Diminished cytokine-induced Jak/STAT signaling is associated with rheumatoid arthritis and disease activity.

Rheumatoid arthritis (RA) is a systemic and incurable autoimmune disease characterized by chronic inflammation in synovial lining of joints. To identify the signaling pathways involved in RA, its disease activity, and treatment response, we adapted a systems immunology approach to simultaneously quantify 42 signaling nodes in 21 immune cell subsets (e.g., IFNα→p-STAT5 in B cells) in peripheral blood mononuclear cells (PBMC) from 194 patients with longstanding RA (including 98 patients before and after treatment), and 41 healthy controls (HC). We found multiple differences between patients with RA compared to HC, predominantly in cytokine-induced Jak/STAT signaling in many immune cell subsets, suggesting pathways that may be associated with susceptibility to RA. We also found that high RA disease activity, compared to low disease activity, was associated with decreased (e.g., IFNα→p-STAT5, IL-10→p-STAT1) or increased (e.g., IL-6→STAT3) response to stimuli in multiple cell subsets. Finally, we compared signaling in patients with established, refractory RA before and six months after initiation of methotrexate (MTX) or TNF inhibitors (TNFi). We noted significant changes from pre-treatment to post-treatment in IFNα→p-STAT5 signaling and IL-10→p-STAT1 signaling in multiple cell subsets; these changes brought the aberrant RA signaling profiles toward those of HC. This large, comprehensive functional signaling pathway study provides novel insights into the pathogenesis of RA and shows the potential of quantification of cytokine-induced signaling as a biomarker of disease activity or treatment response.

Multiplexed ion beam imaging (MIBI) for characterization of the tumor microenvironment across tumor types.

An ability to characterize the cellular composition and spatial organization of the tumor microenvironment (TME) using multiplexed IHC has been limited by the techniques available. Here we show the applicability of multiplexed ion beam imaging (MIBI) for cell phenotype identification and analysis of spatial relationships across numerous tumor types. Formalin-fixed paraffin-embedded (FFPE) samples from tumor biopsies were simultaneously stained with a panel of 15 antibodies, each labeled with a specific metal isotope. Multi-step processing produced images of the TME that were further segmented into single cells. Frequencies of different cell subsets and the distributions of nearest neighbor distances between them were calculated using this data. A total of 50 tumor specimens from 15 tumor types were characterized for their immune profile and spatial organization. Most samples showed infiltrating cytotoxic T cells and macrophages present amongst tumor cells. Spatial analysis of the TME in two ovarian serous carcinoma images highlighted differences in the degree of mixing between tumor and immune cells across samples. Identification of admixed PD-L1+ macrophages and PD-1+ T cells in an urothelial carcinoma sample allowed for the detailed observations of immune cell subset spatial arrangement. These results illustrate the high-parameter capability of MIBI at a sensitivity and resolution uniquely suited to understanding the complex tumor immune landscape including the spatial relationships of immune and tumor cells and expression of immunoregulatory proteins.

High-dimensional analysis of the aging immune system: verification of age-associated differences in immune signaling responses in healthy donors.

BACKGROUND: Single-cell network profiling (SCNP) is a multiparametric flow cytometry-based approach that simultaneously measures evoked signaling in multiple cell subsets. Previously, using the SCNP approach, age-associated immune signaling responses were identified in a cohort of 60 healthy donors. METHODS: In the current study, a high-dimensional analysis of intracellular signaling was performed by measuring 24 signaling nodes in 7 distinct immune cell subsets within PBMCs in an independent cohort of 174 healthy donors [144 elderly (>65 yrs); 30 young (25-40 yrs)]. RESULTS: Associations between age and 9 immune signaling responses identified in the previously published 60 donor cohort were confirmed in the current study. Furthermore, within the current study cohort, 48 additional immune signaling responses differed significantly between young and elderly donors. These associations spanned all profiled modulators and immune cell subsets. CONCLUSIONS: These results demonstrate that SCNP, a systems-based approach, can capture the complexity of the cellular mechanisms underlying immunological aging. Further, the confirmation of age associations in an independent donor cohort supports the use of SCNP as a tool for identifying reproducible predictive biomarkers in areas such as vaccine response and response to cancer immunotherapies.

Association between B-cell receptor responsiveness and disease progression in B-cell chronic lymphocytic leukemia: results from single cell network profiling studies.

While many prognostic markers in B-cell chronic lymphocytic leukemia provide insight into the biology of the disease, few have been demonstrated to be useful in the daily management of patients. B-cell receptor signaling is a driving event in the progression of B-cell chronic lymphocytic leukemia and markers of B-cell receptor responsiveness have been shown to be of prognostic value. Single cell network profiling, a multiparametric flow cytometry-based assay, allows functional signaling analysis at the level of the single cell. B-cell receptor signaling proteins (i.e. p-SYK, p-NF-κB p65, p-ERK, p-p38, p-JNK) were functionally characterized by single cell network profiling in samples from patients with B-cell chronic lymphocytic leukemia in an exploratory study (n=27) after stimulation with anti-IgM. Significant associations of single cell network profiling data with clinical outcome (i.e. time to first treatment), as assessed by Cox regression models, were then confirmed in patients' samples in two other sequential independent studies, i.e. test study 1 (n=30), and test study 2 (n=37). In the exploratory study, higher responsiveness of the B-cell receptor signaling proteins to anti-IgM was associated with poor clinical outcomes. Patients' clustering based on signaling response was at least as powerful in discriminating different disease courses as traditional prognostic markers. In an unselected subgroup of patients with Binet stage A disease (n=21), increased anti-IgM-modulated p-ERK signaling was shown to be a significant, independent predictor of shorter time to first treatment. This result was independently confirmed in two test cohorts from distinct populations of patients. In conclusion, these findings support the utility of the single cell network profiling assay in elucidating signaling perturbations with the potential for the development of a clinically useful prognostic test in patients with early stage B-cell chronic lymphocytic leukemia. These data support the clinical relevance of B-cell receptor signaling in B-cell chronic lymphocytic leukemia, and suggest a key role of ERK activation in the physiopathology of this leukemia.

Single-cell network profiling of peripheral blood mononuclear cells from healthy donors reveals age- and race-associated differences in immune signaling pathway activation.

A greater understanding of the function of the human immune system at the single-cell level in healthy individuals is critical for discerning aberrant cellular behavior that occurs in settings such as autoimmunity, immunosenescence, and cancer. To achieve this goal, a systems-level approach capable of capturing the response of the interdependent immune cell types to external stimuli is required. In this study, an extensive characterization of signaling responses in multiple immune cell subpopulations within PBMCs from a cohort of 60 healthy donors was performed using single-cell network profiling (SCNP). SCNP is a multiparametric flow cytometry-based approach that enables the simultaneous measurement of basal and evoked signaling in multiple cell subsets within heterogeneous populations. In addition to establishing the interindividual degree of variation within a broad panel of immune signaling responses, the possible association of any observed variation with demographic variables including age and race was investigated. Using half of the donors as a training set, multiple age- and race-associated variations in signaling responses in discrete cell subsets were identified, and several were subsequently confirmed in the remaining samples (test set). Such associations may provide insight into age-related immune alterations associated with high infection rates and diminished protection following vaccination and into the basis for ethnic differences in autoimmune disease incidence and treatment response. SCNP allowed for the generation of a functional map of healthy immune cell signaling responses that can provide clinically relevant information regarding both the mechanisms underlying immune pathological conditions and the selection and effect of therapeutics.

Signal transducer and activator of transcription 3 (STAT3) and survivin induction by varicella-zoster virus promote replication and skin pathogenesis.

Varicella-zoster virus (VZV) is a human α-herpesvirus that causes varicella (chickenpox) during primary infection and zoster (shingles) upon reactivation. Like other viruses, VZV must subvert the intrinsic antiviral defenses of differentiated human cells to produce progeny virions. Accordingly, VZV inhibits the activation of the cellular transcription factors IFN regulatory factor 3 (IRF3) and signal transducers and activators of transcription 1 (STAT1), thereby downregulating antiviral factors, including IFNs. Conversely, in this study, we found that VZV triggers STAT3 phosphorylation in cells infected in vitro and in human skin xenografts in SCID mice in vivo and that STAT3 activation induces the anti-apoptotic protein survivin. Small-molecule inhibitors of STAT3 phosphorylation and survivin restrict VZV replication in vitro, and VZV infection of skin xenografts in vivo is markedly impaired by the administration of the phospho-STAT3 inhibitor S3I-201. STAT3 and survivin are required for malignant transformation caused by γ-herpesviruses, such as Kaposi's sarcoma virus. We show that STAT3 activation is also critical for VZV, a nononcogenic herpesvirus, via a survivin-dependent mechanism. Furthermore, STAT3 activation is critical for the life cycle of the virus because VZV skin infection is necessary for viral transmission and persistence in the human population. Therefore, we conclude that takeover of this major cell-signaling pathway is necessary, independent of cell transformation, for herpesvirus pathogenesis and that STAT3 activation and up-regulation of survivin is a common mechanism important for the pathogenesis of lytic as well as tumorigenic herpesviruses.

Kinase substrate interactions.

Kinases have become popular therapeutic targets primarily due to their integral role in cell cycle and tumor progression. The efficacy of high-throughput screening efforts is dependent on the development of high quality multiplex tools capable of replacing lower-throughput technologies such as mass spectroscopy or solution-based assays for the study of kinase-substrate interactions. Functional protein microarrays are comprised of thousands of immobilized proteins on glass slides that have been used successfully to identify protein-protein interactions. Here, we describe the application of functional protein microarrays for the identification of the phosphorylation targets of individual protein kinases using highly sensitive radioactive detection and robust informatics algorithms.

Protein microarray technology.

Protein chips have emerged as a promising approach for a wide variety of applications including the identification of protein-protein interactions, protein-phospholipid interactions, small molecule targets, and substrates of proteins kinases. They can also be used for clinical diagnostics and monitoring disease states. This article reviews current methods in the generation and applications of protein microarrays.

Charging it up: global analysis of protein phosphorylation.

Protein phosphorylation affects most, if not all, cellular activities in eukaryotes and is essential for cell proliferation and development. An estimated 30% of cellular proteins are phosphorylated, representing the phosphoproteome, and phosphorylation can alter a protein's function, activity, localization and stability. Recent studies for large-scale identification of phosphosites using mass spectrometry are revealing the components of the phosphoproteome. The development of new tools, such as kinase assays using modified kinases or protein microarrays, enables rapid kinase substrate identification. The dynamics of specific phosphorylation events can now be monitored using mass spectrometry, single-cell analysis of flow cytometry, or fluorescent reporters. Together, these techniques are beginning to elucidate cellular processes and pathways regulated by phosphorylation, in addition to global regulatory networks.

Global analysis of protein phosphorylation in yeast.

Protein phosphorylation is estimated to affect 30% of the proteome and is a major regulatory mechanism that controls many basic cellular processes. Until recently, our biochemical understanding of protein phosphorylation on a global scale has been extremely limited; only one half of the yeast kinases have known in vivo substrates and the phosphorylating kinase is known for less than 160 phosphoproteins. Here we describe, with the use of proteome chip technology, the in vitro substrates recognized by most yeast protein kinases: we identified over 4,000 phosphorylation events involving 1,325 different proteins. These substrates represent a broad spectrum of different biochemical functions and cellular roles. Distinct sets of substrates were recognized by each protein kinase, including closely related kinases of the protein kinase A family and four cyclin-dependent kinases that vary only in their cyclin subunits. Although many substrates reside in the same cellular compartment or belong to the same functional category as their phosphorylating kinase, many others do not, indicating possible new roles for several kinases. Furthermore, integration of the phosphorylation results with protein-protein interaction and transcription factor binding data revealed novel regulatory modules. Our phosphorylation results have been assembled into a first-generation phosphorylation map for yeast. Because many yeast proteins and pathways are conserved, these results will provide insights into the mechanisms and roles of protein phosphorylation in many eukaryotes.

Substrate specificity analysis of protein kinase complex Dbf2-Mob1 by peptide library and proteome array screening.

BACKGROUND: The mitotic exit network (MEN) is a group of proteins that form a signaling cascade that is essential for cells to exit mitosis in Saccharomyces cerevisiae. The MEN has also been implicated in playing a role in cytokinesis. Two components of this signaling pathway are the protein kinase Dbf2 and its binding partner essential for its kinase activity, Mob1. The components of MEN that act upstream of Dbf2-Mob1 have been characterized, but physiological substrates for Dbf2-Mob1 have yet to be identified. RESULTS: Using a combination of peptide library selection, phosphorylation of optimal peptide variants, and screening of a phosphosite array, we found that Dbf2-Mob1 preferentially phosphorylated serine over threonine and required an arginine three residues upstream of the phosphorylated serine in its substrate. This requirement for arginine in peptide substrates could not be substituted with the similarly charged lysine. This specificity determined for peptide substrates was also evident in many of the proteins phosphorylated by Dbf2-Mob1 in a proteome chip analysis. CONCLUSION: We have determined by peptide library selection and phosphosite array screening that the protein kinase Dbf2-Mob1 preferentially phosphorylated substrates that contain an RXXS motif. A subsequent proteome microarray screen revealed proteins that can be phosphorylated by Dbf2-Mob1 in vitro. These proteins are enriched for RXXS motifs, and may include substrates that mediate the function of Dbf2-Mob1 in mitotic exit and cytokinesis. The relatively low degree of sequence restriction at the site of phosphorylation suggests that Dbf2 achieves specificity by docking its substrates at a site that is distinct from the phosphorylation site.

Global analysis of protein function using protein microarrays.

Protein microarrays containing thousands of proteins arrayed at high density can be prepared and probed for a wide variety of activities, thereby allowing the large scale analysis of many proteins simultaneously. In addition to identifying the activities of many previously uncharacterized proteins, protein microarrays can reveal new activities of well-characterized proteins, thus providing new insights about the functions of these proteins. Below, we describe the construction and use of protein microarrays and their applications using yeast as a model system.

C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression.

To verify the genome annotation and to create a resource to functionally characterize the proteome, we attempted to Gateway-clone all predicted protein-encoding open reading frames (ORFs), or the 'ORFeome,' of Caenorhabditis elegans. We successfully cloned approximately 12,000 ORFs (ORFeome 1.1), of which roughly 4,000 correspond to genes that are untouched by any cDNA or expressed-sequence tag (EST). More than 50% of predicted genes needed corrections in their intron-exon structures. Notably, approximately 11,000 C. elegans proteins can now be expressed under many conditions and characterized using various high-throughput strategies, including large-scale interactome mapping. We suggest that similar ORFeome projects will be valuable for other organisms, including humans.