Immunological risk identified by single-cell multi-omics and cardiovascular outcomes

BACKGROUND AND AIMS: While inflammation and (auto-)immunity are modifiers of atherosclerosis, immunological determinants of cardiovascular risk beyond the established inflammatory markers, high-sensitivity C-reactive protein (hsCRP) and interleukin-6 (IL-6) remain incompletely defined in humans. This exploratory proof-of-concept study aimed to detect immunological signals enriched in patients with increased cardiovascular risk. METHODS: A nested case-control study was performed within the Ludwigshafen Risk and Cardiovascular Health (LURIC) cohort, a single-centre prospective study of 3316 patients. Forty-four individuals with and without angiographically confirmed coronary artery disease (CAD) and defined survival status were selected for comprehensive immunophenotyping employing bulk RNA, single-cell RNA (scRNA), single-cell T-cell receptor sequencing, mass cytometry, spatial imaging, and cytokine secretion in blood immune cells. Multi-omics factor analysis was applied to identify shared transcriptional programmes. Readouts were tested for their association with CAD status and cardiovascular mortality and validated in two independent cohorts. RESULTS: The integrated analysis strategy revealed immunological signals not detectable with conventional biomarkers, including a prominent signature of cellular proliferation, linked to activation, memory formation, clonal expansion, interferon signalling, and cytokine secretion in T cells. Cell types and transcriptional programmes associated with CAD status and poor long-term survival were identified. These associations were independent of established inflammatory markers, hsCRP and IL-6 that originated from myeloid cells. Key findings were replicated across independent cohorts using 136 scRNA sequencing datasets. Cellular proliferation was confirmed by protein validation in atherosclerotic plaques, and signatures were evaluated as stratification tools to predict short-term outcomes in acute coronary syndromes. CONCLUSIONS: Multi-omic profiling of blood immune cells revealed novel immune signatures associated with CAD, short- and long-term cardiovascular disease outcomes that are independent of systemic inflammation and may be used as risk stratification tools in the future.