Immunopeptidomic analysis of human atherosclerosis identifies novel ApoB100-derived antigenic drivers of atherosclerosis

Background and Aims: Recent work suggests atherosclerosis has an auto-immune component, however the main antigenic drivers of T cell responses in this disease are still not well defined. ApoB100 might have an important role but previous attempts to identify immunogenic epitopes of ApoB100 have been based on the screening of the protein sequence and in silico prediction of strong HLA binders. Methods: We used an immunopeptidomic approach to study the peptide repertoire presented by HLA-DR in human atherosclerosis plaques. Selected peptides were used to stimulate patients PBMCs, followed by analysis of CD40L expression in CD4 T cells using flow cytometry and cytokine production using multiplex ELISA. Results: We identified ApoB100 as one of the main sources of peptides presented by HLA-DR in the plaque and selected 20 epitopes from this protein based on their predicted binding affinity for a wide range of HLA-DR isotypes. Next, we studied the presence of antigen-specific CD4 T cells against these epitopes in PBMCs of patients. Results revealed a subgroup of patients (25%) that presented significant CD4 T cell activation in response to these ApoB100 peptides. Furthermore, this T cell response correlated positively with plaque vulnerability, a parameter histologically determined by taking into account neovascularization and necrotic core, calcified, foam cell, cholesterol crystal and inflammatory cell content. Finally, the analysis of the cytokines produced by the stimulated PBMCs showed increased production of IL10 and IL17A but not IFNγ. Conclusions: In conclusion, we identified 20 plaque derived epitopes from ApoB100 which could be used as biomarkers of disease progression and potential targets for therapeutic manipulation.