Differential compartmentalization of myeloid cell phenotypes and responses towards the CNS in Alzheimer's disease.

Myeloid cells are suggested as an important player in Alzheimer´s disease (AD). However, its continuum of phenotypic and functional changes across different body compartments and their use as a biomarker in AD remains elusive. Here, we perform multiple state-of-the-art analyses to phenotypically and metabolically characterize immune cells between peripheral blood (n = 117), cerebrospinal fluid (CSF, n = 117), choroid plexus (CP, n = 13) and brain parenchyma (n = 13). We find that CSF cells increase expression of markers involved in inflammation, phagocytosis, and metabolism. Changes in phenotype of myeloid cells from AD patients are more pronounced in CP and brain parenchyma and upon in vitro stimulation, suggesting that AD-myeloid cells are more vulnerable to environmental changes. Our findings underscore the importance of myeloid cells in AD and the detailed characterization across body compartments may serve as a resource for future studies focusing on the assessment of these cells as biomarkers in AD.

The use and limitations of single-cell mass cytometry for studying human microglia function.

Microglia, the resident innate immune cells of the central nervous system (CNS), play an important role in brain development and homoeostasis, as well as in neuroinflammatory, neurodegenerative and psychiatric diseases. Studies in animal models have been used to determine the origin and development of microglia, and how these cells alter their transcriptional and phenotypic signatures during CNS pathology. However, little is known about their human counterparts. Recent studies in human brain samples have harnessed the power of multiplexed single-cell technologies such as single-cell RNA sequencing (scRNA-seq) and mass cytometry (cytometry by time-of-flight [CyTOF]) to provide a comprehensive molecular view of human microglia in healthy and diseased brains. CyTOF is a powerful tool to study high-dimensional protein expression of human microglia (huMG) at the single-cell level. This technology widens the possibilities of high-throughput quantification (of over 60 targeted molecules) at a single-cell resolution. CyTOF can be combined with scRNA-seq for comprehensive analysis, as it allows single-cell analysis of post-translational modifications of proteins, which provides insights into cell signalling dynamics in targeted cells. In addition, imaging mass cytometry (IMC) has recently become commercially available, and will be useful for analysing multiple cell types in human brain sections. IMC leverages mass spectrometry to acquire spatial data of cell-cell interactions on tissue sections, using (theoretically) over 40 markers at the same time. In this review, we summarise recent studies of huMG using CyTOF and IMC analyses. The uses and limitations as well as future directions of these technologies are discussed.

Single-cell mass cytometry reveals complex myeloid cell composition in active lesions of progressive multiple sclerosis.

Myeloid cells contribute to inflammation and demyelination in the early stages of multiple sclerosis (MS), but it is still unclear to what extent these cells are involved in active lesion formation in progressive MS (PMS). Here, we have harnessed the power of single-cell mass cytometry (CyTOF) to compare myeloid cell phenotypes in active lesions of PMS donors with those in normal-appearing white matter from the same donors and control white matter from non-MS donors. CyTOF measurements of a total of 74 targeted proteins revealed a decreased abundance of homeostatic and TNFhi microglia, and an increase in highly phagocytic and activated microglia states in active lesions of PMS donors. Interestingly, in contrast to results obtained from studies of the inflammatory early disease stages of MS, infiltrating monocyte-derived macrophages were scarce in active lesions of PMS, suggesting fundamental differences of myeloid cell composition in advanced stages of PMS.

Brick plots: an intuitive platform for visualizing multiparametric immunophenotyped cell clusters.

BACKGROUND: The advent of mass cytometry has dramatically increased the parameter limit for immunological analysis. New approaches to analysing high parameter cytometry data have been developed to ease analysis of these complex datasets. Many of these methods assign cells into population clusters based on protein expression similarity. RESULTS: Here we introduce an additional method, termed Brick plots, to visualize these cluster phenotypes in a simplified and intuitive manner. The Brick plot method generates a two-dimensional barcode that displays the phenotype of each cluster in relation to the entire dataset. We show that Brick plots can be used to visualize complex mass cytometry data, both from fundamental research and clinical trials, as well as flow cytometry data. CONCLUSION: Brick plots represent a new approach to visualize complex immunological data in an intuitive manner.

High-Dimensional Mass Cytometric Analysis Reveals an Increase in Effector Regulatory T Cells as a Distinguishing Feature of Colorectal Tumors.

T cell infiltration of tumors plays an important role in determining colorectal cancer disease progression and has been incorporated into the Immunoscore prognostic tool. In this study, mass cytometry was used to demonstrate a significant increase in the frequency of both conventional CD25+FOXP3+CD127lo regulatory T cells (Tregs) as well as BLIMP-1+ Tregs in the tumor compared with nontumor bowel (NTB) of the same patients. Network cluster analyses using SCAFFoLD, VorteX, and CITRUS revealed that an increase in BLIMP-1+ Tregs was a single distinguishing feature of the tumor tissue compared with NTB. BLIMP-1+ Tregs represented the most significantly enriched T cell population in the tumor compared with NTB. The enrichment of ICOS, CD45RO, PD-1, PDL-1, LAG-3, CTLA-4, and TIM-3 on BLIMP-1+ Tregs suggests that BLIMP-1+ Tregs have a more activated phenotype than conventional Tregs and may play a role in antitumor immune responses.