The research team, based at Leiden University, focused on a fundamental problem in cardiovascular medicine: most patients with atherosclerosis are elderly, yet most preclinical studies are performed in young animals. Aging transforms the immune system through a process called immunosenescence, in which hematopoietic stem cells skew toward myeloid lineages, naive lymphocytes dwindle, and senescent cells accumulate and secrete a persistent cocktail of inflammatory signals known as the senescence-associated secretory phenotype. This chronic low-grade inflammation, termed inflammaging, drives a shift toward pro-atherogenic immunity, including Th1 cells, inflammatory macrophages, and age-associated B cells that the same group previously identified inside human and mouse plaques. The result is plaques that are not just larger, but potentially more vulnerable to rupture.

Rapamycin was a logical candidate to counter this process. The drug forms a complex with the FK506-binding protein 12 and allosterically inhibits mTORC1, a central cellular growth regulator that integrates nutrient and growth factor signals. Because mTORC1 activity is tied to cellular senescence, proliferation, and effector T cell differentiation, blocking it can suppress inflammatory responses while paradoxically allowing regulatory T cells, which resist mTOR inhibition through PTEN and PIM2 pathways, to expand. Rapamycin has already been shown to rejuvenate aged hematopoietic stem cells, alleviate oxidized LDL-induced senescence in vascular smooth muscle cells, and increase regulatory T cell numbers in patients receiving rapamycin-eluting stents. What remained unknown was how the drug affects the age-altered immune landscape within established atherosclerotic disease.

To establish relevance, the researchers first mapped mTOR expression in human disease. Using single-cell RNA sequencing data from carotid artery plaques of 18 patients, they found that the MTOR gene was broadly expressed across immune cell clusters, with B cells showing among the highest levels. In mouse aortas, both aging and a Western diet elevated Mtor expression in T cells and macrophages, while B cells again displayed the strongest average expression. When the team cultured splenocytes from 80-week-old atherosclerotic mice with rapamycin, the drug reduced B cell frequency, impaired CD4 T cell proliferation, and shifted the remaining cells toward a more tolerogenic phenotype, with fewer T-bet-positive cells and more FoxP3-positive regulatory T cells, all without compromising cell viability.

For the central experiment, the team treated 80- to 90-week-old male Ldlr-deficient mice, which had developed atherosclerosis gradually over a lifetime on a standard chow diet, with 1 mg/kg rapamycin three times a week for eight weeks. The treatment left overall plaque size, collagen content, and necrotic core area unchanged, but it significantly reduced macrophage content within the aortic root plaques, both in absolute terms and as a percentage of plaque area. Flow cytometry of aortic arch immune cells revealed a significant drop in neutrophils and CD8 T cells, along with a reduced total CD4 T cell pool. Crucially, within the CD4 compartment, the relative frequency of atheroprotective regulatory T cells rose from roughly 57 percent to nearly 71 percent, while pro-inflammatory Th1 cells were unaltered. Notably, serum cholesterol actually increased with treatment, yet plaque burden held steady, suggesting the immunomodulatory effects offset the pro-atherogenic lipid shift.

Single-cell RNA sequencing of more than 32,000 aortic immune cells provided a high-resolution view of these changes. The analysis resolved 45 subclusters spanning myeloid cells, NK cells, B cells, plasma cells, conventional T cells, gamma-delta T cells, and extrathymic double-positive CD4 CD8 T cells. Rapamycin reshaped macrophage composition, reducing Nlrp3-high and Ccr2-high inflammatory subsets and Trem2-high macrophages, while inflammatory gene signatures within key macrophage populations declined. Among B cells, the most striking finding was the near-complete disappearance of a proliferating germinal center-like cluster, accompanied by reduced plasma cells and lower expression of immunoglobulin-related genes, indicating a broad suppression of antigen-driven humoral responses. T cell subclustering showed that effector memory CD4 and cytotoxic Gzmk- and Gzmb-expressing CD8 subsets were drastically reduced, while naive and central memory subsets expanded. Regulatory T cells were preserved and showed elevated Foxp3 and Tgfb1 expression, together with enrichment of IL-10 and CTLA-4 signaling pathways, pointing to enhanced immunoregulatory activity.

The effects extended well beyond the vessel wall. In the mediastinal lymph nodes that drain the heart and aorta, rapamycin diminished the total immune cell population by nearly six-fold, affecting myeloid cells, B cells, and T cells alike. Proliferating CD8 T cells declined, central memory CD8 cells increased, and the frequency of regulatory T cells rose from about 37 percent to nearly 50 percent. Among B cells, age-associated B cells, a pro-inflammatory subset previously implicated in atherosclerosis, dropped significantly, and germinal center B cell frequencies were almost abolished, falling from roughly 1.2 percent to 0.04 percent. This lymphoid shutdown was mirrored in the blood: serum levels of IgM and several IgG subclasses fell substantially, as did antibodies against oxidation-specific epitopes such as malondialdehyde and oxidized LDL, which are hallmark targets of the humoral response in atherosclerosis.

The spleen told a partially different story. Total immune cell numbers there were unaffected, but CD4 T cells declined, myeloid cells increased, and regulatory T cells along with IL-10-producing CD4 T cells rose, consistent with a systemic shift toward a tolerogenic profile. Follicular helper T cells, which orchestrate B cell activation, dropped markedly, and splenic Il21 gene expression fell in parallel, providing a mechanistic link between mTOR inhibition and the collapse of germinal center responses seen in plaques and lymph nodes. The team also examined senescence markers directly: SenMayo enrichment scores, a gene set marking senescent phenotypes, were significantly reduced in aortic B cells, double-positive T cells, NK cells, and conventional T cells after treatment, and qPCR confirmed downregulation of the key cell-cycle arrest genes Btg2 and Cdkn2a, which encodes p16. Markers such as p21 and p53 were unchanged, indicating that rapamycin dampens part, but not all, of the molecular signature of immune aging.

The authors are careful about what these results do and do not mean. Because the aortic arch was used for immune profiling, lesion size there was not assessed, so local differences in plaque burden cannot be excluded as a contributor to the altered immune composition. Germinal center structures were evaluated at the cellular and transcriptional levels rather than by histology, and the increase in Il1b-high macrophages alongside reduced inflammatory gene signatures highlights the complexity of interpreting macrophage polarization under mTOR inhibition. The eight-week treatment window in mice with advanced, established disease was sufficient to remodel immunity but not to shrink plaques, raising the possibility that earlier intervention or longer treatment, perhaps combined with statins to counter the cholesterol increase, could deliver greater structural benefit.

Nevertheless, the study makes a compelling case that immune rejuvenation is a realistic therapeutic goal for age-related cardiovascular disease. By simultaneously reducing plaque macrophage burden, expanding regulatory T cells, suppressing age-associated B cells and germinal center responses, and dialing down senescence-associated gene expression, rapamycin acted as a broad immunological reset in aged atherosclerotic mice. The same breadth cuts both ways: the drug’s suppression of adaptive immunity, including plasma cells and antigen-driven antibodies, could impair protective host defense in elderly patients, and its metabolic side effects would need careful management. The authors argue that future work should define how aging alters mTOR pathway activity across immune cell populations at the protein level, so that the anti-inflammatory benefits of mTOR inhibition can be harnessed while minimizing risks to immunity and metabolic homeostasis. For a population in which cardiovascular disease remains the leading cause of death, that balance could prove decisive.

Subject of Research: Immune reprogramming by mTOR inhibition with rapamycin in aged atherosclerotic mice

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

Article References: de Mol, J., de Korte, D. H., Depuydt, M. A. C., Smit, V., Kleijn, M. N. A. B., van Santbrink, P. J., Binder, C. J., Porsch, F., Schaftenaar, F. H., & Foks, A. C. (2026). Therapeutic Immune Reprogramming by Rapamycin Attenuates Plaque Inflammation and Lymphoid Immune Responses in Aged Atherosclerotic Mice. Aging Cell, 25(10), Article e70730. https://doi.org/10.1111/acel.70730

Image Credits: AI Generated

DOI: 10.1111/acel.70730

Keywords: rapamycin, mTOR, atherosclerosis, immunosenescence, regulatory T cells, age-associated B cells, germinal center, plaque inflammation, macrophages, single-cell RNA sequencing, aging, cardiovascular disease

The central actors in this model are B lymphocytes, the white blood cells that produce antibodies. Throughout life, the B cell compartment is governed by an elaborate series of quality-control checkpoints, often described as tolerance mechanisms, whose job is to prevent cells that recognize the body’s own molecules from surviving and maturing. During development in the bone marrow, immature B cells whose receptors bind self-antigens too strongly are either deleted, rendered inert through a state called anergy, or rescued by receptor editing, a process in which the cell reconfigures its antigen receptor to change its specificity. Later, in peripheral tissues, additional checkpoints eliminate or silence self-reactive cells that escaped the first round of screening. These mechanisms are among the most intensely studied in immunology, and classic work by Goodnow and colleagues and by Nemazee and Weigert established the cellular and genetic logic behind them.

Zouali’s first step concerns what happens to this quality-control apparatus as B cells themselves age. The review argues that aging drives a progressive, cell-intrinsic relaxation of both central and peripheral tolerance. In other words, the checkpoints do not simply disappear; they become leakier, allowing autoreactive clones that would have been purged in youth to survive and accumulate. Several lines of evidence converge on this conclusion. The diversity of the B cell repertoire shrinks markedly in old age, a change correlated with poor health status, and studies of the antibody repertoire in aged animals reveal skewing toward self-reactive specificities. Early developmental checkpoints are also vulnerable: inflammatory immune cells in aged organisms appear to impair the pre-B cell receptor checkpoint, the very gate that screens newly generated B cells, reducing the production of fresh B cells and altering the antibody repertoire in ways that favor autoreactivity.

Critically, this relaxation is not a random decay. The review emphasizes that intrinsic B cell aging involves deep remodeling of the cell’s transcriptional programs, epigenetic landscape, metabolism, and signaling circuitry. Epigenetic modifications, such as changes in DNA methylation, are known to be dynamically reshaped during B cell responses, and epigenetic dysregulation is increasingly implicated in autoimmune pathogenesis. B cells from elderly donors display distinct metabolic phenotypes, with altered mitochondrial activity and nutrient sensing that change how these cells respond to stimulation. Signaling through the B cell receptor and through innate immune receptors such as Toll-like receptors also changes with age, tilting the balance of intracellular pathways toward survival and activation of cells that would previously have been eliminated. Together, these changes durably reprogram B cell responsiveness, establishing a substrate on which autoimmunity can later build.

The second step of the model introduces inflammaging, the chronic, low-grade inflammatory state that characterizes old age. Elevated levels of inflammatory cytokines circulate throughout the aging body, driven by sources as varied as senescent cells, dysfunctional mitochondria, an altered gut microbiota, and persistent viral infections such as cytomegalovirus. Rather than uniformly activating the entire B cell compartment, Zouali argues that inflammaging acts as a selective pressure, preferentially expanding particular inflammation-adapted B cell subsets that are uniquely equipped to thrive under chronic inflammatory conditions. The most prominent of these are age-associated B cells, known as ABCs, and related double-negative 2, or DN2, populations, both characterized by expression of the transcription factor T-bet and the marker CD11c.

These inflammation-adapted subsets are far from innocuous bystanders. Landmark studies showed that Toll-like receptor 7 signaling drives the accumulation of a novel CD11c-positive B cell population that is important for the development of autoimmunity, and that unregulated Toll-like receptor 7 responses generate distinct effector B cells contributing to pathogenic outcomes in systemic lupus erythematosus. In humans, T-bet-positive B cells are induced by viral infections and can dominate antibody responses, illustrating how antiviral programs and autoimmune risk can become entangled. Single-cell analyses of patients with acute lupus have revealed massive expansions of antibody-secreting cells that are diverse in origin and rich in autoreactive specificities, and DN2 B cells have been identified as the major synovial plasma cell precursors in rheumatoid arthritis joints. Elevated expression of BAFF, a survival factor for B cells, in early rheumatoid arthritis further illustrates how inflammatory milieus tilt the competition among B cell subsets.

A key insight of the two-step model concerns the route by which these cells differentiate. In a healthy immune response, B cells that encounter antigen typically migrate into germinal centers, specialized structures where antibody genes undergo mutation and selection, and where tolerance checkpoints operate to weed out cells that have acquired self-reactivity. The review proposes that the synergy between intrinsic B cell aging and inflammaging instead promotes extrafollicular differentiation, a faster, less regulated pathway in which activated B cells become antibody-secreting cells outside germinal centers. Because this route bypasses key germinal-center tolerance checkpoints, it enriches for autoreactive B cell states, allowing self-reactive clones that survived the first-step relaxation to expand and differentiate without encountering the final quality controls. Work defining the extrafollicular B cell response roadmap has highlighted how distinct this pathway is from canonical germinal-center immunity, and how readily it can generate pathogenic antibodies.

The model also explains why conventional B cell-depleting therapies sometimes fall short in older patients. Age-associated B cells are notably resilient: recent studies in murine lupus models demonstrated that T-bet-positive, CD11c-positive ABCs resist ablation by therapies targeting BLyS, the survival factor BAFF, and CD20, the molecule targeted by rituximab. Chronic B cell receptor signaling has been shown to be critical for the differentiation of anergic B cells into age-associated B cells during aging and autoimmunity, suggesting that these cells adopt activation states that shield them from current interventions. Understanding the two-step logic of their generation therefore points toward more selective therapeutic targets, including the innate signaling pathways, metabolic programs, and epigenetic regulators that distinguish inflammation-adapted B cells from the protective repertoire that vaccines and therapies must preserve.

The broader implications reach beyond autoimmunity. Immunosenescence and inflammaging have been implicated in the hyperinflammatory syndrome seen in some older patients with COVID-19, and the same B cell subsets that promote autoreactivity can also shape responses to tumors, since tumor-infiltrating B cells are now recognized as a widespread feature of human cancers. Age-related changes in the immune system also pose major challenges for the development of vaccines tailored to older adults, because the very shifts that favor autoreactive, inflammation-adapted clones undermine the naive and germinal-center responses on which most vaccines depend. A framework that links B cell-intrinsic aging with inflammaging thus offers a conceptual bridge between immune aging, autoimmunity, vaccine failure, and chronic inflammatory disease.

Zouali’s two-step model does not claim that either mechanism alone is sufficient. Intrinsic aging establishes susceptibility by loosening tolerance and accumulating autoreactive clones, while inflammaging supplies the selective pressure and the activation signals that amplify pathogenic B cell programs into clinically meaningful autoimmunity. Disentangling these contributions experimentally, and identifying which of the transcriptional, epigenetic, metabolic, or signaling changes are druggable, now stands as the central challenge. If the model holds, the payoff could be a new generation of interventions that restore tolerance in the aging immune system without dismantling its remaining defenses, turning one of biology’s most stubborn paradoxes into a tractable therapeutic problem.

Subject of Research: Age-associated B cell autoreactivity driven by intrinsic B cell aging and inflammaging

Article Title: A two-step model of age-associated autoreactivity: B cell–intrinsic aging meets inflammaging

Article References: A two-step model of age-associated autoreactivity: B cell–intrinsic aging meets inflammaging. (n.d.). https://doi.org/10.1007/s11357-026-02557-5

Image Credits: AI Generated

DOI: 10.1007/s11357-026-02557-5

Keywords: immunosenescence, B cell tolerance, inflammaging, age-associated B cells, autoreactivity, autoimmunity, extrafollicular differentiation, T-bet, Toll-like receptor 7, epigenetics, B cell metabolism, geroscience