Literature study: high-end applications of mass cytometry (CyTOF)

Mass cytometry involves labeling antibodies with stable heavy metal isotopes (mainly lanthanides) in place of fluorophores

Figure 1.

Although mass cytometry is generally considered to be an alteration based on fluorescence flow cytometry, mass cytometry differs from fluorescence cytometry in several ways, including detection parameters, sensitivity,

Figure 2.

Current innovations in functional assays can be grouped into 12 categories: (A) phenotypic characterization; (B) intracellular cytokine assays; (C) intracellular signaling state characterization; (D) cell volume and size measurements; (E) cellular (F) cell cycle recognition; (G) proliferation tracking; (H) receptor occupancy assay; (I) tetramer-based antigen-specific T cell screening; (J) chromatin modification analysis; (K) ) RNA and protein co-detection; (L) Imaging mass flow cytometry

It is not difficult to find that the benefits of CyTOF in the field of immunology are obvious

Next, the editor will introduce the application of mass spectrometry in the study of single-cell metabolism and covid-19 pathogenesis in detail through two high-scoring papers

1.

Cellular metabolism regulates the activation, differentiation and effector functions of immune cells

In this study, we developed a method to characterize the regulation of single-cell metabolism and its phenotypic characteristics, the single-cell metabolic regulation map (scMEP) (Fig.

Figure 1.
1.
Overview of the scMEP method
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We obtained whole blood cells and identified major immune cell lineages by phenotypic markers, thus enabling in silico subset screening and comparison of metabolic regulation of cell lineages without prior isolation or enrichment
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We observed lineage-specific metabolic states consistent with previously identified functional roles
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Metabolic signatures were also found to be largely capable of isolating immune cell lineages and predicting the immune cell identity of most cells (Fig.
1.
2)
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demonstrated the suitability and robustness of scMEP for studying metabolic regulation and its relationship to single-cell specification, and revealed remarkable cell-type-specific metabolic diversification in the human immune system
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1.
2.
Single-cell metabolic regulation profiles organize the human immune system
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To establish the relationship between antibody-based single-cell metabolic signatures and pathway activity, we activated human naive and memory T cells, including CD4+ and CD8+, and analyzed them by mass flow cytometry and extracellular flux analysis.
Analysis
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Metabolic activity was found to be elevated upon pathway activation
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scMEP scores calculated independently for each cell showed that both glycolytic and oxidative mechanisms were simultaneously up-regulated at the single-cell level
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(As shown in Figure 1.
3)

These data validate the close relationship between scMEP-based quantification of cellular metabolic regulators and pathway activity assessed by a well-established orthogonal approach, and demonstrate that scMEP is capable of uncovering metabolic heterogeneity at the single-cell level
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Figure 1.
3.
Single-cell metabolome profiles of T-cell activation kinetics

To study the metabolic remodeling of human T cells and their relationship to cell activation, differentiation, and proliferation, we used mass cytometry (CyTOF) to benchmark scMEP in a large number of metabolic assays, observing that signaling molecules such as ribose Early phosphorylation of somatic protein S6) and induction of transcription factors such as hypoxia-inducible factor HIF1A, followed by upregulation of metabolic enzymes (GLUT1, HK2 and LDHA), indicate the onset of metabolic remodeling in activated naive T cells
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Using changes in metabolic signature expression over pseudotime, we define three inflection points during metabolic remodeling of human naive CD8+ T cells
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(As shown in Figure 1.
4)

Figure 1.
4.
An integrative model of metabolic reorganization reveals determinants of human T cell activation

In addition to cell-intrinsic factors, metabolic state is also influenced by its tissue, underscoring the importance of directly determining metabolic state in in vitro and clinical samples
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Applying this approach to clinical samples, we identified tissue-specific, metabolically suppressed cytotoxic T cells in human rectal cancer (Figure 1.
5)
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Figure 1.
5.
The metabolic state of cytotoxic T cells reflects their resident tissue

Combining this method with time-of-flight multiplexed ion beam imaging (MIBI-TOF), the researchers revealed the spatial organization of metabolic motifs in human tissues and showed that metabolically suppressed immune cells are excluded from tumor immunity
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(Figure 1.
6.
)

Figure 1.
6 Imaging-based scMEP analysis reveals the effect of space on the organization of metabolic signatures
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In conclusion, here we present a robust method to study single-cell metabolism using antibody-based multiplexing techniques
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The application of scMEP should contribute to a better understanding of human immune cell biology and to identify disease-associated metabolic alterations that serve as potential biomarkers and therapeutic targets for a variety of human diseases
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2.
The research group of Bali Pulendran of Stanford University School of Medicine revealed the pathogenesis and potential therapeutic targets of covid-19 (IF: 47.
7)

The novel coronavirus is spreading rapidly around the world, and research into isolating, sequencing and cloning the virus has progressed rapidly, including developing diagnostic kits and testing vaccine candidates
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However, little is known about the dynamic interactions between the human immune system and the SARS-CoV-2 virus
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In this study, the authors used mass cytometry, single-cell transcriptomics, and multifactorial assays to analyze the immune responses of 76 COVID-19 patients and 69 age-sex-matched controls from two regions
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The results show that SARS-CoV-2 infection leads to T cell exhaustion and apoptosis and impaired peripheral type I IFN responses, and plasma cytokine data can be used to differentiate severe and moderate COVID-19 symptoms
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In addition, severe COVID-19 infection is associated with systemic release of bacterial products, and molecules such as EN-RAGE, TNFSF14, OSM, and IL-6 and their receptors may be attractive therapeutic targets
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Figure 2.
1 Mass cytometry analysis of peripheral blood leukocytes from patients with COVID-19
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To determine immune cell typing in PBMCs, the authors used mass cytometry to analyze 22 cell surface markers and 12 intracellular markers, as well as phosphorylation-specific epitopes and histones against various kinases and signaling molecules Modification marker H3K27ac
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Phospho-CyTOF identified 12 major subtypes of innate and adaptive immune cells, with significantly increased frequencies of plasmablasts and effector CD8 T cells in all infected individuals in both cohorts (Fig.
2.
1B), and pDCs in PBMCs The frequency of pDC was significantly reduced (Fig.
2.
1C), and the kinetics of pDC responses were independent of the timing of symptoms and clinical severity
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In addition, expression of pS6, a canonical target of mTOR activation, was decreased, and IKBα was decreased in pDCs and mDCs (Fig.
2.
1D and E)
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mTOR signaling can mediate IFN-α production in pDCs, suggesting that the ability of pDCs to produce IFN-α may be impaired in COVID-19 patients
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Figure 2.
2 Flow cytometry analysis of peripheral blood leukocytes from patients with COVID-19 after in vitro stimulation
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Intracellular staining assay results showed that compared with healthy controls, IFN-α was reduced in pDCs stimulated by TLR in patients (Fig.
2.
2A), and TNF-α responses were also significantly reduced, suggesting that pDCs are involved in COVID-19 infection Impaired function
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Responses of mDCs and monocytes were also significantly reduced to stimulation with either bacterial ligand cocktails (consisting of TLR2, TLR4, and TLR5 ligands) or viral TLR cocktails (Fig.
2.
2B)
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Furthermore, decreased IKBα levels did not translate into enhanced NF-κβ subunit p65 phosphorylation (Fig.
2.
2C)
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The results suggest that the response of innate immune cells surrounding COVID-19 patients to TLR stimulation is suppressed regardless of clinical severity
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Figure 2.
3 Analysis of cytokine detection in plasma of COVID-19 patients
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Multifactorial assay analysis of plasma samples from the Atlanta cohort found that 43 cytokines, including IL-6, MCP-3, and CXCL10, were significantly upregulated in COVID-19 patients (Fig.
2.
3, top)
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Although cytokine responses in blood myeloid cells and pDCs were impaired, plasma levels of inflammatory molecules were significantly upregulated, suggesting that the tissue source of cytokines in plasma is controversial
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In addition, both multifactorial and ELISA confirmed that TNFSF14, EN-RAGE and OSM were also significantly enhanced in COVID-19 patients and were closely related to clinical severity (Fig.
2.
3 bottom)
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The results suggest that COVID-19 infection induces a unique inflammatory program that may be characterized by the release of cytokines from the lungs but suppresses the peripheral innate immune system
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Figure 2.
4 Transient expression of ISG early in COVID-19 infection
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Data from the single-cell large-scale multidimensional analysis tool CITE-seq revealed several clusters in COVID-19 patients including plasmablasts, platelets, erythrocytes, and granulocyte populations (Fig.
2.
4B)
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An overrepresentational analysis of DEGs showed that many ISGs were upregulated in monocytes and dendritic cells (Fig.
2.
4C), with the exception of modest levels of IFN-γ expression in T and NK cells, without any detectable IFN-α and - The expression of β gene (Fig.
2.
4D), the concentration of IFN-α in plasma was verified by high-sensitivity ELISA, and it was found that the concentration of IFN-α in the plasma gradually increased to a peak around the 8th day after infection, and fell back to the baseline level on the 20th day (Fig.
2.
4E)
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Both CITE-seq and RNA-seq data showed elevated ISG levels in patients, but were also influenced by clinical severity (Fig.
2.
4)
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In the early stage of SARS-CoV-2 infection, circulating IFN-α levels that can induce ISG in the periphery are low, and thus the ability of peripheral innate immune cells to produce inflammatory cytokines is impaired
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The higher-resolution quantitative detection of protein markers on the surface of single cells by mass spectrometry, combined with other single-cell-related technologies, can make new discoveries more rapid and accurate
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More detailed article content can be viewed by downloading the references attached at the end of the article~

Literature-related antibodies and heavy metal markers (some products):

In order to carry out more effective antibody labeling, mass spectrometry experiments require that the antibody buffer does not contain any carrier, that is, does not contain glycerol, sodium azide and other components
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However, most of the antibodies selected in the two literatures contain glycerol components.
If such antibodies are selected, they can be removed through the glycerol removal program (contact Unionville to obtain them)
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In addition, the CST and RnD brands of Univitamin provide you with ready-to-use high-quality carrier-free protein antibodies.
The product advantages are shown in the following figure: