Fluorescent activated cell sorting: An effective approach to study dendritic cell subsets in human atherosclerotic plaques

Abstract

Different immune cell types are present within atherosclerotic plaques. Dendritic cells (DC) are of special interest, since they are considered as the 'center of the immuniverse'. Identifying inflammatory DC subtypes within plaques is important for a better understanding of the lesion pathogenesis and pinpoints their contribution to the atherosclerotic process. We have developed a flow cytometry-based method to characterize and isolate different DC subsets (i.e. CD11b(+), Clec9A(+) and CD16(+) conventional (c)DC and CD123(+) plasmacytoid (p)DC) in human atherosclerotic plaques. We revealed a predominance of pro-inflammatory CD11b(+) DC in advanced human lesions, whereas atheroprotective Clec9A(+) DC were almost absent. CD123(+) pDC and CD16(+) DC were also detectable in plaques. Remarkably, plaques from distinct anatomical locations exhibited different cellular compositions: femoral plaques contained less CD11b(+) and Clec9A(+) DC than carotid plaques. Twice as many monocytes/macrophages were observed compared to DC. Moreover, relative amounts of T cells/B cells/NK cells were 6 times as high as DC numbers. For the first time, fluorescent activated cell sorting analysis of DC subsets in human plaques indicated a predominance of CD11b(+) cDC, in comparison with other DC subsets. Isolation of the different subsets will facilitate detailed functional analysis and may have significant implications for tailoring appropriate therapy.

Hypercholesterolemia Induces a Mast Cell–CD4+ T Cell Interaction in Atherosclerosis

“…The handling of all human samples was performed in accordance with the Code for Proper Secondary Use of Human Tissue. All atherosclerotic samples were processed in single-cell suspensions, as described previously (35). In short, cell suspensions from human atherosclerotic plaques were obtained upon digestion with collagenase IV (Life Technologies) and DNase (Sigma-Aldrich) for 2 h at 37˚C prior to single-cell separation through a 70-mm cell strainer.…”

CD39 identifies a microenvironment-specific anti-inflammatory CD8+ T-cell population in atherosclerotic lesions

“…The handling of all human samples complied with the "Code for Proper Secondary Use of Human Tissue" and are in accordance with the declaration of Helsinki regarding ethical principles for medical research involving human subjects (METC registration number 17-046). Single-cell suspensions were obtained from human plaques as described previously [13] (see Supplementary Data for a full description).…”

Isolation of dendritic cells from umbilical cord blood using magnetic activated cell sorting or adherence

“…In practice, DCs can be generated from two primary cell types: CD34 + cells and monocytes (13). There are a number of ways to isolate monocytes, including plastic adherence and specific marker-based separation techniques, such as MACS and fluorescent activated cell sorting (15). However, DCs derived .…”