Aging exacerbated T-regulatory cells dysfunction and loss of protective immunity in murine atherosclerosis progression

Abstract Background Atherosclerosis is driven by an autoimmune component with autoreactive T cells recognizing Apolipoprotein B (ApoB). Experimental and clinical data suggest that antigen-specific T regulatory (Treg) cells switch to pathogenic T effector (Teff) cells during the natural course of the disease, undermining initially protective immunity. This study investigates the cellular and functional mechanisms underlying the dynamics of T-cell phenotypes in murine atherosclerosis. Methods Female 8-week-old C57BL/6 wild type (WT) and Apoe⁻/⁻ mice were fed a chow diet (CD) or a Western diet (WD) for up to 16 weeks, additional Apoe⁻/⁻ mice were maintained on a chow diet for 52 weeks to study the effects of aging. T cell phenotypes and cytokine expression across tissues, including lymph nodes, spleen, aorta, and blood, were analyzed by flow cytometry. ApoB-specific CD4⁺ T cell responses were evaluated using a Triple-Color Flurospot assay to detect IL-10, IFN-γ, and IL-17. Female Foxp3 lineage-tracking mice (Foxp3-YFP-Cre-Rosa26-loxp-td-RFP-loxp-Apoe⁻/⁻) were placed on chow diet CD and WD and were used to track Tregs and assess their stability and function over time. Results Across all time points, total T cell and Treg numbers increased, indicating persistent immune activation. Atherosclerosis progression led to fewer naïve T cells, an increase in central memory and effector memory T cells, along with elevated pro-inflammatory cytokines (TNF-α, IL-17), primarily in the aortic adventitia, similar dynamics were seen in draining, non-draining lymph nodes and blood, highlighting atherosclerosis as a systemic disease. Aging further exacerbated this immune response, intensifying the inflammatory profile as the disease progressed. Tregs showed significant activation in both draining and non-draining lymph nodes and blood, accompanied by increased secretion of cytokines such as IFN-γ, TNF-α, and IL-17, as well as increased expression of effector T cell-associated transcription factors (T-bet and RORγt). This was accompanied by a downregulation of anti-inflammatory cytokine (IL-10). In aged mice, Tregs exhibited further increased inflammatory cytokines, greater reduction in IL-10, and increased PD-1 expression, suggesting a functional shift towards immune suppression and dysfunction. Foxp3 Lineage-tracking mice revealed Treg phenotype switching, supporting the idea that as atherosclerosis progresses, Tregs lose their regulatory functions and adopt a pro-inflammatory role. Flurospot assays revealed ApoB-specific CD4⁺ T-cell responses showing IL-10 secretion, suggesting a protective phenotype early in the disease. Conclusions Our findings underscore the dynamic nature of Tregs in atherosclerosis, revealing a critical temporal shift from protective to pathogenic immune responses. These results provide a foundation for therapeutic immunomodulatory approaches in the future.Triple-Color Fluorospot IL-10 Response