Multi-parameter immune profiling of peripheral blood mononuclear cells by multiplexed single-cell mass cytometry in patients with early multiple sclerosis

Abstract

Multiple sclerosis (MS) is an inflammatory demyelinating and neurodegenerative disease of the central nervous system (CNS). Studies in rodent models demonstrated an association of CNS-infiltrating monocyte-derived macrophages with disease severity. However, little is known about humans. Here, we performed an exploratory analysis of peripheral blood mononuclear cells (PBMCs) isolated from healthy controls and drug-naïve patients with early MS using multiplexed single-cell mass cytometry and algorithm-based data analysis. Two antibody panels comprising a total of 64 antibodies were designed to comprehensively analyse diverse immune cell populations, with particular emphasis on monocytes. PBMC composition and marker expression were overall similar between the groups. However, an increased abundance of CCR7+ and IL-6+ T cells was detected in early MS-PBMCs, whereas NFAT1hiT-bethiCD4+ T cells were decreased. Similarly, we detected changes in the subset composition of the CCR7+ and MIPβhi HLA-DR+ lymphocyte compartment. Only mild alterations were detected in monocytes/myeloid cells of patients with early MS, namely a decreased abundance of CD141hiIRF8hiCXCR3+CD68− dendritic cells. Unlike in Crohn’s disease, no significant differences were found in the monocyte fraction of patients with early MS compared to healthy controls. This study provides a valuable resource for future studies designed to characterise and target diverse PBMC subsets in MS.

Single-cell mass cytometry of microglia in major depressive disorder reveals a non-inflammatory phenotype with increased homeostatic marker expression

“…For the bead-based compensation of the signal spillover, AbC total antibody compensation beads (Thermo Fisher Scientific) were stained with each of the antibodies used in all three antibody panels according to the manufacturer’s instructions. Stained beads were then measured with CyTOF and the compensation matrix was generated as described previously 40 .…”

“…Following the workflow from our previous study 40 , Cytobank ( www.cytobank.org ) was used for initial manual gating on live single cells and Boolean gating for de-barcoding. Nucleated single intact cells were manually gated according to the signals of DNA intercalators 191 Ir/ 193 Ir and event length.…”

“…We then performed visual inspection of cluster-colored tSNE plots and phenotypic heat maps for a more detailed profile of each cluster, and we determined the number of meta-clusters with consistent phenotypes for statistical testing. Based on visual inspection of t-SNE plots and heat maps generated at the merging step, a final number of meta-clusters was chosen that merged clusters into populations with consistent phenotypes (with a minimal mean frequency of 0.1% of parent) 40 .…”

Mass Cytometry—A Tool for the Curious: Networking in Berlin

“…This development parallels the results from mass cytometry workshops held at CYTO . Many projects presented at the Forum were later published in peer‐reviewed journals .…”

“…GerMaNet also helps the swift distribution of new reagents and data analysis tools among the centers. This approach has proven highly effective, as reflected by several joint publications within the GerMaNet .…”