Flow cytometry is an essential tool for dissecting the functional complexity of hematopoiesis. We used single-cell “mass cytometry” to examine healthy human bone marrow, measuring 34 parameters simultaneously in single cells (binding of 31 antibodies, viability, DNA content, and relative cell size). The signaling behavior of cell subsets spanning a defined hematopoietic hierarchy was monitored with 18 simultaneous markers of functional signaling states perturbed by a set of ex vivo stimuli and inhibitors. The data set allowed for an algorithmically driven assembly of related cell types defined by surface antigen expression, providing a superimposable map of cell signaling responses in combination with drug inhibition. Visualized in this manner, the analysis revealed previously unappreciated instances of both precise signaling responses that were bounded within conventionally defined cell subsets and more continuous phosphorylation responses that crossed cell population boundaries in unexpected manners yet tracked closely with cellular phenotype. Collectively, such single-cell analyses provide system-wide views of immune signaling in healthy human hematopoiesis, against which drug action and disease can be compared for mechanistic studies and pharmacologic intervention.

Altered growth factor responses in phospho-protein-driven signaling networks are crucial to cancer cell survival and pathology. Profiles of cancer cell signaling networks might therefore identify mechanisms by which such cells interpret environmental cues for continued growth. Using multiparameter flow cytometry, we monitored phospho-protein responses to environmental cues in acute myeloid leukemia at the single cell level. By exposing cancer cell signaling networks to potentiating inputs, rather than relying upon the basal levels of protein phosphorylation alone, we could discern unique cancer network profiles that correlated with genetics and disease outcome. Strikingly, individual cancers manifested multiple cell subsets with unique network profiles, reflecting cancer heterogeneity at the level of signaling response. The results revealed a dramatic remodeling of signaling networks in cancer cells. Thus, single cell measurements of phospho-protein responses reveal shifts in signaling potential of a phospho-protein network, allowing for categorizing of cell network phenotypes by multidimensional molecular profiles of signaling.

The ability to comprehensively explore the impact of bio-active molecules on human samples at the single-cell level can provide great insight for biomedical research. Mass cytometry enables quantitative single-cell analysis with deep dimensionality, but currently lacks high-throughput capability. Here we report a method termed mass-tag cellular barcoding (MCB) that increases mass cytometry throughput by sample multiplexing. 96-well format MCB was used to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics, cell-to-cell communication, the signaling variability between 8 donors, and to define the impact of 27 inhibitors on this system. For each compound, 14 phosphorylation sites were measured in 14 PBMC types, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors, and revealed off-target effects. MCB enables high-content, high-throughput screening, with potential applications for drug discovery, pre-clinical testing, and mechanistic investigation of human disease.

Flow cytometry allows high-content, multiparameter analysis of single cells, making it a promising tool for drug discovery and profiling of intracellular signaling. To add high-throughput capacity to flow cytometry, we developed a cell-based multiplexing technique called fluorescent cell barcoding (FCB). In FCB, each sample is labeled with a different signature, or barcode, of fluorescence intensity and emission wavelengths, and mixed with other samples before antibody staining and analysis by flow cytometry. Using three FCB fluorophores, we were able to barcode and combine entire 96-well plates, reducing antibody consumption 100-fold and acquisition time to 5-15 min per plate. Using FCB and phospho-specific flow cytometry, we screened a small-molecule library for inhibitors of T cell-receptor and cytokine signaling, simultaneously determining compound efficacy and selectivity. We also analyzed IFN-gamma signaling in multiple cell types from primary mouse splenocytes, revealing differences in sensitivity and kinetics between B cells, CD4+ and CD4- T cells and CD11b-hi cells.

Background: Recent advances in intracellular staining techniques, cytometer technology, fluorescent reagents, and antibody production have expanded the number of intracellular antigens that can be analyzed by flow cytometry. Measurement of protein phosphorylation with phospho-specific antibodies has given insight into kinase signaling cascades. However, available techniques for phospho-epitope staining can differ greatly, making it necessary to understand the differences between the outcomes when such techniques are applied and to develop robust and reproducible methods of application. Methods: Ten different cellular fixation and permeabilization techniques were tested for their ability to provide phospho-specific staining. Combinations of formaldehyde, methanol, ethanol, acetone, Triton X-100, and saponin were used as fixation and permeabilization reagents. Phospho-specific antibodies were labeled with Alexa Fluor dyes to provide multicolor analysis of different signaling events simultaneously within individual cells. Results: Fixing cells with 1.5% formaldehyde followed by permeabilization in methanol gave optimal results for pERK, pp38, pJNK, pStat1, pStat5, and pStat6 staining.

Summary Type I Interferons (IFNs) are important cytokines for innate immunity against viruses and cancer. Sixteen human IFN variants signal through the same cell surface receptors, IFNAR1 and IFNAR2, yet they can evoke markedly different physiological effects. The crystal structures of two human type I IFN ternary signaling complexes containing IFNα2 and IFNω reveal recognition modes and heterotrimeric architectures that are unique amongst the cytokine receptor superfamily, but conserved between different type I IFNs. Receptor-ligand cross-reactivity is enabled by conserved receptor-ligand "anchor-points" interspersed amongst ligand-specific interactions that ‘tune’ the relative IFN binding affinities, in an apparent extracellular ‘ligand proofreading’ mechanism that modulates biological activity. Functional differences between IFNs are linked to their respective receptor recognition chemistries, in concert with a ligand-induced conformational change in IFNAR1, that collectively control signal initiation and complex stability, ultimately regulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns.

Cytobank is a Web‐based application for storage, analysis, and sharing of flow cytometry experiments. Researchers use a Web browser to log in and use a wide range of tools developed for basic and advanced flow cytometry. In addition to providing access to standard cytometry tools from any computer, Cytobank creates a platform and community for developing new analysis and publication tools. Figure layouts created on Cytobank are designed to allow transparent access to the underlying experiment annotation and data processing steps. Since all flow cytometry files and analysis data are stored on a central server, experiments and figures can be viewed or edited by anyone with the proper permission, from any computer with Internet access. Once a primary researcher has performed the initial analysis of the data, collaborators can engage in experiment analysis and make their own figure layouts using the gated, compensated experiment files. Cytobank is available to the scientific community at http://www.cytobank.org. Curr. Protoc. Cytom. 53:10.17.1‐10.17.24. © 2010 by John Wiley & Sons, Inc.

SUMMARY Most cell surface receptors for cytokines and growth factors signal as dimers, but it is unclear if remodeling receptor dimer topology is a viable strategy to ‘tune’ signaling output. We utilized diabodies (DA) as surrogate ligands in a prototypical dimeric receptor-ligand system, the cytokine Erythropoietin and its receptor (EpoR), to dimerize EpoR ectodomains in non-native architectures. Diabody-induced signaling amplitude varied from full to minimal agonism, and structures of the DA/EpoR complexes differed in EpoR dimer orientation and proximity. Diabodies also elicited biased, or differential activation of signaling pathways and gene expression profiles compared to EPO. Non-signaling diabodies inhibited proliferation of erythroid precursors from patients with a myeloproliferative neoplasm due to a constitutively active JAK2V617F mutation. Thus, intracellular oncogenic mutations causing ligand-independent receptor activation can be counteracted by extracellular ligands that re-orient receptors into inactive dimer topologies. This approach has broad applications for tuning signaling output for many dimeric receptor systems.