The cellular complexity and functional diversity of the human immune system necessitate the use of high-dimensional single-cell tools to uncover its role in multifaceted diseases such as rheumatic ...diseases, as well as other autoimmune and inflammatory disorders. Proteomic technologies that use elemental (heavy metal) reporter ions, such as mass cytometry (also known as CyTOF) and analogous high-dimensional imaging approaches (including multiplexed ion beam imaging (MIBI) and imaging mass cytometry (IMC)), have been developed from their low-dimensional counterparts, flow cytometry and immunohistochemistry, to meet this need. A growing number of studies have been published that use these technologies to identify functional biomarkers and therapeutic targets in rheumatic diseases, but the full potential of their application to rheumatic disease research has yet to be fulfilled. This Review introduces the underlying technologies for high-dimensional immune monitoring and discusses aspects necessary for their successful implementation, including study design principles, analytical tools and future developments for the field of rheumatology.
Tissue regeneration is an orchestrated progression of cells from an immature state to a mature one, conventionally represented as distinctive cell subsets. A continuum of transitional cell states ...exists between these discrete stages. We combine the depth of single-cell mass cytometry and an algorithm developed to leverage this continuum by aligning single cells of a given lineage onto a unified trajectory that accurately predicts the developmental path de novo. Applied to human B cell lymphopoiesis, the algorithm (termed Wanderlust) constructed trajectories spanning from hematopoietic stem cells through to naive B cells. This trajectory revealed nascent fractions of B cell progenitors and aligned them with developmentally cued regulatory signaling including IL-7/STAT5 and cellular events such as immunoglobulin rearrangement, highlighting checkpoints across which regulatory signals are rewired paralleling changes in cellular state. This study provides a comprehensive analysis of human B lymphopoiesis, laying a foundation to apply this approach to other tissues and “corrupted” developmental processes including cancer.
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•Wanderlust aligns single cells on a trajectory according to their developmental path•Mass cytometry plus Wanderlust infers path from HSCs to naive B cells•Trajectory identifies precursor B cell populations where rearrangement occurs•Discovery of coordination points dictating cell fate decisions
Combining single-cell mass cytometry data from a population of cells from human tissue with a new algorithm that leverages the single-cell data to order the cells along their developmental trajectory provides insight into B cell development and paves the for similar analyses in other tissues.
Cytotoxic CD8
+ T lymphocytes directly kill infected or aberrant cells and secrete proinflammatory cytokines. By using metal-labeled probes and mass spectrometric analysis (cytometry by ...time-of-flight, or CyTOF) of human CD8
+ T cells, we analyzed the expression of many more proteins than previously possible with fluorescent labels, including surface markers, cytokines, and antigen specificity with modified peptide-MHC tetramers. With 3-dimensional principal component analysis (3D-PCA) to display phenotypic diversity, we observed a relatively uniform pattern of variation in all subjects tested, highlighting the interrelatedness of previously described subsets and the continuous nature of CD8
+ T cell differentiation. These data also showed much greater complexity in the CD8
+ T cell compartment than previously appreciated, including a nearly combinatorial pattern of cytokine expression, with distinct niches occupied by virus-specific cells. This large degree of functional diversity even between cells with the same specificity gives CD8
+ T cells a remarkable degree of flexibility in responding to pathogens.
► T cell specificity, phenotype, and function can be assessed by mass spectrometry ► Principal component analysis revealed a common pattern of phenotypic progression ► Expression of cytokines by CD8
+ T cells showed large combinatorial diversity ► Viral-specific cells occupied distinct niches of phenotypic and functional diversity
Advances in single-cell mass cytometry have increasingly improved highly multidimensional characterization of immune cell heterogeneity. The immunoassay multiplexing capacity relies on monoclonal ...antibodies labeled with stable heavy-metal isotopes. To date, a variety of rare-earth elements and noble and post-transition metal isotopes have been used in mass cytometry; nevertheless, the methods used for antibody conjugation differ because of the individual metal coordination chemistries and distinct stabilities of various metal cations. Herein, we provide three optimized protocols for conjugating monoclonal IgG antibodies with 48 high-purity heavy-metal isotopes: (i) 38 isotopes of lanthanides, 2 isotopes of indium, and 1 isotope of yttrium; (ii) 6 isotopes of palladium; and (iii) 1 isotope of bismuth. Bifunctional chelating agents containing coordinative ligands of monomeric DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or polymeric pentetic acid (DTPA) were used to stably sequester isotopic cations in aqueous solutions and were subsequently coupled to IgG antibodies using site-specific biorthogonal reactions. Furthermore, quantification methods based on antibody inherent absorption at 280 nm and on extrinsic absorption at 562 nm after staining with bicinchoninic acid (BCA) are reported to determine metal-isotope-tagged antibodies. In addition, a freeze-drying procedure to prepare palladium isotopic mass tags is described. To demonstrate the utility, experiments using six palladium-tagged CD45 antibodies for barcoding assays of live immune cells in cytometry by time-of-flight (CyTOF) are described. Conjugation of pure isotopes of lanthanides, indium, or yttrium takes ~3.5 h. Conjugation of bismuth takes ~4 h. Preparation of palladium mass tags takes ~8 h. Conjugation of pure isotopes of palladium takes ~2.5 h. Antibody titration takes ~4 h.
Elucidating the spectrum of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) states in clinical samples promises insights on cancer progression and drug resistance. ...Using mass cytometry time-course analysis, we resolve lung cancer EMT states through TGFβ-treatment and identify, through TGFβ-withdrawal, a distinct MET state. We demonstrate significant differences between EMT and MET trajectories using a computational tool (TRACER) for reconstructing trajectories between cell states. In addition, we construct a lung cancer reference map of EMT and MET states referred to as the EMT-MET PHENOtypic STAte MaP (PHENOSTAMP). Using a neural net algorithm, we project clinical samples onto the EMT-MET PHENOSTAMP to characterize their phenotypic profile with single-cell resolution in terms of our in vitro EMT-MET analysis. In summary, we provide a framework to phenotypically characterize clinical samples in the context of in vitro EMT-MET findings which could help assess clinical relevance of EMT in cancer in future studies.
The immune system is critical in modulating cancer progression, but knowledge of immune composition, phenotype, and interactions with tumor is limited. We used multiplexed ion beam imaging by ...time-of-flight (MIBI-TOF) to simultaneously quantify in situ expression of 36 proteins covering identity, function, and immune regulation at sub-cellular resolution in 41 triple-negative breast cancer patients. Multi-step processing, including deep-learning-based segmentation, revealed variability in the composition of tumor-immune populations across individuals, reconciled by overall immune infiltration and enriched co-occurrence of immune subpopulations and checkpoint expression. Spatial enrichment analysis showed immune mixed and compartmentalized tumors, coinciding with expression of PD1, PD-L1, and IDO in a cell-type- and location-specific manner. Ordered immune structures along the tumor-immune border were associated with compartmentalization and linked to survival. These data demonstrate organization in the tumor-immune microenvironment that is structured in cellular composition, spatial arrangement, and regulatory-protein expression and provide a framework to apply multiplexed imaging to immune oncology.
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•Multiplexed Imaging of subcellular expression of 36 proteins in 41 TNBC patients•Enriched co-occurrence of immune populations defines a structured immune environment•Tumor infiltration by immune cells varies from compartmentalized to mixed patients•Expression of PD-1 and PD-L1 by distinct cells relate to tumor histology and survival
A combination of multiplexed imaging and machine learning reveals that spatial organization of immune phenotypes within triple-negative breast tumors is linked to survival.
Acute myeloid leukemia (AML) manifests as phenotypically and functionally diverse cells, often within the same patient. Intratumor phenotypic and functional heterogeneity have been linked primarily ...by physical sorting experiments, which assume that functionally distinct subpopulations can be prospectively isolated by surface phenotypes. This assumption has proven problematic, and we therefore developed a data-driven approach. Using mass cytometry, we profiled surface and intracellular signaling proteins simultaneously in millions of healthy and leukemic cells. We developed PhenoGraph, which algorithmically defines phenotypes in high-dimensional single-cell data. PhenoGraph revealed that the surface phenotypes of leukemic blasts do not necessarily reflect their intracellular state. Using hematopoietic progenitors, we defined a signaling-based measure of cellular phenotype, which led to isolation of a gene expression signature that was predictive of survival in independent cohorts. This study presents new methods for large-scale analysis of single-cell heterogeneity and demonstrates their utility, yielding insights into AML pathophysiology.
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•PhenoGraph partitions high-dimensional single-cell data into subpopulations•PhenoGraph plus mass cytometry elucidate intra- and intertumor heterogeneity in AML•Surface phenotypes and regulatory intercellular signaling are decoupled in leukemia•Signaling-based definition of primitive cells correlates with clinical outcome
The PhenoGraph algorithm robustly partitions high-parameter single-cell data into phenotypically distinct subpopulations, aiding the study of complex tissues and disease cohorts. Applying PhenoGraph to a pediatric acute myeloid leukemia dataset revealed a recurrent population of leukemic cells with variable cell surface markers, but consistent signaling dynamics that mimicked normal hematopoietic progenitors.
A deep profiler's guide to cytometry Bendall, Sean C; Nolan, Garry P; Roederer, Mario ...
Trends in immunology,
07/2012, Letnik:
33, Številka:
7
Journal Article
Recenzirano
Odprti dostop
In recent years, advances in technology have provided us with tools to quantify the expression of multiple genes in individual cells. The ability to measure simultaneously multiple genes in the same ...cell is necessary to resolve the great diversity of cell subsets, as well as to define their function in the host. Fluorescence-based flow cytometry is the benchmark for this; with it, we can quantify 18 proteins per cell, at >10 000 cells/s. Mass cytometry is a new technology that promises to extend these capabilities significantly. Immunophenotyping by mass spectrometry provides the ability to measure >36 proteins at a rate of 1000 cells/s. We review these cytometric technologies, capable of high-content, high-throughput single-cell assays.
Single-cell barcoding enables the combined processing and acquisition of multiple individual samples as one. This maximizes assay efficiency and eliminates technical variability in both sample ...preparation and analysis. Remaining challenges are the barcoding of live, unprocessed cells to increase downstream assay performance combined with the flexibility of the approach towards a broad range of cell types. To that end, we developed a novel antibody-based platform that allows the robust barcoding of live human cells for mass cytometry (CyTOF). By targeting both the MHC class I complex (beta-2-microglobulin) and a broadly expressed sodium-potassium ATPase-subunit (CD298) with platinum-conjugated antibodies, human immune cells, stem cells as well as tumor cells could be multiplexed in the same single-cell assay. In addition, we present a novel palladium-based covalent viability reagent compatible with this barcoding strategy. Altogether, this platform enables mass cytometry-based, live-cell barcoding across a multitude of human sample types and provides a scheme for multiplexed barcoding of human single-cell assays in general.
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