Structural linkage between ligand discrimination and receptor activation by type I interferons.

Type I Interferons (IFNs) are important cytokines for innate immunity against viruses and cancer. Sixteen human type I 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 among the cytokine receptor superfamily but conserved between different type I IFNs. Receptor-ligand cross-reactivity is enabled by conserved receptor-ligand "anchor points" interspersed among 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.

Functional comparison of PBMCs isolated by Cell Preparation Tubes (CPT) vs. Lymphoprep Tubes.

BACKGROUND: Cryopreserved human peripheral blood mononuclear cells (PBMCs) are a commonly used sample type for a variety of immunological assays. Many factors can affect the quality of PBMCs, and careful consideration and validation of an appropriate PBMC isolation and cryopreservation method is important for well-designed clinical studies. A major point of divergence in PBMC isolation protocols is the collection of blood, either directly into vacutainers pre-filled with density gradient medium or the use of conical tubes containing a porous barrier to separate the density gradient medium from blood. To address potential differences in sample outcome, we isolated, cryopreserved, and compared PBMCs using parallel protocols differing only in the use of one of two common tube types for isolation. METHODS: Whole blood was processed in parallel using both Cell Preparation Tubes™ (CPT, BD Biosciences) and Lymphoprep™ Tubes (Axis-Shield) and assessed for yield and viability prior to cryopreservation. After thawing, samples were further examined by flow cytometry for cell yield, cell viability, frequency of 10 cell subsets, and capacity for stimulation-dependent CD4+ and CD8+ T cell intracellular cytokine production. RESULTS: No significant differences in cell recovery, viability, frequency of immune cell subsets, or T cell functionality between PBMC samples isolated using CPT or Lymphoprep tubes were identified. CONCLUSION: CPT and Lymphoprep tubes are effective and comparable methods for PBMC isolation for immunological studies.

The initial phase of an immune response functions to activate regulatory T cells.

An early reaction of CD4(+) T lymphocytes to Ag is the production of cytokines, notably IL-2. To detect cytokine-dependent responses, naive Ag-specific T cells were stimulated in vivo and the presence of phosphorylated STAT5 molecules was used to identify the cell populations responding to IL-2. Within hours of T cell priming, IL-2-dependent STAT5 phosphorylation occurred primarily in Foxp3(+) regulatory T cells. In contrast, the Ag-specific T cells received STAT5 signals only after repeated Ag exposure or memory differentiation. Regulatory T cells receiving IL-2 signals proliferated and developed enhanced suppressive activity. These results indicate that one of the earliest events in a T cell response is the activation of endogenous regulatory cells, potentially to prevent autoimmunity.

Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry.

Intracellular flow cytometry permits quantitation of diverse molecular targets at the single-cell level. However, limitations in detection sensitivity inherently restrict the method, sometimes resulting in the inability to measure proteins of very low abundance or to differentiate cells expressing subtly different protein concentrations. To improve these measurements, an enzymatic amplification approach called tyramide signal amplification (TSA) was optimized for assessment of intracellular kinase cascades. First, Pacific Blue, Pacific Orange, and Alexa Fluor 488 tyramide reporters were shown to exhibit low nonspecific binding in permeabilized cells. Next, the effects of antibody concentration, tyramide concentration, and reaction time on assay resolution were characterized. Use of optimized TSA resulted in a 10-fold or greater improvement in measurement resolution of endogenous Erk and Stat cell signaling pathways relative to standard, nonamplified detection. TSA also enhanced assay sensitivity and, in conjunction with fluorescent cell barcoding, improved assay performance according to a metric used to evaluate high-throughput drug screens. TSA was used to profile Stat1 phosphorylation in primary immune system cells, which revealed heterogeneity in various populations, including CD4+ FoxP3+ regulatory T cells. We anticipate the approach will be broadly applicable to intracellular flow cytometry assays with low signal-to-noise ratios.

High-content single-cell drug screening with phosphospecific flow cytometry.

Drug screening is often limited to cell-free assays involving purified enzymes, but it is arguably best applied against systems that represent disease states or complex physiological cellular networks. Here, we describe a high-content, cell-based drug discovery platform based on phosphospecific flow cytometry, or phosphoflow, that enabled screening for inhibitors against multiple endogenous kinase signaling pathways in heterogeneous primary cell populations at the single-cell level. From a library of small-molecule natural products, we identified pathway-selective inhibitors of Jak-Stat and MAP kinase signaling. Dose-response experiments in primary cells confirmed pathway selectivity, but importantly also revealed differential inhibition of cell types and new druggability trends across multiple compounds. Lead compound selectivity was confirmed in vivo in mice. Phosphoflow therefore provides a unique platform that can be applied throughout the drug discovery process, from early compound screening to in vivo testing and clinical monitoring of drug efficacy.

WebFlow: a software package for high-throughput analysis of flow cytometry data.

Flow cytometry has emerged as a powerful tool for quantitative, single-cell analysis of both surface markers and intracellular antigens, including phosphoproteins and kinase signaling cascades, with the flexibility to process hundreds of samples in multiwell plate format. Quantitative flow cytometric analysis is being applied in many areas of biology, from the study of immunology in animal models or human patients to high-content drug screening of pharmacologically active compounds. However, these experiments generate thousands of data points per sample, each with multiple measured parameters, leading to data management and analysis challenges. We developed WebFlow (http://webflow.stanford.edu), a web server-based software package to manage, analyze, and visualize data from flow cytometry experiments. WebFlow is accessible via standard web browsers and does not require users to install software on their personal computers. The software enables plate-based annotation of large data sets, which provides the basis for exploratory data analysis tools and rapid visualization of multiple different parameters. These tools include custom user-defined statistics to normalize data to other wells or other channels, as well as interactive, user-selectable heat maps for viewing the underlying single-cell data. The web-based approach of WebFlow allows for sharing of data with collaborators or the general public. WebFlow provides a novel platform for quantitative analysis of flow cytometric data from high-throughput drug screening or disease profiling experiments.

Cellular Signaling Analysis shows antiviral, ribavirin-mediated ribosomal signaling modulation.

As antiviral drug resistance develops and new viruses emerge there is a pressing need to develop strategies to rapidly develop antiviral therapeutics. Here we use phospho-specific flow cytometry to assess perturbations of many different cellular signaling pathways during treatment with drug combinations that are highly effective in blocking Herpes simplex virus type 1 (HSV-1) infection. We discovered two antiviral drug combinations act on distinct signaling pathways, either STAT1 or S6 phosphorylation, to block HSV-1 infection. We focused on upregulation of S6 phosphorylation by HSV-1 infection, and our subsequent finding that ribavirin antagonizes this upregulation of S6 phosphorylation. We go on to show that the S6 kinase inhibitor SL0101 blocks HSV-1 replication in vitro and in an in vivo animal model of HSV-1 infection. Overall, we have used an unbiased analysis of cellular signaling pathways during treatment by antiviral drug combinations to discover a novel antiviral drug target against HSV-1 infection. The outcomes of the approach we present highlight the importance of analyzing how antiviral drugs modulate cellular and pathogen-induced signaling as a method to discover new drug therapy targets.

Phospho-specific flow cytometry in drug discovery.

Here we present phospho-specific flow cytometry as a new tool for drug discovery with applications throughout the drug development pipeline, from target identification to library screening, disease model assessment and clinical screening and diagnostics. The single cell, multiparameter nature of flow cytometry generates high-content datasets, and current improvements in the technology are rapidly increasing its high-throughput capacity, making it a valuable platform in modern drug discovery.:

Flow cytometric analysis of kinase signaling cascades.

Flow cytometry offers the capability to assess the heterogeneity of cellular subsets that exist in complex populations, such as peripheral blood, based on immunophenotypes. We describe methodologies to measure phospho-epitopes in single cells as determinants of intracellular kinase activity. Multiparametric staining, using both surface and intracellular stains, allows for the study of discrete biochemical events in readily discernible lymphocyte subsets. As such, the usage of multiparameter flow cytometry to obtain proteomic information provides several major advantages: (1) the ability to perform multiparametric experiments to identify distinct signaling profiles in defined lymphocyte populations, (2) simultaneous correlation of multiple active kinases involved in signaling cascades, (3) profiling of active kinase states to identify signaling signatures of interest rapidly, and (4) biochemical access to rare cell subsets such as those from clinically derived samples or populations that comprise too few in numbers for conventional biochemical analysis.

COP9 signalosome component JAB1/CSN5 is necessary for T cell signaling through LFA-1 and HIV-1 replication.

To determine critical host factors involved in HIV-1 replication, a dominant effector genetics approach was developed to reveal signaling pathways on which HIV-1 depends for replication. A large library of short peptide aptamers was expressed via retroviral delivery in T cells. Peptides that interfered with T cell activation-dependent processes that might support HIV-1 replication were identified. One of the selected peptides altered signaling, lead to a difference in T cell activation status, and inhibited HIV-1 replication. The target of the peptide was JAB1/CSN5, a component of the signalosome complex. JAB1 expression overcame the inhibition of HIV-1 replication in the presence of peptide and also promoted HIV-1 replication in activated primary CD4(+) T cells. This peptide blocked physiological release of JAB1 from the accessory T cell surface protein LFA-1, downstream AP-1 dependent events, NFAT activation, and HIV-1 replication. Thus, genetic selection for intracellular aptamer inhibitors of host cell processes proximal to signals at the immunological synapse of T cells can define unique mechanisms important to HIV-1 replication.

Single-cell phospho-protein analysis by flow cytometry.

This protocol describes methods for monitoring intracellular phosphorylation-dependent signaling events on a single-cell basis. This approach measures cell signaling by treating cells with exogenous stimuli, fixing cells with formaldehyde, permeabilizing with methanol, and then staining with phospho-specific antibodies. Thus, cell signaling states can be determined as a measure of how cells interact with their environment. This method has applications in clinical research as well as mechanistic studies of basic biology. In clinical research, diagnostic or drug efficacy information can be retrieved by discovering how a disease affects the ability of cells to respond to growth factors. Basic scientists can use this technique to analyze signaling events in cell lines and human or murine primary cells, including rare populations, like B1 cells or stem cells. This technique has broad applications bringing standard biochemical analysis into primary cells in order to garner valuable information about signaling events in physiologic settings.

Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators.

Mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on human samples at single-cell resolution, but instruments process only one sample at a time. Here we describe mass-tag cellular barcoding (MCB), which increases mass cytometry throughput by using n metal ion tags to multiplex up to 2n samples. We used seven tags to multiplex an entire 96-well plate, and applied MCB to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics and cell-to-cell communication, signaling variability between PBMCs from eight human donors, and the effects of 27 inhibitors on this system. For each inhibitor, we measured 14 phosphorylation sites in 14 PBMC types at 96 conditions, 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. High-content, high-throughput screening with MCB should be useful for drug discovery, preclinical testing and mechanistic investigation of human disease.

Fluorescent cell barcoding for multiplex flow cytometry.

Fluorescent cell barcoding (FCB) enables high throughput, high content flow cytometry by multiplexing samples prior to staining and acquisition on the cytometer. Individual cell samples are barcoded, or labeled, with unique signatures of fluorescent dyes so that they can be mixed together, stained, and analyzed as a single sample. By mixing samples prior to staining, antibody consumption is typically reduced 10- to 100-fold. In addition, data robustness is increased through the combination of control and treated samples, which minimizes pipetting error, staining variation, and the need for normalization. Finally, speed of acquisition is enhanced, enabling large profiling experiments to be run with standard cytometer hardware. In this unit, we outline the steps necessary to apply the FCB method to cell lines, as well as primary peripheral blood samples. Important technical considerations, such as choice of barcoding dyes, concentrations, labeling buffers, compensation, and software analysis, are discussed.

Phospho flow cytometry methods for the analysis of kinase signaling in cell lines and primary human blood samples.

Phospho-specific flow cytometry, or phospho flow, measures the phosphorylation state of intracellular proteins at the single cell level. Many phosphorylation events can be analyzed simultaneously in each cell, along with cell surface markers, enabling complex biochemical signaling networks to be resolved in heterogeneous cell populations. The method has been applied to many diverse areas of biology, including the characterization of signaling pathways in normal immune responses to antigenic stimulation and microbial challenge, alteration of signaling networks that occur in cancer and autoimmune diseases, and high-throughput, high-content drug discovery. In this chapter, we provide detailed experimental protocols for performing phospho flow in cell lines, Ficoll-purified peripheral blood mononuclear cells, and whole blood. These protocols are applicable to both human and murine samples. We also provide methods for the validation of surface marker antibodies for use in phospho flow. Finally, we discuss data analysis methods, in particular, how to quantify changes in phosphorylation and how to visualize the large data sets that can result from experiments in primary cells.

Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum.

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.

Web-based analysis and publication of flow cytometry experiments.

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.

Alternate mechanisms of initial pattern recognition drive differential immune responses to related poxviruses.

Vaccinia immunization was pivotal to successful smallpox eradication. However, the early immune responses that distinguish poxvirus immunization from pathogenic infection remain unknown. To address this, we developed a strategy to map the activation of key signaling networks in vivo and applied this approach to define and compare the earliest signaling events elicited by immunizing (vaccinia) and lethal (ectromelia) poxvirus infections in mice. Vaccinia induced rapid TLR2-dependent responses, leading to IL-6 production, which then initiated STAT3 signaling in dendritic and T cells. In contrast, ectromelia did not induce TLR2 activation, and profound mouse strain-dependent responses were observed. In resistant C57BL/6 mice, the STAT1 and STAT3 pathways were rapidly activated, whereas in susceptible BALB/c mice, IL-6-dependent STAT3 activation did not occur. These data link early immune signaling events to infection outcome and suggest that activation of different pattern-recognition receptors early after infection may be important in determining vaccine efficacy.

Stage dependent aberrant regulation of cytokine-STAT signaling in murine systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease of unknown etiology that involves multiple interacting cell types driven by numerous cytokines and autoimmune epitopes. Although the initiating events leading to SLE pathology are not understood, there is a growing realization that dysregulated cytokine action on immune cells plays an important role in promoting the inflammatory autoimmune state. We applied phospho-specific flow cytometry to characterize the extent to which regulation of cytokine signal transduction through the STAT family of transcription factors is disturbed during the progression of SLE. Using a panel of 10 cytokines thought to have causal roles in the disease, we measured signaling responses at the single-cell level in five immune cell types from the MRLlpr murine model. This generated a highly multiplexed view of how cytokine stimuli are processed by intracellular signaling networks in adaptive and innate immune cells during different stages of SLE pathogenesis. We report that robust changes in cytokine signal transduction occur during the progression of SLE in multiple immune cell subtypes including increased T cell responsiveness to IL-10 and ablation of Stat1 responses to IFNalpha, IFNgamma, IL-6, and IL-21, Stat3 responses to IL-6, Stat5 responses to IL-15, and Stat6 responses to IL-4. We found increased intracellular expression of Suppressor of Cytokine Signaling 1 protein correlated with negative regulation of Stat1 responses to inflammatory cytokines. The results provide evidence of negative feedback regulation opposing inflammatory cytokines that have self-sustaining activities and suggest a cytokine-driven oscillator circuit may drive the periodic disease activity observed in many SLE patients.

Single-cell phospho-protein analysis by flow cytometry.

This protocol describes methods for monitoring intracellular phosphorylation-dependent signaling events on a single-cell basis. This approach measures cell signaling by treating cells with exogenous stimuli, fixing cells with formaldehyde, permeabilizing with methanol, and then staining with phospho-specific antibodies. Thus, cell signaling states can be determined as a measure of how cells interact with their environment. This method has applications in clinical research as well as mechanistic studies of basic biology. In clinical research, diagnostic or drug efficacy information can be retrieved by discovering how a disease affects the ability of cells to respond to growth factors. Basic scientists can use this technique to analyze signaling events in cell lines and human or murine primary cells, including rare populations, like B1 cells or stem cells. This technique has broad applications to take standard biochemical analysis into primary cells to garner valuable information about signaling events in physiologic settings.