Cyclone: an accessible pipeline to analyze, evaluate, and optimize multiparametric cytometry data

In the past decade, high-dimensional single-cell technologies have revolutionized basic and translational immunology research and are now a key element of the toolbox used by scientists to study the immune system. However, analysis of the data generated by these approaches often requires clustering algorithms and dimensionality reduction representation, which are computationally intense and difficult to evaluate and optimize. Here, we present Cytometry Clustering Optimization and Evaluation (Cyclone), an analysis pipeline integrating dimensionality reduction, clustering, evaluation, and optimization of clustering resolution, and downstream visualization tools facilitating the analysis of a wide range of cytometry data. We benchmarked and validated Cyclone on mass cytometry (CyTOF), full-spectrum fluorescence-based cytometry, and multiplexed immunofluorescence (IF) in a variety of biological contexts, including infectious diseases and cancer. In each instance, Cyclone not only recapitulates gold standard immune cell identification but also enables the unsupervised identification of lymphocytes and mononuclear phagocyte subsets that are associated with distinct biological features. Altogether, the Cyclone pipeline is a versatile and accessible pipeline for performing, optimizing, and evaluating clustering on a variety of cytometry datasets, which will further power immunology research and provide a scaffold for biological discovery

Single-cell mapping of progressive fetal-to-adult transition in human naïve T cells

Whereas the human fetal immune system is poised to generate immune tolerance and suppress inflammation in utero, an adult-like immune system emerges to orchestrate anti-pathogen immune responses in post-natal life. Compared to their adult counterparts, fetal naïve T cells more readily differentiate into tolerance-prone regulatory T cells (Tregs) upon stimulation, and fetal monocytes exhibit distinct responses to cytokine stimulation and impaired antigen presentation capacity. It is believed that tolerogenic responses across various immune cells are adaptations that allow the fetal immune system to suppress responses to non-inherited maternal antigens that might otherwise lead to pregnancy complications. After birth, the balance between tolerance versus protection must shift. It has been posited that cells of the adult immune system arise as a discrete ontological “layer” of hematopoietic stem-progenitor cells (HSPCs) and their progeny; evidence supporting this model in humans has, however, been inconclusive. Although it has been shown that various immune cell transitions during early gestation may be modeled by the layering hypothesis, it is unknown whether the transition from fetal-to-adult follows a similar mechanism. Here, we combine bulk and single-cell transcriptional profiling of lymphoid, myeloid, and HSPCs from fetal, perinatal, and adult developmental stages to demonstrate that the fetal-to-adult transition occurs progressively along a continuum of maturity—with a substantial degree of interindividual variation at the time of birth—rather than via a transition between discrete waves. We find that (1) newborn immune cells are relatively homogenous, each with an intermediate transitional phenotype, rather than having either a fetal or adult phenotype, (2) UCB HSPCs are not fully adult-transitioned, (3) the progression of transition at birth shows a high degree of inter-individual variability, and (4) pathways known to affect T cell polarization are among those expressed more highly in newborn than adult naïve CD4 T cells. These findings have important implications in the design of strategies for prophylaxis against infection in the newborn, and for the use of umbilical cord blood (UCB) in the setting of transplantation. I also present dittoSeq, a universal bulk and single-cell visualization toolkit that powered the analysis of these data. dittoSeq visualizations are color blindness-friendly by default, robustly documented to power ease-of-use by both novice and experienced coders, and allow highly customizable generation of both daily-use and publication-quality figures

Single-cell mapping of progressive fetal-to-adult transition in human naïve T cells

Whereas the human fetal immune system is poised to generate immune tolerance and suppress inflammation in utero, an adult-like immune system emerges to orchestrate anti-pathogen immune responses in post-natal life. Compared to their adult counterparts, fetal naïve T cells more readily differentiate into tolerance-prone regulatory T cells (Tregs) upon stimulation, and fetal monocytes exhibit distinct responses to cytokine stimulation and impaired antigen presentation capacity. It is believed that tolerogenic responses across various immune cells are adaptations that allow the fetal immune system to suppress responses to non-inherited maternal antigens that might otherwise lead to pregnancy complications. After birth, the balance between tolerance versus protection must shift. It has been posited that cells of the adult immune system arise as a discrete ontological “layer” of hematopoietic stem-progenitor cells (HSPCs) and their progeny; evidence supporting this model in humans has, however, been inconclusive. Although it has been shown that various immune cell transitions during early gestation may be modeled by the layering hypothesis, it is unknown whether the transition from fetal-to-adult follows a similar mechanism. Here, we combine bulk and single-cell transcriptional profiling of lymphoid, myeloid, and HSPCs from fetal, perinatal, and adult developmental stages to demonstrate that the fetal-to-adult transition occurs progressively along a continuum of maturity—with a substantial degree of interindividual variation at the time of birth—rather than via a transition between discrete waves. We find that (1) newborn immune cells are relatively homogenous, each with an intermediate transitional phenotype, rather than having either a fetal or adult phenotype, (2) UCB HSPCs are not fully adult-transitioned, (3) the progression of transition at birth shows a high degree of inter-individual variability, and (4) pathways known to affect T cell polarization are among those expressed more highly in newborn than adult naïve CD4 T cells. These findings have important implications in the design of strategies for prophylaxis against infection in the newborn, and for the use of umbilical cord blood (UCB) in the setting of transplantation. I also present dittoSeq, a universal bulk and single-cell visualization toolkit that powered the analysis of these data. dittoSeq visualizations are color blindness-friendly by default, robustly documented to power ease-of-use by both novice and experienced coders, and allow highly customizable generation of both daily-use and publication-quality figures

ratioNext Scores

Final 'ratioNext' scores of xCell signatures, as shown in Fig.2B and elsewher

xCell signature gene sets

Lists of genes used for each of the 64 cell type signatures used by xCel

Signature-Cell Type Target Map

Targeting relationship between xCell cell type signatures and cell types identified in healthy human endometrium scRNAseq dataset, used in Fig.2

Color Palette

Color palette used for immune subtypes in Fig.3A, and violin plots in 3C, 5C, 5D, S7

Normalized signature scores of xCell signatures

Medium normalized signature score between each xCell cell type signature and each cell type identified in healthy human endometrium scRNAseq dataset, as shown in Fig.2