged Atherosclerotic Mice

Abstract

Aging is a major risk factor for atherosclerosis and is accompanied by profound changes in the immune system, including effector T cell expansion, senescent cell accumulation, and increased pro-inflammatory signaling. Rapamycin, a promising rejuvenation therapeutic that inhibits mTORC1 and modulates immune aging, was investigated for its potential to attenuate pro-atherogenic immune responses in aged atherosclerotic mice. 80- to 90-week-old male Ldlr^−/− mice with established atherosclerotic lesions were treated intraperitoneally with rapamycin or control triweekly for 8 weeks. Systemic and plaque immunity was assessed by flow cytometry or single-cell RNA-sequencing. Plaque morphology was evaluated by histology. Rapamycin treatment reduced plaque macrophage content (0.73 ± 0.07 × 10⁵ μm²) compared to control (ctrl: 0.95 ± 0.09 × 10⁵ μm², p < 0.05) and decreased total T cell numbers in vascular and lymphoid tissues. This was accompanied by a shift from effector to central memory phenotypes, particularly within the CD8⁺ compartment, and a relative enrichment of regulatory CD4⁺ T cells (Tregs). Single-cell transcriptomics of aortic Tregs revealed increased expression of suppressive genes (Foxp3, Tgfb1) upon rapamycin. Rapamycin also reduced T follicular helper cells, IL-21 expression, germinal center B cells, plasma cells, and total and oxidation-specific epitope-specific immunoglobulin levels. Additionally, age-associated B cells were diminished in spleen and lymph nodes, reflecting a broader anti-inflammatory effect on B cell immunity. Finally, aortic leukocytes displayed profoundly reduced senescent enrichment scores. Rapamycin remodels the immune landscape in aged atherosclerotic mice, promoting regulatory lymphocyte subsets and suppressing antigen-driven and inflammatory immune responses. These findings highlight the potential of immune-targeted rejuvenation to mitigate age-related atherogenic inflammation.