Background:

Acute coronary syndromes (ACS) remain the most devastating clinical manifestation of cardiovascular disease, the most common cause of global mortality. ACS frequently arises from rupture of the coronary fibrous cap (RFC = ruptured fibrous cap), in which a highly thrombogenic necrotic core is exposed to circulating blood, thereby triggering thrombus formation and impairing myocardial perfusion. While inflammation has been implicated as a key mechanism contributing to atherosclerotic plaque vulnerability and rupture, the underlying mechanisms leading to coronary plaque erosion (IFC = Intact fibrous cap), another form of ACS, are not well understood. In the recent findings of the OPTICO-ACS study, flow cytometric (FACS) analysis of blood from the site of IFC culprit lesion (CL) showed enrichment of both CD4⁺ and CD8⁺ T cells (+8.1% and +11.2%, respectively, both P<0.05) as well as effector molecules such as granzyme A (+22.4%), perforin (+58.8%), and granulysin (+75.4%) as compared with RFC-CL sites. The proximity of IFC lesions to coronary bifurcations seen by optical coherence tomography (OCT) provides further indications linking shear stress alterations to the occurrence of IFC type ACS. We aim to further explore the underlying immune cell mechanisms of coronary plaque erosion via a novel single cell multi-omic approach, never before established from samples aspirated along the arterial sheath.

Methods and Results:

Patients presenting to the Charité University Hospital with Myocardial Infarction (MI) underwent emergent coronary angiography & percutaneous coronary intervention. MI was characterized as either IFC or RFC by OCT, in which then blood samples were obtained by aspiration directly along the arterial sheath (right radial artery: n=155/91%; femoral artery: n=25/9%). Peripheral blood mononuclear cells were isolated via density gradient within four hours of sample aspiration and cryopreserved. Samples from 24 patients were promptly thawed, washed and incubated with 137 oligonucleotide barcoded antibodies (Totaseq-C-Kit, Biolegend) to detect surface protein expression as well as a cellular hashtag antibody (Biolegend) – an antibody-oligonucleotide conjugate allowing for sample multiplexing. After cell surface staining, viable cells were sorted by FACS and single cell libraries were prepared via the 10x Chromium Controller and 5’-V(D)J reagents (both 10x Genomics) to simultaneously measure transcriptomes, epitopes and T cell receptor (TCR) clonality (CITE+TCR) by sequencing with the NovaSeq6000 (Illumina). To this end, bioinformatic integration of single cell epitope and transcriptome information – via weighted nearest neighbor analysis (WNN) – found 32 unique leukocyte clusters as well as heterogeneous T cell oligoclonality indicative of antigen specific expansion. In  ICF-ACS patients, more T cell clonotypes expanded, yet the size of few individual clones was higher in controls and RFC-ACS. As an excerpt and in complement to our published FACS data, we observed increased CCL4 expression in CD8⁺ T-lymphocytes within RFC-ACS as well as increased GNLY (encoding for granulysin) in CD8⁺ T-lymphocytes within IFC-ACS.

Conclusion:

Our high-parametric immune cell analysis shows distinct differences in the adaptive immune system, particularly CD8⁺ T-lymphocytes and their effector molecules, in the pathogenesis of IFC-ACS vs. RFC-ACS, providing novel insights in the underlying mechanisms leading in either ACS form.