ApoB-Specific CD4+ T Cells in Mouse and Human Atherosclerosis

Abstract

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 (T_H) 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 T_H 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 T_H 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.

Keywords: LDL; T cells; apolipoprotein B; atherosclerosis; autoimmunity; immunity.

Figure 1

Approaches to detect Apolipoprotein B (ApoB)-specific CD4⁺ T cells in mice and humans. Functional restimulation with ApoB-peptides requires a peptide mapping of the entire ApoB-sequence and a selection of peptides with the highest predicted and tested affinity towards the MHC-II complex. Antigen-presenting cells are loaded with selected peptides and co-incubated with CD4⁺ T cells. Expression of activation markers is subsequently used to identify activated, ApoB-reactive T cells (A) Design of tetramers of MHC-II loaded with the peptide of interest. Tetramers are labelled with fluorochromes for the detection of tetramer-binding T cells in flow cytometry. (B) In simultaneous T cell receptor (TCR) and gene expression RNA-sequencing, T cells with an oligoclonal repertoire of the TCR are detected on a single cell level, which allows to selectively analyze gene expression in these cells. (C) Workflow for the generation of a TCR-transgenic mouse: peptide-specific T cells are expanded in vivo by immunization with LDL or a self-peptide from ApoB, isolated, and further expanded ex vivo by restimulation with LDL or peptide-loaded APC. Resulting clones undergo TCR-sequencing and the most promising TCR-sequences are used as templates for the generation of a TCR-transgenic mouse (D).

Figure 2

Activation and differentiation of ApoB-specific CD4⁺ T cells in mice. The ApoB-containing apolipoproteins LDL, VLDL, and chylomicrons (CM) are taken up by antigen-presenting cells (APCs) by endocytosis. After their intracellular processing, apolipoprotein-derived peptides are loaded on MHC-II molecules, before the entire MHC-II-peptide complex is transposed to the cell membrane. There, MHC-II-peptide complexes can be recognized and bound by a specific T cell receptor (TCR). In combination with sufficient co-stimulatory signaling events provided by the APC, a naïve CD4⁺ T cell is activated and may differentiate into distinct, partially overlapping T_H-types of immunity: Most ApoB-specific CD4⁺ T cells (ApoB⁺) express transcriptomes and markers of T_H17 and T-regulatory cells (T_reg). They express CCR5 and CXCR6, two known chemokine receptors (CCRs) required for aortic homing. Over time, ApoB⁺ cells acquire additional pro-inflammatory transcriptional programs and express the T_H1 transcription factor T-bet, as well as the CCRs CXCR5 and CCR6. The initially detectable protective T_reg signature is lost in this process. After vaccination, IL-10 secreting FoxP3⁺ApoB⁺ cells have been described at the site of vaccination. In transgenic mice, only expressing a TCR that recognizes a specific ApoB-peptide, a part of ApoB⁺ cells differentiates into T_FH that promote plasma cells generation and the production of LDL-lowering anti-LDL antibodies.