Pathogenic Autoimmunity in Atherosclerosis Evolves From Initially Protective Apolipoprotein B ₁₀₀ –Reactive CD4 ⁺ T-Regulatory Cells

Abstract

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.

Heterogeneity of T Cells in Atherosclerosis Defined by Single-Cell RNA-Sequencing and Cytometry by Time of Flight

“… 47 Ultimately, novel immunophenotyping workflows may be combined with reagents to detect antigen-specific T H cells recognizing particular antigenic peptide laden MHC multimers labeled with fluorochromes. 58 Recently, we have demonstrated the existence of human and mouse ApoB-specific T H cells on a single-cell level by MHC-II tetramers 3 , 5 and linked these to single-cell transcriptomes. 5 These technical developments helped to describe several novel layers of T-cell diversity in atherosclerosis with respect to spatial, temporal, phenotypic, antigen-specific, and transcriptional resolution that fundamentally differs from traditional discrimination and nomenclature of CD4 + T cells.…”

“…Traditional immunophenotyping by fluorescence-activated cell sorting is per se limited by the usage of predefined sets of antibodies: in most studies, staining for CD62L and CD44, which discriminates between naive, effector-memory T cells, and central-memory T cells in the mouse, has been combined with intracellular expression of T H -defining TFs or cytokines for canonical gating and identification strategies. 2 , 3 , 5 , 28 , 69 Contrastingly, in most available scRNA-seq studies, T H -cell phenotypes were inferred from dimensionality reduction algorithms to detect clusters of cells with similar transcriptome without the bias of preselecting markers. Several T-cell populations that partially overlap have been identified by these screening approaches: 4 in Apoe −/− 39 and in Ldlr −/− mice, 46 3 populations in adventitial cell preparations from Apoe −/− and wild-type mice, 52 and 5 clusters in an integrative analysis of published datasets.…”

“… 2 Increasing evidence argues for the presence of CD4 + T cells recognizing peptides from ApoB (apolipoprotein B)-100, an established self-antigen in atherosclerosis. 3 – 5 The presentation of cognate peptides by antigen-presenting cells (APCs) on surface-expressed MHC-II induces the activation and differentiation of naive CD4 + T cells into functionally and phenotypically distinct T-helper (T H ) types that express exclusive sets of transcription factors (TFs) and cytokines. 6 , 7 T H type 1 (T H 1) CD4 + T cells are proatherogenic and express the TF T-bet and the lead-cytokine IFN-γ (interferon-gamma).…”

“… 29 While numbers of T regs decrease during acute coronary syndromes in humans, 30 – 32 frequencies of blood T regs are higher in patients with angiographically documented atherosclerosis compared with healthy individuals. 5 Other studies have failed to establish that frequencies of blood T regs predict future myocardial infarction. 33 Traditional TF staining in mouse atherosclerotic aortas by intracellular flow cytometry (fluorescence-activated cell sorting) and a canonical gating strategy has suggested a mix of FoxP3 + (T reg ), RORγT + (T H 17), and T-bet + (T H 1) CD4 + T cells, 5 , 29 , 34 – 36 while the quantification of T H -associated surface-expressed chemokine-receptor patterns in humans suggests a predominant T H 1 and T H 2 phenotype.…”

“… 5 Other studies have failed to establish that frequencies of blood T regs predict future myocardial infarction. 33 Traditional TF staining in mouse atherosclerotic aortas by intracellular flow cytometry (fluorescence-activated cell sorting) and a canonical gating strategy has suggested a mix of FoxP3 + (T reg ), RORγT + (T H 17), and T-bet + (T H 1) CD4 + T cells, 5 , 29 , 34 – 36 while the quantification of T H -associated surface-expressed chemokine-receptor patterns in humans suggests a predominant T H 1 and T H 2 phenotype. 8 , 37 These partially conflicting findings have sparked several fundamental questions: are patterns of cytokines, chemokine receptors, and transcription factors sufficient to define functionally relevant phenotypes of lesional and circulating T-helper cells?…”