Single-cell mass cytometry significantly increases the dimensionality of cytometry analysis as compared to fluorescence flow cytometry, providing unprecedented resolution of cellular diversity in ...tissues. However, analysis and interpretation of these high-dimensional data poses a significant technical challenge. Here, we present cytofkit, a new Bioconductor package, which integrates both state-of-the-art bioinformatics methods and in-house novel algorithms to offer a comprehensive toolset for mass cytometry data analysis. Cytofkit provides functions for data pre-processing, data visualization through linear or non-linear dimensionality reduction, automatic identification of cell subsets, and inference of the relatedness between cell subsets. This pipeline also provides a graphical user interface (GUI) for ease of use, as well as a shiny application (APP) for interactive visualization of cell subpopulations and progression profiles of key markers. Applied to a CD14-CD19- PBMCs dataset, cytofkit accurately identified different subsets of lymphocytes; applied to a human CD4+ T cell dataset, cytofkit uncovered multiple subtypes of TFH cells spanning blood and tonsils. Cytofkit is implemented in R, licensed under the Artistic license 2.0, and freely available from the Bioconductor website, https://bioconductor.org/packages/cytofkit/. Cytofkit is also applicable for flow cytometry data analysis.
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
Immunologists are being compelled to develop new high-dimensional perspectives of cellular heterogeneity and to determine which applications best exploit the power of mass cytometry and associated ...multiplex approaches.
Advances in single-cell technologies have enabled high-resolution dissection of tissue composition. Several tools for dimensionality reduction are available to analyze the large number of parameters ...generated in single-cell studies. Recently, a nonlinear dimensionality-reduction technique, uniform manifold approximation and projection (UMAP), was developed for the analysis of any type of high-dimensional data. Here we apply it to biological data, using three well-characterized mass cytometry and single-cell RNA sequencing datasets. Comparing the performance of UMAP with five other tools, we find that UMAP provides the fastest run times, highest reproducibility and the most meaningful organization of cell clusters. The work highlights the use of UMAP for improved visualization and interpretation of single-cell data.
Flow cytometry is used increasingly in clinical research for cancer, immunology and vaccines. Technological advances in cytometry instrumentation are increasing the size and dimensionality of data ...sets, posing a challenge for traditional data management and analysis. Automated analysis methods, despite a general consensus of their importance to the future of the field, have been slow to gain widespread adoption. Here we present OpenCyto, a new BioConductor infrastructure and data analysis framework designed to lower the barrier of entry to automated flow data analysis algorithms by addressing key areas that we believe have held back wider adoption of automated approaches. OpenCyto supports end-to-end data analysis that is robust and reproducible while generating results that are easy to interpret. We have improved the existing, widely used core BioConductor flow cytometry infrastructure by allowing analysis to scale in a memory efficient manner to the large flow data sets that arise in clinical trials, and integrating domain-specific knowledge as part of the pipeline through the hierarchical relationships among cell populations. Pipelines are defined through a text-based csv file, limiting the need to write data-specific code, and are data agnostic to simplify repetitive analysis for core facilities. We demonstrate how to analyze two large cytometry data sets: an intracellular cytokine staining (ICS) data set from a published HIV vaccine trial focused on detecting rare, antigen-specific T-cell populations, where we identify a new subset of CD8 T-cells with a vaccine-regimen specific response that could not be identified through manual analysis, and a CyTOF T-cell phenotyping data set where a large staining panel and many cell populations are a challenge for traditional analysis. The substantial improvements to the core BioConductor flow cytometry packages give OpenCyto the potential for wide adoption. It can rapidly leverage new developments in computational cytometry and facilitate reproducible analysis in a unified environment.
Summary Lung cancer is a leading cause of cancer-related mortality worldwide, and is classically divided into two major histological subtypes: non-small-cell lung cancer (NSCLC) and small-cell lung ...cancer (SCLC). Although NSCLC and SCLC are considered distinct entities with different genomic landscapes, emerging evidence highlights a convergence in therapeutically relevant targets for both histologies. In adenocarcinomas with defined alterations such as EGFR mutations and ALK translocations, targeted therapies are now first-line standard of care. By contrast, many experimental and targeted agents remain largely unsuccessful for SCLC. Intense preclinical research and clinical trials are underway to exploit unique traits of lung cancer, such as oncogene dependency, DNA damage response, angiogenesis, and cellular plasticity arising from presence of cancer stem cell lineages. In addition, the promising clinical activity observed in NSCLC in response to immune checkpoint blockade has spurred great interest in the field of immunooncology, with the scope to develop a diverse repertoire of synergistic and personalised immunotherapeutics. In this Review, we discuss novel therapeutic agents for lung cancer that are in early-stage development, and how prospective clinical trials and drug development may be shaped by a deeper understanding of this heterogeneous disease.
Neutrophils are specialized innate cells that require constant replenishment from proliferative bone marrow (BM) precursors as a result of their short half-life. Although it is established that ...neutrophils are derived from the granulocyte-macrophage progenitor (GMP), the differentiation pathways from GMP to functional mature neutrophils are poorly defined. Using mass cytometry (CyTOF) and cell-cycle-based analysis, we identified three neutrophil subsets within the BM: a committed proliferative neutrophil precursor (preNeu) which differentiates into non-proliferating immature neutrophils and mature neutrophils. Transcriptomic profiling and functional analysis revealed that preNeu require the C/EBPε transcription factor for their generation from the GMP, and their proliferative program is substituted by a gain of migratory and effector function as they mature. preNeus expand under microbial and tumoral stress, and immature neutrophils are recruited to the periphery of tumor-bearing mice. In summary, our study identifies specialized BM granulocytic populations that ensure supply under homeostasis and stress responses.
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•Proliferation activity identifies committed neutrophil precursor in mice and humans•Neutrophil subsets possess distinct transcriptomic and functional signatures•Defect in neutrophil development leads to impaired neutrophil-mediated responses•Increased circulating immature neutrophils are associated with cancer progression
The neutrophil differentiation pathway is poorly defined. Evrard et. al. demonstrate a workflow of characterizing bone marrow neutrophil subsets on the basis of their proliferative capacity and molecular signatures and thereby define the developmental trajectory and functional properties of neutrophils.
Chronic HBV (CHB) infection suppresses virus-specific T cells, but its impact on humoral immunity has been poorly analyzed. Here, we developed a dual-staining method that utilizes hepatitis B virus ...(HBV) surface antigens (HBsAg) labeled with fluorochromes as "baits" for specific ex vivo detection of HBsAg-specific B cells and analysis of their quantity, function, and phenotype. We studied healthy vaccinated subjects (n = 18) and patients with resolved (n = 21), acute (n = 11), or chronic (n = 96) HBV infection and observed that frequencies of circulating HBsAg-specific B cells were independent of HBV infection status. In contrast, the presence of serum HBsAg affected function and phenotype of HBsAg-specific B cells that were unable to mature in vitro into Ab-secreting cells and displayed an increased expression of markers linked to hyperactivation (CD21lo) and exhaustion (PD-1). Importantly, B cell alterations were not limited to HBsAg-specific B cells, but affected the global B cell population. HBsAg-specific B cell maturation could be partially restored by a method involving the combination of the cytokines IL-2 and IL-21 and CD40L-expressing feeder cells and was further boosted by the addition of anti-PD-1 Abs. In conclusion, HBV infection has a marked impact on global and HBV-specific humoral immunity, yet HBsAg-specific B cells are amenable to a partial rescue by B cell-maturing cytokines and PD-1 blockade.
Summary
Innate lymphoid cells (ILCs) have been divided into three distinct groups based on functional capacities, cytokine profiles and transcription factor expression. Studies performed mainly in ...mice have demonstrated the importance of ILCs in chronic inflammation, infection, allergy and cancer. In this review, we discuss the heterogeneity of human ILC and focus primarily on the taxonomy of human ILC cell subsets and their phenotypical and functional diversity. We summarize recent findings concerning the diversity of ILCs between and within the major subsets natural killer (NK), ILC1, intra‐epithelial ILC1 (ieILC1), ILC2, ILC3, lymphoid tissues inducer (LTi) and ILC progenitor (ILCP), as well as the abundance of each in human tissues. We also discuss the similarities observed between groups of cells in term of receptors expressed and cytokines produced, and how these relate to the pleiotropic properties of each subset.
Dissecting human ILC heterogeneity: more than just three subsets. We summarize recent findings about the diversity of ILCs between and within the major subsets (NK, ILC1, ieILC1, ILC2, ILC3, LTi, ILCP), as well as the abundance of each in human tissues. We also discuss the similarities observed between groups of cells in term of receptors expressed and cytokines produced, and how these relate to the pleiotropic properties of each subset.
Human mononuclear phagocytes comprise phenotypically and functionally overlapping subsets of dendritic cells (DCs) and monocytes, but the extent of their heterogeneity and distinct markers for subset ...identification remains elusive. By integrating high-dimensional single-cell protein and RNA expression data, we identified distinct markers to delineate monocytes from conventional DC2 (cDC2s). Using CD88 and CD89 for monocytes and HLA-DQ and FcεRIα for cDC2s allowed for their specific identification in blood and tissues. We also showed that cDC2s could be subdivided into phenotypically and functionally distinct subsets based on CD5, CD163, and CD14 expression, including a distinct subset of circulating inflammatory CD5−CD163+CD14+ cells related to previously defined DC3s. These inflammatory DC3s were expanded in systemic lupus erythematosus patients and correlated with disease activity. These findings further unravel the heterogeneity of DC subpopulations in health and disease and may pave the way for the identification of specific DC subset-targeting therapies.
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•InfinityFlow protein expression analysis reveals DC- and monocyte-specific markers•Monocytes are CD88+CD89+, while cDC2s are HLA-DQ+FcεRIα+•cDC2s comprise CD5+ DC2s and CD5−CD163+/−CD14+/− DC3s•Pro-inflammatory CD14+ DC3 expansion correlates with disease activity in SLE patients
Using high-dimensional protein and RNA single-cell analyses, Dutertre et al. analyze human dendritic cell and monocyte subsets and identify markers that delineate them and unravel their heterogeneity. They also reveal the presence of inflammatory CD14+ DC3s, a subset of cDC2s, that correlate with disease progression and may be functionally involved in systemic lupus erythematosus immunopathology.
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