Frank H. Schaftenaar

BACKGROUND: Tissue resident memory T (T _RM ) cells are a T-cell subset that resides at the site of prior antigen recognition to protect the body against reoccurring encounters. Besides their protective function, T _RM cells have also been implicated in inflammatory disorders. T _RM cells are characterized by the expression of CD69 and transcription factors Hobit (homolog of Blimp-1 [B lymphocyte–induced maturation protein 1] in T cells) and Blimp-1. As the majority of T cells in the arterial intima expresses CD69, T _RM cells may contribute to the pathogenesis of atherosclerosis as well. Here, we aimed to assess the presence and potential role of T _RM cells in atherosclerosis. METHODS: To identify T _RM cells in human atherosclerotic lesions, a single-cell RNA-sequencing data set was interrogated, and T-cell phenotypes were compared with that of integrated predefined T _RM cells. The presence and phenotype of T _RM in atherosclerotic lesions was corroborated using a mouse model that enabled tracking of Hobit-expressing T _RM cells. To explore the function of T _RM cells during atherogenesis, RAG1 ^−/− (recombination activating gene 1 deficient) LDLr ^−/− (low-density lipoprotein receptor knockout) mice received a bone marrow transplant from Hobit ^KO/CRE Blimp-1 ^flox/flox mice, which exhibit abrogated T _RM cell formation, whereafter the mice were fed a Western-type diet for 10 weeks. RESULTS: Human atherosclerotic lesions contained T cells that exhibited a T _RM cell–associated gene signature. Moreover, a fraction of these T cells clustered together with predefined T _RM cells upon integration. The presence of Hobit-expressing T _RM cells in the atherosclerotic lesion was confirmed in mice. These lesion-derived T _RM cells were characterized by the expression of CD69 and CD49α. Moreover, we demonstrated that this small T-cell subset significantly affects lesion composition, by reducing the amount of intralesional macrophages and increasing collagen content. CONCLUSIONS: T _RM cells, characterized by the expression of CD69 and CD49α, constitute a minor population in atherosclerotic lesions and are associated with increased lesion stability in a Hobit and Blimp-1 knockout mouse model.

INTRODUCTION: Viral infections have been associated with the progression of atherosclerosis and CD8 ⁺ T-cells directed against common viruses, such as influenza, Epstein-Barr virus, and cytomegalovirus, have been detected inside human atherosclerotic lesions. These virus-specific CD8 ⁺ T-cells have been hypothesized to contribute to the development of atherosclerosis; however, whether they affect disease progression directly remains unclear. In this study, we aimed to characterize the activation status of virus-specific CD8 ⁺ T-cells in the atherosclerotic lesion. METHODS: The presence, clonality, tissue enrichment, and phenotype of virus-associated CD8 ⁺ T-cells in atherosclerotic lesions were assessed by exploiting bulk T-cell receptor-β sequencing and single-cell T-cell receptor (α and β) sequencing datasets on human endarterectomy samples and patient-matched blood samples. To investigate if virus-specific CD8 ⁺ T-cells can be activated through T-cell receptor stimulation in the atherosclerotic lesion, the immunopeptidome of human plaques was determined. RESULTS: Virus-associated CD8 ⁺ T-cells accumulated more in the atherosclerotic lesion (mean=2.0%), compared with patient-matched blood samples (mean=1.4%; P =0.05), and were more clonally expanded and tissue enriched in the atherosclerotic lesion in comparison with nonassociated CD8 ⁺ T-cells from the lesion. Single-cell T-cell receptor sequencing and flow cytometry revealed that these virus-associated CD8 ⁺ T-cells were phenotypically highly similar to other CD8 ⁺ T-cells in the lesion and that both exhibited a more activated phenotype compared with circulating T-cells. Interestingly, virus-associated CD8 ⁺ T-cells are unlikely to be activated through antigen-specific interactions in the atherosclerotic lesion, as no virus-derived peptides were detected on HLA-I in the lesion. CONCLUSIONS: This study suggests that virus-specific CD8 ⁺ T-cells are tissue enriched in atherosclerotic lesions; however, their potential contribution to inflammation may involve antigen-independent mechanisms.

The bone marrow has emerged as a potentially important target in cardiovascular disease as it generates all leukocytes involved in atherogenesis. In the current study, we evaluated whether a change in bone marrow functionality underlies the increased atherosclerosis susceptibility associated with high-density lipoprotein (HDL) deficiency. We found that HDL deficiency in mice due to the genetic lack of hepatocyte-derived apolipoprotein A1 (APOA1) was associated with an increase in the Lin−Sca-1+Kit+ (LSK) bone marrow stem cell population and lymphoid-primed multipotent progenitor numbers, which translated into a higher production and systemic flux of T cell subsets. In accordance with APOA1 deficiency-associated priming of stem cells to increase T lymphocyte production, atherogenic diet-fed low-density lipoprotein receptor knockout mice transplanted with bone marrow from APOA1-knockout mice displayed marked lymphocytosis as compared to wild-type bone marrow recipients. However, atherosclerotic lesion sizes and collagen contents were similar in the two groups of bone marrow recipients. In conclusion, systemic lack of APOA1 primes bone marrow stem cells for T cell lymphopoiesis. Our data provide novel evidence for a regulatory role of HDL in bone marrow functioning in normolipidemic mice.

A hallmark of advanced atherosclerosis is inadequate immunosuppression by regulatory T (Treg) cells inside atherosclerotic lesions. Dyslipidemia has been suggested to alter Treg cell migration by affecting the expression of specific membrane proteins, thereby decreasing Treg cell migration towards atherosclerotic lesions. Besides membrane proteins, cellular metabolism has been shown to be a crucial factor in Treg cell migration. We aimed to determine whether dyslipidemia contributes to altered migration of Treg cells, in part, by affecting cellular metabolism. Dyslipidemia was induced by feeding Ldlr^−/− mice a western-type diet for 16–20 weeks and intrinsic changes in Treg cells affecting their migration and metabolism were examined. Dyslipidemia was associated with altered mTORC2 signalling in Treg cells, decreased expression of membrane proteins involved in migration, including CD62L, CCR7, and S1Pr1, and decreased Treg cell migration towards lymph nodes. Furthermore, we discovered that diet-induced dyslipidemia inhibited mTORC1 signalling, induced PPARδ activation and increased fatty acid (FA) oxidation in Treg cells. Moreover, mass-spectrometry analysis of serum from Ldlr^−/− mice with normolipidemia or dyslipidemia showed increases in multiple PPARδ ligands during dyslipidemia. Treatment with a synthetic PPARδ agonist increased the migratory capacity of Treg cells in vitro and in vivo in an FA oxidation-dependent manner. Furthermore, diet-induced dyslipidemia actually enhanced Treg cell migration into the inflamed peritoneum and into atherosclerotic lesions in vitro. Altogether, our findings implicate that dyslipidemia does not contribute to atherosclerosis by impairing Treg cell migration as dyslipidemia associated with an effector-like migratory phenotype in Treg cells.