Atherosclerotic lesions are populated by cells of the innate and adaptive immune system, including CD8⁺ T cells. The CD8⁺ T cell infiltrate has recently been characterized in mouse and human atherosclerosis and revealed activated, cytotoxic, and possibly dysfunctional and exhausted cell phenotypes. In mouse models of atherosclerosis, antibody-mediated depletion of CD8⁺ T cells ameliorates atherosclerosis. CD8⁺ T cells control monopoiesis and macrophage accumulation in early atherosclerosis. In addition, CD8⁺ T cells exert cytotoxic functions in atherosclerotic plaques and contribute to macrophage cell death and necrotic core formation. CD8⁺ T cell activation may be antigen-specific, and epitopes of atherosclerosis-relevant antigens may be targets of CD8⁺ T cells and their cytotoxic activity. CD8⁺ T cell functions are tightly controlled by costimulatory and coinhibitory immune checkpoints. Subsets of regulatory CD25⁺CD8⁺ T cells with immunosuppressive functions can inhibit atherosclerosis. Importantly, local cytotoxic CD8⁺ T cell responses may trigger endothelial damage and plaque erosion in acute coronary syndromes. Understanding the complex role of CD8⁺ T cells in atherosclerosis may pave the way for defining novel treatment approaches in atherosclerosis. In this review article, we discuss these aspects, highlighting the emerging and critical role of CD8⁺ T cells in atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of the vessel wall controlled by local and systemic immune responses. The role of interleukin-23 receptor (IL-23R), expressed in adaptive immune cells (mainly T-helper 17 cells) and γδ T cells, in atherosclerosis is only incompletely understood. Here, we investigated the vascular cell types expressing IL-23R and addressed the function of IL-23R and γδ T cells in atherosclerosis. IL-23R⁺ cells were frequently found in the aortic root in contrast to the aorta in low-density lipoprotein receptor deficient IL-23R reporter mice (Ldlr^−/−Il23r^gfp/+), and mostly identified as γδ T cells that express IL-17 and GM-CSF. scRNA-seq confirmed γδ T cells as the main cell type expressing Il23r and Il17a in the aorta. Ldlr^−/−Il23r^gfp/gfp mice deficient in IL-23R showed a loss of IL-23R⁺ cells in the vasculature, and had reduced atherosclerotic lesion formation in the aortic root compared to Ldlr^−/− controls after 6 weeks of high-fat diet feeding. In contrast, Ldlr^−/−Tcrδ^−/− mice lacking all γδ T cells displayed unaltered early atherosclerotic lesion formation compared to Ldlr^−/− mice. In both HFD-fed Ldlr^−/−Il23r^gfp/gfp and Ldlr^−/−Tcrδ^−/− mice a reduction in the plaque necrotic core area was noted as well as an expansion of splenic regulatory T cells. In vitro, exposure of bone marrow-derived macrophages to both IL-17A and GM-CSF induced cell necrosis, and necroptotic RIP3K and MLKL expression, as well as inflammatory mediators. IL-23R⁺ γδ T cells are predominantly found in the aortic root rather than the aorta and promote early atherosclerotic lesion formation, plaque necrosis, and inflammation at this site. Targeting IL-23R may thus be explored as a therapeutic approach to mitigate atherosclerotic lesion development.

BACKGROUND: Atherosclerosis is driven by the infiltration of the arterial intima by diverse immune cells and smooth muscle cells (SMCs). CD8 ⁺ T cells promote lesion growth during atherosclerotic lesion development, but their role in advanced atherosclerosis is less clear. Here, we studied the role of CD8 ⁺ T cells and their effects on SMCs in established atherosclerosis. METHODS: CD8 ⁺ T cells were depleted in (SMC reporter) low-density lipoprotein receptor-deficient ( Ldlr ^−/− ) mice with established atherosclerotic lesions. Atherosclerotic lesion formation was examined, and single-cell RNA sequencing of aortic SMCs and their progeny was performed. Additionally, coculture experiments with primary aortic SMCs and CD8 ⁺ T cells were conducted. RESULTS: Although we could not detect differences in atherosclerotic lesion size, an increased plaque SMC content was noted in mice after CD8 ⁺ T-cell depletion. Single-cell RNA sequencing of aortic lineage-traced SMCs revealed contractile SMCs and a modulated SMC cluster, expressing macrophage- and osteoblast-related genes. CD8 ⁺ T-cell depletion was associated with an increased contractile but decreased macrophage and osteoblast-like gene signature in this modulated aortic SMC cluster. Conversely, exposure of isolated aortic SMCs to activated CD8 ⁺ T cells decreased the expression of genes indicative of a contractile SMC phenotype and induced a macrophage and osteoblast-like cell state. Notably, CD8 ⁺ T cells triggered calcium deposits in SMCs under osteogenic conditions. Mechanistically, we identified transcription factors highly expressed in modulated SMCs, including Runx1 , to be induced by CD8 ⁺ T cells in cultured SMCs in an IFNγ (interferon-γ)-dependent manner. CONCLUSIONS: We here uncovered CD8 ⁺ T cells to control the SMC phenotype in atherosclerosis. CD8 ⁺ T cells promote SMC dedifferentiation and drive SMCs to adopt features of macrophage-like and osteoblast-like, procalcifying cell phenotypes. Given the critical role of SMCs in atherosclerotic plaque stability, CD8 ⁺ T cells could thus be explored as therapeutic target cells during lesion progression.

Endothelial dysfunction is cause and consequence of cardiovascular diseases. The endothelial hormone C-type natriuretic peptide (CNP) regulates vascular tone and the vascular barrier. Its cGMP-synthesizing guanylyl cyclase-B (GC-B) receptor is expressed in endothelial cells themselves. To characterize the role of endothelial CNP/cGMP signaling, we studied mice with endothelial-selective GC-B deletion. Endothelial EC GC-B KO mice had thicker, stiffer aortae and isolated systolic hypertension. This was associated with increased proinflammatory E-selectin and VCAM-1 expression and impaired nitric oxide bioavailability. Atherosclerosis susceptibility was evaluated in such KO and control littermates on Ldlr (low-density lipoprotein receptor)-deficient background fed a Western diet for 10 weeks. Notably, the plaque areas and heights within the aortic roots were markedly increased in the double EC GC-B/Ldlr KO mice. This was accompanied by enhanced macrophage infiltration and greater necrotic cores, indicating unstable plaques. Finally, we found that EC GC-B KO mice had diminished vascular regeneration after critical hind-limb ischemia. Remarkably, all these genotype-dependent changes were only observed in female and not in male mice. Auto/paracrine endothelial CNP/GC-B/cGMP signaling protects from arterial stiffness, systolic hypertension, and atherosclerosis and improves reparative angiogenesis. Interestingly, our data indicate a sex disparity in the connection of diminished CNP/GC-B activity to endothelial dysfunction.

Aims: Vascular endothelial growth factors (VEGFs) and their endothelial tyrosine kinase receptors are crucial mediators of vasculogenesis and angiogenesis, with FLT1 (VEGFR1) acting as a decoy receptor for VEGFs. Alternative polyadenylation (APA) of Flt1 transcripts produces a truncated mRNA transcript encoding soluble (s) FLT1, which contains only the extracellular ligand-binding domain. The relative roles of full-length (fl) FLT1 and sFLT1 in angiogenesis are incompletely understood. Alternative polyadenylation is an important post-transcriptional mechanism of gene regulation that is increasingly recognized to affect the expression of many genes related to cardiovascular diseases. However, there are no in vivo models available to target APA and to examine the physiological roles of gene isoforms. Methods and results: We here generated mice by introducing silent mutations into the Flt1 exon 13/intron 13 5′ splice site through homology-directed repair with CRISPR/Cas9 gene editing. Embryonic development, postnatal retinal vascular development, and reparative angiogenesis were evaluated in models of hindlimb ischaemia and myocardial infarction, complemented by assessments of cardiac functional recovery and cell phenotypes through snRNAseq, as well as in vitro assays examining endothelial cell phenotype and function. The introduction of specific splice site mutations within Flt1 to activate APA led to a complete loss of flFLT1 and an increase in sFLT1 expression. Using this mouse model, we demonstrate that sFLT1 alone is sufficient for developmental vasculogenesis, whereas the specific loss of flFLT1 enhances reparative revascularization and angiogenesis and improves cardiac recovery after myocardial infarction. Conclusion: Our findings show that APA can be effectively targeted to modulate FLT1 isoform expression in vivo. Our mouse model may help to understand diseases driven by an imbalance in FLT1 isoform expression. Given the widespread presence of APA signals, our approach could furthermore serve as a model for studying the function of other truncated gene isoforms.

Aims: Patients undergoing allogeneic haematopoietic cell transplantation (allo-HCT) are at a risk of developing graft-vs.-host disease (GvHD) and are afflicted with an increased incidence of cardiovascular events. Whether allo-HCT contributes to atherosclerosis progression has not been addressed experimentally. Methods and results: Here, we applied a novel minor histocompatibility antigen mismatch allo-HCT mouse model by transplanting C57BL/6 mice deficient for the LDL receptor (B6.Ldlr−/−) with bone marrow (BM) or BM and T cells (BMT) from BALB/b donors (BALB/b, H-2b → B6.Ldlr−/−, and H-2b), and feeding recipients a western diet. Mild clinical GvHD symptoms ensued with low disease activity in typical GvHD target organs. However, allogeneic BMT recipients developed increased atherosclerotic lesions compared with mice receiving BM only or syngeneic BMT recipients. Atherosclerotic lesions showed a heightened infiltration of effector CD8+ T cells in the aorta of BMT recipients. Furthermore, BMT recipients exhibited significantly higher serum cholesterol levels than BM-recipients. Notably, CD8+ T-cell depletion in B6.Ldlr−/− BMT recipients reduced atherosclerotic lesion formation and decreased cholesterol levels. Conclusion: These data may provide a novel mechanistic underpinning for the clinically observed increased incidence of cardiovascular disease in long-term allo-HCT survivors. Moreover, we have identified CD8+ T cells as potential targets for mitigating GvHD-induced atherosclerosis.