Showing papers by "Garry P. Nolan published in 2017"

The Human Cell Atlas

Abstract: The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites

Abstract: The human gut microbiota produces dozens of metabolites that accumulate in the bloodstream, where they can have systemic effects on the host. Although these small molecules commonly reach concentrations similar to those achieved by pharmaceutical agents, remarkably little is known about the microbial metabolic pathways that produce them. Here we use a combination of genetics and metabolic profiling to characterize a pathway from the gut symbiont Clostridium sporogenes that generates aromatic amino acid metabolites. Our results reveal that this pathway produces twelve compounds, nine of which are known to accumulate in host serum. All three aromatic amino acids (tryptophan, phenylalanine and tyrosine) serve as substrates for the pathway, and it involves branching and alternative reductases for specific intermediates. By genetically manipulating C. sporogenes, we modulate serum levels of these metabolites in gnotobiotic mice, and show that in turn this affects intestinal permeability and systemic immunity. This work has the potential to provide the basis of a systematic effort to engineer the molecular output of the gut bacterial community.

An immune clock of human pregnancy.

Abstract: The maintenance of pregnancy relies on finely tuned immune adaptations. We demonstrate that these adaptations are precisely timed, reflecting an immune clock of pregnancy in women delivering at term. Using mass cytometry, the abundance and functional responses of all major immune cell subsets were quantified in serial blood samples collected throughout pregnancy. Cell signaling-based Elastic Net, a regularized regression method adapted from the elastic net algorithm, was developed to infer and prospectively validate a predictive model of interrelated immune events that accurately captures the chronology of pregnancy. Model components highlighted existing knowledge and revealed previously unreported biology, including a critical role for the interleukin-2-dependent STAT5ab signaling pathway in modulating T cell function during pregnancy. These findings unravel the precise timing of immunological events occurring during a term pregnancy and provide the analytical framework to identify immunological deviations implicated in pregnancy-related pathologies.

Expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states

Abstract: Low-grade, chronic inflammation has been associated with many diseases of aging, but the mechanisms responsible for producing this inflammation remain unclear. Inflammasomes can drive chronic inflammation in the context of an infectious disease or cellular stress, and they trigger the maturation of interleukin-1β (IL-1β). Here we find that the expression of specific inflammasome gene modules stratifies older individuals into two extremes: those with constitutive expression of IL-1β, nucleotide metabolism dysfunction, elevated oxidative stress, high rates of hypertension and arterial stiffness; and those without constitutive expression of IL-1β, who lack these characteristics. Adenine and N4-acetylcytidine, nucleotide-derived metabolites that are detectable in the blood of the former group, prime and activate the NLRC4 inflammasome, induce the production of IL-1β, activate platelets and neutrophils and elevate blood pressure in mice. In individuals over 85 years of age, the elevated expression of inflammasome gene modules was associated with all-cause mortality. Thus, targeting inflammasome components may ameliorate chronic inflammation and various other age-associated conditions.

Unifying mechanism for different fibrotic diseases

Abstract: Fibrotic diseases are not well-understood. They represent a number of different diseases that are characterized by the development of severe organ fibrosis without any obvious cause, such as the devastating diseases idiopathic pulmonary fibrosis (IPF) and scleroderma. These diseases have a poor prognosis comparable with endstage cancer and are uncurable. Given the phenotypic differences, it was assumed that the different fibrotic diseases also have different pathomechanisms. Here, we demonstrate that many endstage fibrotic diseases, including IPF; scleroderma; myelofibrosis; kidney-, pancreas-, and heart-fibrosis; and nonalcoholic steatohepatosis converge in the activation of the AP1 transcription factor c-JUN in the pathologic fibroblasts. Expression of the related AP1 transcription factor FRA2 was restricted to pulmonary artery hypertension. Induction of c-Jun in mice was sufficient to induce severe fibrosis in multiple organs and steatohepatosis, which was dependent on sustained c-Jun expression. Single cell mass cytometry revealed that c-Jun activates multiple signaling pathways in mice, including pAkt and CD47, which were also induced in human disease. αCD47 antibody treatment and VEGF or PI3K inhibition reversed various organ c-Jun-mediated fibroses in vivo. These data suggest that c-JUN is a central molecular mediator of most fibrotic conditions.

High-resolution myogenic lineage mapping by single-cell mass cytometry

Abstract: Porpiglia et al. use single-cell mass cytometry to analyse surface markers and key myogenic transcription factors of skeletal muscle stem cells during homeostasis and repair, and identify previously unrecognized myogenic progenitor cell populations.

Mass cytometry analysis reveals hyperactive NF Kappa B signaling in myelofibrosis and secondary acute myeloid leukemia

Abstract: Myeloproliferative neoplasms (MPNs) feature a malignant clone containing the JAK2 V617F mutation, or another mutation causing dysregulated JAK2 kinase activity. The multiple disease phenotypes of MPNs, and their tendency to transform phenotypically, suggest pathophysiologic heterogeneities beyond a common phenomenon of JAK2 hyperactivation. JAK2 has the potential to activate multiple other signaling molecules, either directly through downstream effectors, or indirectly through induction of target gene expression. We have interrogated myeloproliferative signaling in myelofibrosis (MF) and secondary acute myeloid leukemia (sAML) patient samples using mass cytometry, which allows the quantitative measurement of multiple signaling molecules simultaneously at the single-cell level, in cell populations representing a nearly complete spectrum of hematopoiesis. MF and sAML malignant cells demonstrated a high prevalence of hyperactivation of the JAK-STAT, MAP kinase, PI3 kinase and NFκB signaling pathways. Constitutive NFκB signaling was evident across MF and sAML patients. A supporting gene set enrichment analysis (GSEA) of MF showed many NFκB target genes to be expressed above normal levels in MF patient CD34+ cells. NFκB inhibition suppressed colony formation from MF CD34+ cells. This study indicates that NFκB signaling contributes to human myeloproliferative disease and is abnormally activated in MF and sAML.

The Human Cell Atlas

Abstract: The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body, by undertaking a Human Cell Atlas Project as an international collaborative effort. The aim would be to define all human cell types in terms of distinctive molecular profiles (e.g., gene expression) and connect this information with classical cellular descriptions (e.g., location and morphology). A comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, as well as provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas.

Upregulation of Human Endogenous Retrovirus-K Is Linked to Immunity and Inflammation in Pulmonary Arterial Hypertension

Abstract: Background:Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic, or associated with other conditions. Circul...

Atomic mass tag of bismuth‐209 for increasing the immunoassay multiplexing capacity of mass cytometry

Abstract: Mass cytometry (or CyTOF) is an atomic mass spectrometry-based single-cell immunoassay technology, which has provided an increasingly systematic and sophisticated view in basic biological and clinical studies. Using elemental reporters composed of stable heavy metal isotopes, more than 50 cellular parameters are measured simultaneously. However, this current multiplexing does not meet the theoretical capability of CyTOF instrumentation with 135 detectable channels, primarily due to the limitation of available chemistries for conjugating elemental mass tags to affinity reagents. To address this issue, we develop herein additional metallic mass tag based on bismuth-209 (209 Bi) for efficient conjugation to monoclonal antibody. This enables the use of an addtional channel m/z = 209 of CyTOF for single-cell immunoassays. Bismuth has nearly the same charge-to-radius ratio as lanthanide elements; thus, bismuth(III) cations (209 Bi3+ ) could coordinate with DTPA chelators in the same geometry of O- and N-donor groups as that of lanthanide. In this report, the coordination chemistry of 209 Bi3+ with DTPA chelators and Maxpar® X8 polymers were investigated in details. Accordingly, the protocols of conjugating antibody with bismuth mass tag were provided. A method based on UV-Vis absorbance at 280 nm of 209 Bi3+ -labeling DTPA complexes was developed to evaluate the stoichiometric ratio of 209 Bi3+ cations to the conjugated antibody. Side-by-side single-cell analysis experiments with bismuth- and lanthanide-tagged antibodies were carried out to compare the analytical sensitivities. The measurement accuracy of bismuth-tagged antibody was validated within in vitro assay using primary human natural killer cells. Furthermore, bismuth-tagged antibodies were successfully employed in cell cycle measurements and high-dimensional phenotyping immunoassays. © 2017 International Society for Advancement of Cytometry.

The histone deacetylase inhibitor givinostat (ITF2357) exhibits potent anti-tumor activity against CRLF2-rearranged BCP-ALL

Abstract: Leukemias bearing CRLF2 and JAK2 gene alterations are characterized by aberrant JAK/STAT signaling and poor prognosis. The HDAC inhibitor givinostat/ITF2357 has been shown to exert anti-neoplastic activity against both systemic juvenile idiopathic arthritis and myeloproliferative neoplasms through inhibition of the JAK/STAT pathway. These findings led us to hypothesize that givinostat might also act against CRLF2-rearranged BCP-ALL, which lack effective therapies. Here, we found that givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged cell lines, positive for exon 16 JAK2 mutations. Likewise, givinostat killed primary cells, but not their normal hematopoietic counterparts, from patients carrying CRLF2 rearrangements. At low doses, givinostat downregulated the expression of genes belonging to the JAK/STAT pathway and inhibited STAT5 phosphorylation. In vivo, givinostat significantly reduced engraftment of human blasts in patient-derived xenograft models of CRLF2-positive BCP-ALL. Importantly, givinostat killed ruxolitinib-resistant cells and potentiated the effect of current chemotherapy. Thus, givinostat in combination with conventional chemotherapy may represent an effective therapeutic option for these difficult-to-treat subsets of ALL. Lastly, the selective killing of cancer cells by givinostat may allow the design of reduced intensity regimens in CRLF2-rearranged Down syndrome-associated BCP-ALL patients with an overall benefit in terms of both toxicity and related complications.

Highly-multiplexed fluorescent imaging

Abstract: Provided herein is a method and system for analyzing a sample. In some embodiments the method makes use of a plurality of capture agents that are each linked to a different oligonucleotide and a corresponding plurality of labeled nucleic acid probes, wherein each of the labeled nucleic acid probes specifically hybridizes with only one of the oligonucleotides. The sample is labeled with the capture agents en masse, and sub-sets of the capture agents are detected using iterative cycles using corresponding subsets of the labeled nucleic acid probes.

An updated debarcoding tool for mass cytometry with cell type-specific and cell sample-specific stringency adjustment.

Abstract: Pooled sample analysis by mass cytometry barcoding carries many advantages: reduced antibody consumption, increased sample throughput, removal of cell doublets, reduction of cross-contamination by sample carryover, and the elimination of tube-to-tube-variability in antibody staining. A single-cell debarcoding algorithm was previously developed to improve the accuracy and yield of sample deconvolution, but this method was limited to using fixed parameters for debarcoding stringency filtering, which could introduce cell-specific or sample-specific bias to cell yield in scenarios where barcode staining intensity and variance are not uniform across the pooled samples. To address this issue, we have updated the algorithm to output debarcoding parameters for every cell in the sample-assigned FCS files, which allows for visualization and analysis of these parameters via flow cytometry analysis software. This strategy can be used to detect cell type-specific and sample-specific effects on the underlying cell data that arise during the debarcoding process. An additional benefit to this strategy is the decoupling of barcode stringency filtering from the debarcoding and sample assignment process. This is accomplished by removing the stringency filters during sample assignment, and then filtering after the fact with 1- and 2-dimensional gating on the debarcoding parameters which are output with the FCS files. These data exploration strategies serve as an important quality check for barcoded mass cytometry datasets, and allow cell type and sample-specific stringency adjustment that can remove bias in cell yield introduced during the debarcoding process.

Multiplexed single molecule rna visualization with a two-probe proximity ligation system

Abstract: SNAIL provides cost-efficient detection of specific nucleic acids in single cells, and may be combined with flow cytometry to simultaneously analyze large numbers of cells for a plurality of nucleic acids, eg at least one, to up to 5, up to 10, up to 15, up to 20 or more transcripts can be simultaneously analyzed, at a rate of up to about 50, 100, 250, 500 or more cells/second The methods require only two primers for amplification, and may further include a detection primer

In silico modeling identifies CD45 as a regulator of IL-2 synergy in the NKG2D-mediated activation of immature human NK cells.

Abstract: Natural killer (NK) cells perform immunosurveillance of virally infected and transformed cells, and their activation depends on the balance between signaling by inhibitory and activating receptors. Cytokine receptor signaling can synergize with activating receptor signaling to induce NK cell activation. We investigated the interplay between the signaling pathways stimulated by the cytokine interleukin-2 (IL-2) and the activating receptor NKG2D in immature (CD56bright) and mature (CD56dim) subsets of human primary NK cells using mass cytometry experiments and in silico modeling. Our analysis revealed that IL-2 changed the abundances of several key proteins, including NKG2D and the phosphatase CD45. Furthermore, we found differences in correlations between protein abundances, which were associated with the maturation state of the NK cells. The mass cytometry measurements also revealed that the signaling kinetics of key protein abundances induced by NKG2D stimulation depended on the maturation state and the pretreatment condition of the NK cells. Our in silico model, which described the multidimensional data with coupled first-order reactions, predicted that the increase in CD45 abundance was a major enhancer of NKG2D-mediated activation in IL-2-treated CD56bright NK cells but not in IL-2-treated CD56dim NK cells. This dependence on CD45 was verified by measurement of CD107a mobilization to the NK cell surface (a marker of activation). Our mathematical framework can be used to glean mechanisms underlying synergistic signaling pathways in other activated immune cells.

The road ahead: Implementing mass cytometry in clinical studies, one cell at a time

Abstract: Disease in humans involves a complicated interplay of pathology expressed on each individual’s unique genetic and phenotypic backdrop. The exponential growth of genomic, transcriptomic, and proteomic single-cell technologies provides unprecedented opportunities to capture and understand this complexity. Mass cytometry in particular—a flow cytometry platform that enables simultaneous measurement of over 50 parameters per cell (1–3)—holds significant promise to identify molecular signatures that underlie clinical outcomes (4,5), to help monitor disease progression (6), and to predict therapeutic responses (7,8). However, as the dimensionality of mass cytometry datasets increases, the development of appropriate computational approaches and standardized protocols becomes paramount (9). In this special issue published jointly with Cytometry A (10), we explore the necessary steps to transform mass cytometry from a technological tour-de-force to a valuable clinical platform. We highlight six studies that illustrate recent progress. The manuscripts by Yao et al. (11), Corneau et al. (12), and Strauss-Albee et al. (13) emphasize the utility of mass cytometry for identifying cell subsets that capture patient-specific disease attributes in the fields of pulmonology, neonatology, and virology. The studies by Abraham et al. (14) and Vendrame et al. (15) apply visualization and statistical tools in novel combinations to interpret high-dimensional data, while the manuscript by Leelatian et al. (16) describes a standardized protocol to derive isolated cancer cells from solid tissue for analysis by mass cytometry. Yao et al. (11) demonstrate the utility of multiparameter profiling of airway inflammatory cells in patients with cystic fibrosis (CF) to better understand the complex interactions between an individual’s immune state and disease severity. Using a novel assay for the mass cytometry analysis of airway inflammatory cells, the authors identified significant differences in the frequency of immune cell subsets in sputum collected from patients with CF, patients with asthma, and healthy controls. Interestingly, within each group substantial variability was observed when stimulating airway monocytes with lipopolysaccharide. These findings provide the basis for future work relating patient-specific signatures of airway inflammatory cells to disease progression and severity. Strauss-Albee et al. (13) focus on the deep immune profiling of umbilical cord blood samples to better define the natural killer (NK) cell repertoire and functional capacity of newborns. The authors provide a comprehensive overview of neonatal NK cell subsets and their functional attributes. Such characterization is important to better understand disease processes that engage the innate immune system of neonates who are uniquely vulnerable to infections and are unable to mount an efficient immune response to vaccination. In a third example emphasizing a translational application of mass cytometry, Corneau et al. (12) examine the cell cycle of CD41 T cells in the setting of HIV infection. Using a combination of cell cycle, differentiation, activation and exhaustion markers, they provide a more nuanced view of the effects of HIV infection on CD41 T cell cycling and question the traditional definitions of “resting” CD41 T cells. These results have potential clinical implications, as resting CD4 1 T cells are a reservoir for HIV infection difficult to target with commonly used treatments for HIV. Visualization of multiple cellular attributes across many cell subsets and robust statistical interpretation of high dimensional data remain critical challenges in implementing mass cytometry in clinical studies. The studies by Vendrame et al. (15) and Abraham et al. (14) illustrate these points. Vendrame et al. leverage the distinct strengths of existing computational approaches (i.e., viSNE (17) and citrus (18)] combined with custommade statistical tools (i.e., correspondence analysis and Friedman–Rafsky significance test) to extensively explore the effects of cytokines on human NK cell phenotype and function. Abraham et al. (14) introduce a radial visualization method called RADVIS, as an elegant

Deep Immune Profiling of an Arginine-Enriched Nutritional Intervention in Patients Undergoing Surgery.

Abstract: Application of high-content immune profiling technologies has enormous potential to advance medicine. Whether these technologies reveal pertinent biology when implemented in interventional clinical trials is an important question. The beneficial effects of preoperative arginine-enriched dietary supplements (AES) are highly context specific, as they reduce infection rates in elective surgery, but possibly increase morbidity in critically ill patients. This study combined single-cell mass cytometry with the multiplex analysis of relevant plasma cytokines to comprehensively profile the immune-modifying effects of this much-debated intervention in patients undergoing surgery. An elastic net algorithm applied to the high-dimensional mass cytometry dataset identified a cross-validated model consisting of 20 interrelated immune features that separated patients assigned to AES from controls. The model revealed wide-ranging effects of AES on innate and adaptive immune compartments. Notably, AES increased STAT1 and STAT3 signaling responses in lymphoid cell subsets after surgery, consistent with enhanced adaptive mechanisms that may protect against postsurgical infection. Unexpectedly, AES also increased ERK and P38 MAPK signaling responses in monocytic myeloid-derived suppressor cells, which was paired with their pronounced expansion. These results provide novel mechanistic arguments as to why AES may exert context-specific beneficial or adverse effects in patients with critical illness. This study lays out an analytical framework to distill high-dimensional datasets gathered in an interventional clinical trial into a fairly simple model that converges with known biology and provides insight into novel and clinically relevant cellular mechanisms.

High‐throughput precision measurement of subcellular localization in single cells

Abstract: To quantify visual and spatial information in single cells with a throughput of thousands of cells per second, we developed Subcellular Localization Assay (SLA). This adaptation of Proximity Ligation Assay expands the capabilities of flow cytometry to include data relating to localization of proteins to and within organelles. We used SLA to detect the nuclear import of transcription factors across cell subsets in complex samples. We further measured intranuclear re-localization of target proteins across the cell cycle and upon DNA damage induction. SLA combines multiple single-cell methods to bring about a new dimension of inquiry and analysis in complex cell populations. © 2017 International Society for Advancement of Cytometry.

Mass cytometry: The time to settle down.

Abstract: MASS cytometry (CyTOF) technology was first described by Bandura et al. in 2009 (1) boosting the number of measurable markers per single cell and revolutionizing the flow cytometry field toward a horizon of a theoretical 100 measurements This revolution also boosted the development of other single cell multi-parameter technologies such as spectral flow cytometry (2) and chip-based cytometry (3), together with conventional flow cytometry that can now reach 50 theoretical parameters (4). The aim of this special issue is to mark the point that mass cytometry is presently a well-established technology with a large community of scientists committed to its development. It represents also a “settling moment” as described in a previous editorial (5) to next bring the technology to full maturity. This special Cytometry Part A issue includes the first OMIP describing a mass cytometry panel for the immune phenotype of human peripheral leukocytes together with a series of manuscripts introducing new reagents, protocols, quality controls and, also, a nice example of how the multidimensional nature of mass cytometry can address important biological question such as the status of a “challenged” immune system in comparison to a system kept in the clean environment of a pathogen-free laboratory. For those accustomed to traditional flow cytometry, one of the main drawbacks of mass cytometry is the absence of forward and side light scatter measurements to appreciate cell size and internal complexity. In this issue Stern et al. (this issue, page 14) use two plasma membrane staining assays based on wheat germ agglutinin and osmium tetroxide to evaluate cell size in mass cytometry experiments. Resolution is not comparable to conventional flow cytometry light scatter measurements; nevertheless, the combined use of these new membrane specific moieties, combined with phenotypic markers and the use of algorithms able to simultaneously evaluate multiple measurements hold promise for an extended use of these two reagents. To increase the assortment of available labels, Schulz et al. (this issue, page 25) introduce streptavidin coupled silver nanoparticles that can be used to include biotinylated reagents in mass cytometry panels. Of note, silver isotopes are detected in channels where at the moment no other reagents are available and hence the new reagent can be easily integrated in existing antibody panels. Wheat germ agglutinin and osmium were previously used to stain plasma membranes whereas silver nanoparticles are already in use in a wide range of immunoassays. Hence, the manuscripts by Stern et al. and Schulz et al. are nice examples of innovation created by changing the domain of usage. In the near future, mass cytometry will be used for clinical and longitudinal studies requiring an improvement of standardized protocols, methods to facilitate longitudinal analysis, and to accelerate the time needed for sample acquisition. Four manuscripts go in this direction. The OMIP-034 by Baumgart et al. (this issue, page 34) describes a basic 26 antibody panel able to identify neutrophils, eosinophils, basophils, monocytes, dendritic cells, T and B lymphocytes. Of note, the panel leaves several channels free to be completed with additional “drop-in” markers, and therefore a common backbone can be shared for multiple purposes. The panel described in OMIP-34 was designed keeping in account of the minimal but significant signal interference described by Takahashi et al. (this issue, page 39). Of interest for longitudinal studies and

EBI3 regulates the NK cell response to mouse cytomegalovirus infection

Abstract: Natural killer (NK) cells are key mediators in the control of cytomegalovirus infection. Here, we show that Epstein-Barr virus-induced 3 (EBI3) is expressed by human NK cells after NKG2D or IL-12 plus IL-18 stimulation and by mouse NK cells during mouse cytomegalovirus (MCMV) infection. The induction of EBI3 protein expression in mouse NK cells is a late activation event. Thus, early activation events of NK cells, such as IFNγ production and CD69 expression, were not affected in EBI3-deficient (Ebi3-/- ) C57BL/6 (B6) mice during MCMV infection. Furthermore, comparable levels of early viral replication in spleen and liver were observed in MCMV-infected Ebi3-/- and wild-type (WT) B6 mice. Interestingly, the viral load in salivary glands and oral lavage was strongly decreased in the MCMV-infected Ebi3-/- B6 mice, suggesting that EBI3 plays a role in the establishment of MCMV latency. We detected a decrease in the sustained IL-10 production by NK cells and lower serum levels of IL-10 in the MCMV-infected Ebi3-/- B6 mice. Furthermore, we observed an increase in dendritic cell maturation markers and an increase in activated CD8+ T cells. Thus, EBI3 dampens the immune response against MCMV infection, resulting in prolonged viral persistence.

Scalable multi-sample single-cell data analysis by Partition-Assisted Clustering and Multiple Alignments of Networks.

Abstract: Mass cytometry (CyTOF) has greatly expanded the capability of cytometry. It is now easy to generate multiple CyTOF samples in a single study, with each sample containing single-cell measurement on 50 markers for more than hundreds of thousands of cells. Current methods do not adequately address the issues concerning combining multiple samples for subpopulation discovery, and these issues can be quickly and dramatically amplified with increasing number of samples. To overcome this limitation, we developed Partition-Assisted Clustering and Multiple Alignments of Networks (PAC-MAN) for the fast automatic identification of cell populations in CyTOF data closely matching that of expert manual-discovery, and for alignments between subpopulations across samples to define dataset-level cellular states. PAC-MAN is computationally efficient, allowing the management of very large CyTOF datasets, which are increasingly common in clinical studies and cancer studies that monitor various tissue samples for each subject.

Meta-analysis of Cytometry Data Reveals Racial Differences in Immune Cells

Abstract: While meta-analysis has demonstrated increased statistical power and more robust estimations in studies, the application of this commonly accepted methodology to cytometry data has been challenging. Different cytometry studies often involve diverse sets of markers. Moreover, the detected values of the same marker are inconsistent between studies due to different experimental designs and cytometer configurations. As a result, the cell subsets identified by existing auto-gating methods cannot be directly compared across studies. We developed MetaCyto for automated meta-analysis of both flow and mass cytometry (CyTOF) data. By combining clustering methods with a silhouette scanning method, MetaCyto is able to identify common cell subsets across studies, thus enabling meta-analysis. Applying MetaCyto on a set of 10 heterogeneous cytometry studies with a total of 5966 samples allowed us to identify multiple cell populations exhibiting differences in phenotype and abundance across races. Software is released to the public through GitHub (github.com/hzc363/MetaCyto).

Microsphere cytometry to interrogate microenvironment-dependent cell signaling.

Abstract: Microenvironmental cues comprising surface-mediated and soluble factors control cellular signaling mechanisms underlying normal cellular responses that define homeostatic and diseased cell states. In order to measure cell signaling in single adherent cells, we developed a novel microsphere-based flow cytometry approach. Single normal or neoplastic cells were adhered to uniform microspheres that display mimetic-microenvironments comprising surface combinations of extracellular matrix (ECM) in the presence of soluble agonists/antagonists. Temporal signaling responses were measured with fluorophore-conjugated antibodies that recognize response-dependent epitopes by multiparametric flow cytometry. Using this approach we demonstrate that microenvironment-mimetic combinations of growth factors and extracellular matrix proteins generate distinct cellular signal networks that reveal unique cell signatures in normal and patient biopsy-derived neoplastic cells.