scholarly journals Reticulated platelet mass cytometry reveals unexplored therapeutic targets in patients with chronic coronary syndrome

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
D Bongiovanni ◽  
M Klug ◽  
O Lazareva ◽  
K Kirmes ◽  
M Biasi ◽  
...  
Keyword(s):  
Single Cell ◽  
Antiplatelet Therapy ◽  
Peripheral Blood ◽  
Therapeutic Targets ◽  
Transmembrane Proteins ◽  
Thrombin Receptor ◽  
Mass Cytometry ◽  
Activation Markers ◽  
Coronary Syndrome ◽  
Custom Made

Abstract Background Reticulated platelets (RPs) are young, hyper-reactive thrombocytes that contain more RNA compared with mature platelets (MPs). The measurement of RPs level in peripheral blood with point-of-care systems is fast, reproducible, and inexpensive. Elevated RPs in peripheral blood predict adverse events in patients with acute and chronic coronary syndrome through unknown mechanisms. Preliminary transcriptome analyses reported an enrichment of pro-thrombotic transcripts. However, proteomic analyses are not available, and the biological features of RPs are largely unknown. Purpose We aimed to perform the largest proteomic characterization of RPs using mass cytometry with single-cell resolution in patients with chronic coronary syndrome (CCS) undergoing dual antiplatelet therapy (DAPT). Methods Thrombocytes from peripheral blood of CCS patients were isolated, prepared for mass cytometry (CyTOF) and stained with a custom-made CyTOF-panel of 20 antibodies targeting important transmembrane proteins (anti-CD9, anti-CD29, anti-CD31, anti-CD36-, anti-CD40, anti-CD41, anti-CD42a, anti-CD42b-, anti-CD47, anti-CD61, anti-CD62P-, anti-CD63, anti-CD69, anti-CD107a, anti-CD154, anti-GPVI, antiGPIIb/GPIIIa complex, anti-Par1, anti-PEAR-1 and the negative control anti-CD3 coupled with different metal isotopes). Two samples were prepared from each donor: one baseline sample (non-stimulated platelets) and one sample stimulated with 10 μM thrombin receptor-activating peptide (TRAP). According to previous experiences and common practice, we detected RPs and MPs based on their RNA content. We analyzed the results with a custom bioinformatic pipeline. Results 13 patients with CCS on DAPT were included in this study. Mass cytometry highlighted an expression heterogeneity of relevant transmembrane proteins in thrombocytes of CCS patients (Figure 1A-B colored according to expression level: from blue-low to red-high). CyTOF detected an upregulation of important transmembrane receptors in RPs compared to MPs in quiescent platelets: GPVI (p<0.0001), PAR-1 (p<0.0001), GPIX (p<0.0001), and GPIbα (p<0.0001, Figure 1C). After TRAP-stimulation, RPs expressed higher levels of the activation markers P-Selectin (p=0.0016) and LAMP-3 (CD63, p<0.0001) compared to MPs confirming RPs hyperactivity (Figure 1D). Conclusion We here describe the first biological proteomic characterization with single-cell resolution of RPs biology in CCS patients. The upregulation of the activation markers P-Selectin and LAMP-3 as well as of specific transmembrane proteins as the collagen receptor GPVI and the thrombin receptor PAR-1 in patients treated with DAPT (schematic overview in Figure 2) provides the first solid biomolecular explanation of RPs hyper-reactivity and involvement in cardiovascular disease. Moreover, these results offer unexplored therapeutic targets to tailor antiplatelet therapy based on platelet protein expression in patients with elevated RPs FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German Center for Cardiovascular Research (DZHK) Figure 1. Platelet expression Figure 2. Schematic overview

scholarly journals Single-Cell Mass Cytometry on Peripheral Blood Identifies Immune Cell Subsets Associated with Primary Biliary Cholangitis

2020 ◽  
Author(s):  
Jin Sung Jang ◽  
Brian Juran ◽  
Kevin Y. Cunningham ◽  
Vinod K. Gupta ◽  
YoungMin Son ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
B Cells ◽  
Single Cell ◽  
Peripheral Blood ◽  
Immune Cell ◽  
Memory B Cells ◽  
Primary Biliary Cholangitis ◽  
Mass Cytometry ◽  
Peripheral Immune System

AbstractThe relationship between Primary Biliary Cholangitis (PBC), a chronic cholestatic autoimmune liver disease, and the peripheral immune system remains to be fully understood. Herein, we performed the first mass cytometry (CyTOF)-based, immunophenotyping analysis of the peripheral immune system in PBC at single-cell resolution. CyTOF was performed on peripheral blood mononuclear cells (PBMCs) from PBC patients (n=33) and age-/sex-matched healthy controls (n=33) to obtain immune cell abundance and marker expression profiles. Hiearchical clustering methods were applied to identify immune cell types and subsets significantly associated with PBC. Subsets of gamma-delta T cells (CD3+TCRgd+), CD8+ T cells (CD3+CD8+CD161+PD1+), and memory B cells (CD3-CD19+CD20+CD24+CD27+) were found to have lower abundance in PBC than in control. In contrast, higher abundance of subsets of monocytes and naïve B cells were observed in PBC compared to control. Furthermore, several naïve B cell (CD3-CD19+CD20+CD24-CD27-) subsets were significantly higher in PBC patients with cirrhosis (indicative of late-stage disease) than in those without cirrhosis. Alternatively, subsets of CD8+CD161+ T cells and memory B cells were lower in abundance in cirrhotic relative to non-cirrhotic PBC patients. Future immunophenotyping investigations could lead to better understanding of PBC pathogenesis and progression, and also to the discovery of novel biomarkers and treatment strategies.

scholarly journals Agile workflow for interactive analysis of mass cytometry data

2020 ◽  
Author(s):  
Julia Casado ◽  
Oskari Lehtonen ◽  
Ville Rantanen ◽  
Katja Kaipio ◽  
Luca Pasquini ◽  
...  
Keyword(s):  
Single Cell ◽  
Peripheral Blood ◽  
Large Scale ◽  
Immune Cell ◽  
Single Cell Analysis ◽  
Supplementary Information ◽  
Mass Cytometry ◽  
Interactive Analysis ◽  
Link Type ◽  
Cell Subpopulations

AbstractMotivationSingle-cell proteomics technologies, such as mass cytometry, have enabled characterization of cell-to-cell variation and cell populations at a single cell resolution. These large amounts of data, however, require dedicated, interactive tools for translating the data into knowledge.ResultsWe present a comprehensive, interactive method called Cyto to streamline analysis of large-scale cytometry data. Cyto is a workflow-based open-source solution that automatizes the use of of state-of-the-art single-cell analysis methods with interactive visualization. We show the utility of Cyto by applying it to mass cytometry data from peripheral blood and high-grade serous ovarian cancer (HGSOC) samples. Our results show that Cyto is able to reliably capture the immune cell sub-populations from peripheral blood as well as cellular compositions of unique immune- and cancer cell subpopulations in HGSOC tumor and ascites samples.AvailabilityThe method is available as a Docker container at https://hub.docker.com/r/anduril/cyto and the user guide and source code are available at https://bitbucket.org/anduril-dev/[email protected] informationSupplementary material is available and FCS files are hosted at flowrepository.org/id/FR-FCM-Z2LW

scholarly journals Single-cell mass cytometry on peripheral blood identifies immune cell subsets associated with primary biliary cholangitis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jin Sung Jang ◽  
Brian D. Juran ◽  
Kevin Y. Cunningham ◽  
Vinod K. Gupta ◽  
Young Min Son ◽  
...  
Keyword(s):  
Single Cell ◽  
Peripheral Blood ◽  
Immune Cell ◽  
Cell Mass ◽  
Primary Biliary Cholangitis ◽  
Mass Cytometry ◽  
Immune Cell Subsets

scholarly journals Agile workflow for interactive analysis of mass cytometry data

2020 ◽  
Author(s):  
Julia Casado ◽  
Oskari Lehtonen ◽  
Ville Rantanen ◽  
Katja Kaipio ◽  
Luca Pasquini ◽  
...  
Keyword(s):  
Single Cell ◽  
Peripheral Blood ◽  
Large Scale ◽  
Immune Cell ◽  
Single Cell Analysis ◽  
Supplementary Information ◽  
Mass Cytometry ◽  
Interactive Analysis ◽  
Cell Subpopulations ◽  
Cell Variation

Abstract Motivation Single-cell proteomics technologies, such as mass cytometry, have enabled characterization of cell-to-cell variation and cell populations at a single-cell resolution. These large amounts of data, require dedicated, interactive tools for translating the data into knowledge. Results We present a comprehensive, interactive method called Cyto to streamline analysis of large-scale cytometry data. Cyto is a workflow-based open-source solution that automates the use of state-of-the-art single-cell analysis methods with interactive visualization. We show the utility of Cyto by applying it to mass cytometry data from peripheral blood and high-grade serous ovarian cancer (HGSOC) samples. Our results show that Cyto is able to reliably capture the immune cell sub-populations from peripheral blood and cellular compositions of unique immune- and cancer cell subpopulations in HGSOC tumor and ascites samples. Availabilityand implementation The method is available as a Docker container at https://hub.docker.com/r/anduril/cyto and the user guide and source code are available at https://bitbucket.org/anduril-dev/cyto. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Single-cell analyses identify tobacco smoke exposure-associated, dysfunctional CD16+ CD8 T cells with high cytolytic potential in peripheral blood

2019 ◽  
Author(s):  
Suzanne N. Martos ◽  
Michelle R. Campbell ◽  
Oswaldo A. Lozoya ◽  
Brian D. Bennett ◽  
Isabel J.B. Thompson ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Tobacco Smoke ◽  
Peripheral Blood ◽  
Cd8 T Cell ◽  
Tobacco Smoke Exposure ◽  
Mass Cytometry

SUMMARYTobacco smoke exposure has been found to impact immune response, leukocyte subtypes, DNA methylation, and gene expression in human whole blood. Analysis with single cell technologies will resolve smoking associated (sub)population compositions, gene expression differences, and identification of rare subtypes masked by bulk fraction data. To characterize smoking-related gene expression changes in primary immune cells, we performed single-cell RNA sequencing (scRNAseq) on >45,000 human peripheral blood mononuclear cells (PBMCs) from smokers (n=4) and nonsmokers (n=4). Major cell type population frequencies showed strong correlation between scRNAseq and mass cytometry. Transcriptomes revealed an altered subpopulation of Natural Killer (NK)-like T lymphocytes in smokers, which expressed elevated levels of FCGR3A (gene encoding CD16) compared to other CD8 T cell subpopulations. Relatively rare in nonsmokers (median: 1.8%), the transcriptionally unique subset of CD8 T cells comprised 7.3% of PBMCs in smokers. Mass cytometry confirmed a significant increase (p = 0.03) in the frequency of CD16+ CD8 T cells in smokers. The majority of CD16+ CD8 T cells were CD45RA positive, indicating an effector memory re-expressing CD45RA T cell (TEMRA) phenotype. We expect that cigarette smoke alters CD8 T cell composition by shifting CD8 T cells toward differentiated functional states. Pseudotemporal ordering of CD8 T cell clusters revealed that smokers’ cells were biased toward later pseudotimes, and characterization of established markers in CD8 T cell subsets indicates a higher frequency of terminally differentiated cells in smokers than in nonsmokers, which corresponded with a lower frequency in naïve CD8 T cells. Consistent with an end-stage TEMRA phenotype, FCGR3A-expressing CD8 T cells were inferred as the most differentiated cluster by pseudotime analysis and expressed markers linked to senescence. Examination of differentially expressed genes in other PBMCs uncovered additional senescence-associated genes in CD4 T cells, NKT cells, NK cells, and monocytes. We also observed elevated Tregs, inducers of T cell senescence, in smokers. Taken together, our results suggest smoking-induced, senescence-associated immune cell dysregulation contributes to smoking-mediated pathologies.

scholarly journals SARS-CoV-2 infection is associated with a pro-thrombotic platelet phenotype

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dario Bongiovanni ◽  
Melissa Klug ◽  
Olga Lazareva ◽  
Simon Weidlich ◽  
Marina Biasi ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Transmembrane Proteins ◽  
Surface Expression ◽  
Cardiovascular Complications ◽  
Healthy Controls ◽  
Mass Cytometry ◽  
Activation Markers ◽  
Median Signal Intensity ◽  
Novel Coronavirus

AbstractNovel coronavirus disease 2019 (COVID-19) is associated with a hypercoagulable state, characterized by abnormal coagulation parameters and by increased incidence of cardiovascular complications. With this study, we aimed to investigate the activation state and the expression of transmembrane proteins in platelets of hospitalized COVID-19 patients. We investigated transmembrane proteins expression with a customized mass cytometry panel of 21 antibodies. Platelets of 8 hospitalized COVID-19 patients not requiring intensive care support and without pre-existing conditions were compared to platelets of healthy controls (11 donors) with and without in vitro stimulation with thrombin receptor-activating peptide (TRAP). Mass cytometry of non-stimulated platelets detected an increased surface expression of activation markers P-Selectin (0.67 vs. 1.87 median signal intensity for controls vs. patients, p = 0.0015) and LAMP-3 (CD63, 0.37 vs. 0.81, p = 0.0004), the GPIIb/IIIa complex (4.58 vs. 5.03, p < 0.0001) and other adhesion molecules involved in platelet activation and platelet–leukocyte interactions. Upon TRAP stimulation, mass cytometry detected a higher expression of P-selectin in COVID-19 samples compared to controls (p < 0.0001). However, we observed a significantly reduced capacity of COVID-19 platelets to increase the expression of activation markers LAMP-3 and P-Selectin upon stimulation with TRAP. We detected a hyperactivated phenotype in platelets during SARS-CoV-2 infection, consisting of highly expressed platelet activation markers, which might contribute to the hypercoagulopathy observed in COVID-19. In addition, several transmembrane proteins were more highly expressed compared to healthy controls. These findings support research projects investigating antithrombotic and antiplatelet treatment regimes in COVID-19 patients, and provide new insights on the phenotypical platelet expression during SARS-CoV-2 infection.

Insights into therapeutic targets and biomarkers using integrated multi-‘omics’ approaches for dilated and ischemic cardiomyopathies

2021 ◽  
Author(s):  
Austė Kanapeckaitė ◽  
Neringa Burokienė
Keyword(s):  
Machine Learning ◽  
Single Cell ◽  
Learning Algorithm ◽  
Expression Profiles ◽  
Therapeutic Targets ◽  
Development Stage ◽  
Biological Data ◽  
Specific Gene ◽  
Tissue Remodelling ◽  
Pharmacological Management

Abstract At present, heart failure (HF) treatment only targets the symptoms based on the left ventricle dysfunction severity; however, the lack of systemic ‘omics’ studies and available biological data to uncover the heterogeneous underlying mechanisms signifies the need to shift the analytical paradigm towards network-centric and data mining approaches. This study, for the first time, aimed to investigate how bulk and single cell RNA-sequencing as well as the proteomics analysis of the human heart tissue can be integrated to uncover HF-specific networks and potential therapeutic targets or biomarkers. We also aimed to address the issue of dealing with a limited number of samples and to show how appropriate statistical models, enrichment with other datasets as well as machine learning-guided analysis can aid in such cases. Furthermore, we elucidated specific gene expression profiles using transcriptomic and mined data from public databases. This was achieved using the two-step machine learning algorithm to predict the likelihood of the therapeutic target or biomarker tractability based on a novel scoring system, which has also been introduced in this study. The described methodology could be very useful for the target or biomarker selection and evaluation during the pre-clinical therapeutics development stage as well as disease progression monitoring. In addition, the present study sheds new light into the complex aetiology of HF, differentiating between subtle changes in dilated cardiomyopathies (DCs) and ischemic cardiomyopathies (ICs) on the single cell, proteome and whole transcriptome level, demonstrating that HF might be dependent on the involvement of not only the cardiomyocytes but also on other cell populations. Identified tissue remodelling and inflammatory processes can be beneficial when selecting targeted pharmacological management for DCs or ICs, respectively.

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