Background: Throughout the inflammatory response that accompanies atherosclerosis, autoreactive CD4 + T-helper cells accumulate in the atherosclerotic plaque. Apolipoprotein B 100 (apoB), the core protein of low-density lipoprotein, is an autoantigen that drives the generation of pathogenic T-helper type 1 (T H 1) cells with proinflammatory cytokine secretion. Clinical data suggest the existence of apoB-specific CD4 + T cells with an atheroprotective, regulatory T cell (T reg ) phenotype in healthy individuals. Yet, the function of apoB-reactive T regs and their relationship with pathogenic T H 1 cells remain unknown. Methods: To interrogate the function of autoreactive CD4 + T cells in atherosclerosis, we used a novel tetramer of major histocompatibility complex II to track T cells reactive to the mouse self-peptide apo B 978-993 (apoB + ) at the single-cell level. Results: We found that apoB + T cells build an oligoclonal population in lymph nodes of healthy mice that exhibit a T reg -like transcriptome, although only 21% of all apoB + T cells expressed the T reg transcription factor FoxP3 (Forkhead Box P3) protein as detected by flow cytometry. In single-cell RNA sequencing, apoB + T cells formed several clusters with mixed T H signatures that suggested overlapping multilineage phenotypes with pro- and anti-inflammatory transcripts of T H 1, T helper cell type 2 (T H 2), and T helper cell type 17 (T H 17), and of follicular-helper T cells. ApoB + T cells were increased in mice and humans with atherosclerosis and progressively converted into pathogenic T H 1/T H 17-like cells with proinflammatory properties and only a residual T reg transcriptome. Plaque T cells that expanded during progression of atherosclerosis consistently showed a mixed T H 1/T H 17 phenotype in single-cell RNA sequencing. In addition, we observed a loss of FoxP3 in a fraction of apoB + T regs in lineage tracing of hyperlipidemic Apoe –/– mice. In adoptive transfer experiments, converting apoB + T regs failed to protect from atherosclerosis. Conclusions: Our results demonstrate an unexpected mixed phenotype of apoB-reactive autoimmune T cells in atherosclerosis and suggest an initially protective autoimmune response against apoB with a progressive derangement in clinical disease. These findings identify apoB autoreactive T regs as a novel cellular target in atherosclerosis.

Atherosclerosis is a chronic inflammatory condition of the arterial wall that leads to the formation of vessel-occluding plaques within the subintimal space of middle-sized and larger arteries. While traditionally understood as a myeloid-driven lipid-storage disease, growing evidence suggests that the accumulation of low-density lipoprotein cholesterol (LDL-C) ignites an autoimmune response with CD4+ T-helper (TH) cells that recognize self-peptides from Apolipoprotein B (ApoB), the core protein of LDL-C. These autoreactive CD4+ T cells home to the atherosclerotic plaque, clonally expand, instruct other cells in the plaque, and induce clinical plaque instability. Recent developments in detecting antigen-specific cells at the single cell level have demonstrated that ApoB-reactive CD4+ T cells exist in humans and mice. Their phenotypes and functions deviate from classical immunological concepts of distinct and terminally differentiated TH immunity. Instead, ApoB-specific CD4+ T cells have a highly plastic phenotype, can acquire several, partially opposing and mixed transcriptional programs simultaneously, and transit from one TH subset into another over time. In this review, we highlight adaptive immune mechanisms in atherosclerosis with a focus on CD4+ T cells, introduce novel technologies to detect ApoB-specific CD4+ T cells at the single cell level, and discuss the potential impact of ApoB-driven autoimmunity in atherosclerosis.

RationaleAtherosclerosis is a chronic inflammatory disease of large arteries that involves an autoimmune response with autoreactive T cells and auto-antibodies recognizing Apolipoprotein B (ApoB), the core protein of low-density lipoprotein (LDL). Here, we aimed to establish a clinical association between circulating human ApoB auto-antibodies with atherosclerosis and its clinical risk factors using a novel assay to detect auto-antibodies against a pool of highly immunogenic ApoB-peptides.Methods and ResultsTo detect polyclonal IgM- and IgG-antibodies recognizing ApoB, we developed a chemiluminescent sandwich ELISA with 30 ApoB peptides selected by an in silico assay for a high binding affinity to MHC-II, which cover more than 80% of known MHC-II variants in a Caucasian population. This pre-selection of immunogenic self-peptides accounted for the high variability of human MHC-II, which is fundamental to allow T cell dependent generation of IgG antibodies. We quantified levels of ApoB-autoantibodies in a clinical cohort of 307 patients that underwent coronary angiography. Plasma anti-ApoB IgG and IgM concentrations showed no differences across healthy individuals (n = 67), patients with coronary artery disease (n = 179), and patients with an acute coronary syndrome (n = 61). However, plasma levels of anti-ApoB IgG, which are considered pro-inflammatory, were significantly increased in patients with obesity (p = 0.044) and arterial hypertension (p < 0.0001). In addition, patients diagnosed with the metabolic syndrome showed significantly elevated Anti-ApoB IgG (p = 0.002). Even when normalized for total plasma IgG, anti-ApoB IgG remained highly upregulated in hypertensive patients (p < 0.0001). We observed no association with triglycerides, total cholesterol, VLDL, or LDL plasma levels. However, total and normalized anti-ApoB IgG levels negatively correlated with HDL. In contrast, total and normalized anti-ApoB IgM, that have been suggested as anti-inflammatory, were significantly lower in diabetic patients (p = 0.012) and in patients with the metabolic syndrome (p = 0.005).ConclusionUsing a novel ELISA method to detect auto-antibodies against ApoB in humans, we show that anti-ApoB IgG associate with cardiovascular risk factors but not with the clinical appearance of atherosclerosis, suggesting that humoral immune responses against ApoB are shaped by cardiovascular risk factors but not disease status itself. This novel tool will be helpful to develop immune-based risk stratification for clinical atherosclerosis in the future.