Sniffing out atherosclerosis Olfactory receptors are best known for their presence in the nose and their role in detecting smells, but they are also present in other tissues and perform additional biological functions. For example, vascular macrophages involved in the pathogenesis of atherosclerosis express multiple subtypes of olfactory receptors. Orecchioni et al . focused on olfactory receptor 2, a receptor for the compound octanal, and identified its contribution to atherosclerosis pathogenesis and the formation of atherosclerotic plaques (see the Perspective by Rayner and Rasheed). The authors show that most of the octanal was not directly derived from the diet, but rather was generated as a by-product of lipid peroxidation, suggesting a potential pathway for intervention. —YN

Regulatory T cells (Tregs) express the lineage-defining transcription factor FoxP3 and play crucial roles in self-tolerance and immune homeostasis. Thymic tTregs are selected based on affinity for self-antigens and are stable under most conditions. Peripheral pTregs differentiate from conventional CD4 T cells under the influence of TGF-β and other cytokines and are less stable. Treg plasticity refers to their ability to inducibly express molecules characteristic of helper CD4 T cell lineages like T-helper (Th)1, Th2, Th17 or follicular helper T cells. Plastic Tregs retain FoxP3 and are thought to be specialized regulators for “their” lineage. Unstable Tregs lose FoxP3 and switch to become exTregs, which acquire pro-inflammatory T-helper cell programs. Atherosclerosis with systemic hyperlipidemia, hypercholesterolemia, inflammatory cytokines, and local hypoxia provides an environment that is likely conducive to Tregs switching to exTregs.

BACKGROUND: CD (cluster of differentiation) 4 + T-cell responses to APOB (apolipoprotein B) are well characterized in atherosclerotic mice and detectable in humans. CD4 + T cells recognize antigenic peptides displayed on highly polymorphic HLA (human leukocyte antigen)-II. Immunogenicity of individual APOB peptides is largely unknown in humans. Only 1 HLA-II-restricted epitope was validated using the DRB1*07:01-APOB 3036 –3050 tetramer. We hypothesized that human APOB may contain discrete immunodominant CD4 + T-cell epitopes that trigger atherosclerosis-related autoimmune responses in donors with diverse HLA alleles. METHODS: We selected 20 APOB-derived peptides (APOB 20 ) from an in silico screen and experimentally validated binding to the most commonly occurring human HLA-II alleles. We optimized a restimulation-based workflow to evaluate antigenicity of multiple candidate peptides in HLA-typed donors. This included activation-induced marker assay, intracellular cytokine staining, IFNγ (interferon gamma) enzyme–linked immunospot and cytometric bead array. High-throughput sequencing delineated TCR (T-cell receptor) clonalities of APOB-reactive CD4 + T cells. RESULTS: Using stringent positive, negative, and crossover stimulation controls, we confirmed specificity of expansion-based protocols to detect CD4 + T cytokine responses to APOB 20 pool. Ex vivo assessment of AIM + CD4 + T cells revealed statistically significant autoimmune response to APOB 20 but not to a ubiquitously expressed negative control protein, actin. Resolution of CD4 + T responses to the level of individual peptides using IFNγ enzyme–linked immunospot led to the discovery of 6 immunodominant epitopes (APOB 6 ) that triggered robust CD4 + T activation in most donors. APOB 6 -specific responding CD4 + T cells were enriched in unique expanded TCR clonotypes and preferentially expressed memory markers. Cytometric bead array analysis detected APOB 6 -induced secretion of both proinflammatory and regulatory cytokines. In clinical samples from patients with angiographically verified coronary artery disease, APOB 6 stimulation induced higher activation and memory phenotypes and augmented secretion of proinflammatory cytokines TNF (tumor necrosis factor) and IFNγ, compared with patients with low coronary artery disease. CONCLUSIONS: Using 3 cohorts, each with ≈20 donors, we discovered and validated 6 immunodominant, HLA-II–restricted APOB epitopes. Immune response to these APOB epitopes correlated with coronary artery disease severity.

Despite the decades-old knowledge that males and people with diabetes mellitus (DM) are at increased risk for coronary artery disease (CAD), the reasons for this association are only partially understood. Among the immune cells involved, recent evidence supports a critical role of T cells as drivers and modifiers of CAD. CD4+ T cells are commonly found in atherosclerotic plaques. We aimed to understand the relationship of CAD with sex and DM by single-cell RNA (scRNA-Seq) and antibody sequencing (CITE-Seq) of CD4+ T cells. Peripheral blood mononuclear cells (PBMCs) of 61 men and women who underwent cardiac catheterization were interrogated by scRNA-Seq combined with 49 surface markers (CITE-Seq). CAD severity was quantified using Gensini scores, with scores above 30 considered CAD+ and below 6 considered CAD−. Four pairs of groups were matched for clinical and demographic parameters. To test how sex and DM changed cell proportions and gene expression, we compared matched groups of men and women, as well as diabetic and non-diabetic subjects. We analyzed 41,782 single CD4+ T cell transcriptomes for sex differences in 16 women and 45 men with and without coronary artery disease and with and without DM. We identified 16 clusters in CD4+ T cells. The proportion of cells in CD4+ effector memory cluster 8 (CD4T8, CCR2+ Em) was significantly decreased in CAD+, especially among DM+ participants. This same cluster, CD4T8, was significantly decreased in female participants, along with two other CD4+ T cell clusters. In CD4+ T cells, 31 genes showed significant and coordinated upregulation in both CAD and DM. The DM gene signature was partially additive to the CAD gene signature. We conclude that (1) CAD and DM are clearly reflected in PBMC transcriptomes, and (2) significant differences exist between women and men and (3) between subjects with DM and non-DM.

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