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.

Immune cell-driven destruction of the aortic wall remains a major contributor of death in patients burdened with aortic aneurysms (AAs). Dendritic cells (DCs) play critical roles in bridging innate and adaptive immunity by orchestrating robust inflammatory responses and concomitantly sustaining immune tolerance. However, the specific roles of DCs in AA pathogenesis remain to be explored. To examine the participation of DCs in AA pathogenesis, we used single-cell RNA sequencing (scRNA-seq) integration analyses to characterize DC heterogeneity and elucidate their putative involvement in AA pathogenesis in several mouse AA models and translate the experimental data to human AAs. Our data reveal that conventional DC2s (cDC2s) constituted the most abundant DC subtypes in both murine and human AAs. Furthermore, cDC1s, plasmacytoid DCs (pDCs) and immunosuppressive mature regulatory DCs (mregDCs) were identified. Within the cDC2 subtypes, the AA tissue environment trained cDC2s and a newly defined DC3s subtype toward highly pro-inflammatory phenotypes. Parallel to the increased prevalence of pro-inflammatory activated cDC2s and DC3s, a significant reduction of the number of mregDCs was observed in mouse AAs. This data revealed that the balance between pro- vs. the anti-inflammatory DCs is disrupted in mouse AAs. Thus, therapeutic reconstitution strategies to correct this dysbalance together with protective measures that are already in use in clinical practice may lead to beneficial AA outcomes before surgical intervention is needed.