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Circ Res. 2018 Jun 8;122(12):1675-1688. doi: 10.1161/CIRCRESAHA.117.312513. Epub 2018 Mar 15.

Atlas of the Immune Cell Repertoire in Mouse Atherosclerosis Defined by Single-Cell RNA-Sequencing and Mass Cytometry.

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From the Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, CA (H.W., E.E., M.V., K.B., H.Q.D., K.K., A.A.J.H., A.B.P., A.K.G., C.C.H., K.L., D.W.).
Institute of Experimental Biomedicine, University Hospital Würzburg, Germany (C.C., A.Z.).
Helmholtz Institute for RNA-based Infection Research, Würzburg, Germany (E.V., A.-E.S.).
Department of Cardiology and Angiology I, University Heart Center Freiburg, Germany (N.A.M., N.H., I.H., A.Z., D.W.).
the Faculty of Medicine, University of Freiburg, Germany (N.A.M., N.H., I.H., A.Z., D.W.).
Department of Bioengineering, University of California, San Diego (K.L.).
Institute of Experimental Biomedicine, University Hospital Würzburg, Germany (C.C., A.Z.)



Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of aortic leukocytes is poorly understood.


We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single-cell RNA-sequencing and mass cytometry (cytometry by time of flight) to define an atlas of the immune cell landscape in atherosclerosis.


Using single-cell RNA-sequencing of aortic leukocytes from chow diet- and Western diet-fed Apoe-/- and Ldlr-/- mice, we detected 11 principal leukocyte clusters with distinct phenotypic and spatial characteristics while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on the single-cell level established that multiple pathways, such as for lipid metabolism, proliferation, and cytokine secretion, were confined to particular leukocyte clusters. Leukocyte populations were differentially regulated in atherosclerotic Apoe-/- and Ldlr-/- mice. We confirmed the phenotypic diversity of these clusters with a novel mass cytometry 35-marker panel with metal-labeled antibodies and conventional flow cytometry. Cell populations retrieved by these protein-based approaches were highly correlated to transcriptionally defined clusters. In an integrated screening strategy of single-cell RNA-sequencing, mass cytometry, and fluorescence-activated cell sorting, we detected 3 principal B-cell subsets with alterations in surface markers, functional pathways, and in vitro cytokine secretion. Leukocyte cluster gene signatures revealed leukocyte frequencies in 126 human plaques by a genetic deconvolution strategy. This approach revealed that human carotid plaques and microdissected mouse plaques were mostly populated by macrophages, T-cells, and monocytes. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients.


The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunologic mechanisms and cell-type-specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis.


atherosclerosis; flow cytometry; immune system; leukocytes; lymphocytes; macrophages; mass cytometry; single-cell RNA-sequencing

[Available on 2019-06-08]

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