Atherosclerotic plaques form in the inner layer of arteries triggering heart attacks and strokes. Although T cells have been detected in atherosclerosis, tolerance dysfunction as a disease driver remains unexplored. Here we examine tolerance checkpoints in atherosclerotic plaques, artery tertiary lymphoid organs and lymph nodes in mice burdened by advanced atherosclerosis, via single-cell RNA sequencing paired with T cell antigen receptor sequencing. Complex patterns of deteriorating peripheral T cell tolerance were observed being most pronounced in plaques followed by artery tertiary lymphoid organs, lymph nodes and blood. Affected checkpoints included clonal expansion of CD4⁺, CD8⁺ and regulatory T cells; aberrant tolerance-regulating transcripts of clonally expanded T cells; T cell exhaustion; T_reg–TH₁₇ T cell conversion; and dysfunctional antigen presentation. Moreover, single-cell RNA-sequencing profiles of human plaques revealed that the CD8⁺ T cell tolerance dysfunction observed in mouse plaques was shared in human coronary and carotid artery plaques. Thus, our data support the concept of atherosclerosis as a bona fide T cell autoimmune disease targeting the arterial wall.

Three systemic biological systems, i.e., the nervous, the immune, and the cardiovascular systems, form a mutually responsive and forward-acting tissue network to regulate acute and chronic cardiovascular function in health and disease. Two sub-circuits within the cardiovascular system have been described, the artery brain circuit (ABC) and the heart brain circuit (HBC), forming a large cardiovascular brain circuit (CBC). Likewise, the nervous system consists of the peripheral nervous system and the central nervous system with their functional distinct sensory and effector arms. Moreover, the immune system with its constituents, i.e., the innate and the adaptive immune systems, interact with the CBC and the nervous system at multiple levels. As understanding the structure and inner workings of the CBC gains momentum, it becomes evident that further research into the CBC may lead to unprecedented classes of therapies to treat cardiovascular diseases as multiple new biologically active molecules are being discovered that likely affect cardiovascular disease progression. Here, we weigh the merits of integrating these recent observations in cardiovascular neurobiology into previous views of cardiovascular disease pathogeneses. These considerations lead us to propose the Neuroimmune Cardiovascular Circuit Hypothesis.

Background: γδ T cells represent a heterogeneous family of innate-like lymphocytes with adaptive immunity features. Although abundant in barrier tissues such as skin and intestine, γδ T cells are rare in the cardiovascular system, which severely limits exploration of their roles in atherosclerosis. Consequently, the spatial localization, functional states, and antigen-driven responses of γδ T cells within atherosclerotic lesions remain poorly defined. Methods: To nevertheless examine γδ T cells in atherosclerosis, we employed an integrative, three-pronged strategy combining γδ T cell single-cell RNA sequencing (scRNA-seq) and single-cell T cell receptor sequencing (scTCR-seq), spatial transcriptomics, and large-scale multi-dataset scRNA-seq integration across multiple hyperlipidemic mouse models. Large-scale multi-dataset scRNA-seq integration was also applied to human atherosclerotic plaque datasets. Results: We found significant enrichment of γδ T cells within both atherosclerotic plaques and artery tertiary lymphoid organs (ATLOs), particularly of the proinflammatory IL17-producing γδ T17 subtype. Unexpectedly, γδ T17 cells containing paired Vγ6Vδ4 TCR chains with identical complementarity-determining region 3 (CDR3) sequences underwent clonal expansion in atherosclerotic plaques and ATLOs. Transcriptomic analysis revealed that plaques and ATLOs locally educate γδ T17 cells - here termed γδ eduT17 cells - towards an anti-apoptotic, tissue-resident, and apparent hypofunctional phenotype. Furthermore, γδ eduT17 cells underwent metabolic reprogramming within the atherosclerotic microenvironment. Spatial transcriptomics revealed that γδ T cells preferentially localize in ATLOs, particularly at the interface between T cell zones and B cell follicles. Multi-dataset integration confirmed the conservation of these features across multiple hyperlipidemic mouse models. In contrast, human atherosclerotic plaques harbored substantially fewer γδ T cells and the human phenotypes were dominated by an effector/cytolytic γδ T cell subtype, characterized by transcriptomes enriched in cytotoxic effector molecules. Conclusions: Our findings identify γδ T cells as a previously underappreciated T cell lineage population in atherosclerosis. Murine atherosclerosis is characterized by the enrichment, education, and clonal expansion of proinflammatory γδ eduT17 cells within plaques and ATLOs. In contrast, human plaques harbor γδ T cells with cytolytic features, suggesting divergent roles of γδ T cells between species. These results highlight the importance of local vascular microenvironments in shaping γδ T cell function and emphasize the need for caution when extrapolating mechanistic insights from mouse models to human atherosclerosis.

Artery tertiary lymphoid organs (ATLOs) emerge in atherosclerosis, which is a chronic inflammatory artery disease with an autoimmune component. However, whether disease-relevant autoimmune B cells emerge in ATLOs remains unknown. In this study, we isolate germinal center (GC) B cells from ATLOs and lymph nodes from healthy and atherosclerosis-burdened mice, expression clone 60 autoantibodies and screen them for arterial wall reactivity. ATLO GC B cell-derived autoantibodies skew to atherosclerosis-relevant autoantigens versus their counterparts in lymph nodes of both genotypes. One ATLO GC B cell-derived autoantibody (termed A6) binds to histone 2B (H2B) with high affinity. Both vaccination with H2B and adoptive transfer of A6 accelerate atherosclerosis, revealing a pathogenic autoantibody−autoantigen pair. Mechanistically, ATLOs specifically show both distorted B cell activation and immune tolerance checkpoint-regulating gene expression profiles. In a human cohort, circulating anti-H2B antibody titers positively correlate with aortic calcification in humans. We suggest that ATLOs harbor a dysregulated immune tolerance environment permissive for autoreactive B cells that express pathogenic autoantibodies promoting atherosclerosis.

SUMMARY: Artery tertiary lymphoid organs (ATLOs) emerge in atherosclerosis which is a chronic inflammatory artery disease with an autoimmune component. However, whether disease-relevant autoimmune B cells emerge in ATLOs and their impacts remains unknown. To map atherosclerosis-specific humoral autoimmunity and define its roles, we isolated germinal-center (GC) B cells from ATLOs and lymph-nodes, expression-cloned 60 autoantibodies and screened them for arterial wall reactivity. ATLO-derived autoantibodies markedly skewed to atherosclerosis-relevant autoantigens versus those of lymph-nodes. One ATLO GC-derived autoantibody bound to histone 2B (H2B) with high-affinity (∼25 nM). Moreover, vaccination with H2B or adoptive transfer of its cognate autoantibody markedly accelerated atherosclerosis suggesting that ATLOs fail to delete pathogenic high-affinity self-reactive B cells. In a human cohort, total circulating anti H2B antibody titers positively correlated with aortic and coronary artery calcification. We conclude that ATLOs harbor a dysregulated immune tolerance environment permissive for autoreactive B cells that express pathogenic autoantibodies driving atherosclerosis.