Garry Nolan

All Publications

• MetaCyto: A Tool for Automated Meta-analysis of Mass and Flow Cytometry Data CELL REPORTS Hu, Z., Jujjavarapu, C., Hughey, J. J., Andorf, S., Lee, H., Gherardini, P., Spitzer, M. H., Thomas, C. G., Campbell, J., Dunn, P., Wiser, J., Kidd, B. A., Dudley, J. T., Nolan, G. P., Bhattacharya, S., Butte, A. J. 2018; 24 (5): 1377–88

  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 commonly labeled cell subsets across studies, thus enabling meta-analysis. Applying MetaCyto across a set of ten heterogeneous cytometry studies totaling 2,926 samples enabled us to identify multiple cell populations exhibiting differences in abundance between demographic groups. Software is released to the public through Bioconductor (http://bioconductor.org/packages/release/bioc/html/MetaCyto.html).

  View details for DOI 10.1016/j.celrep.2018.07.003

  View details for Web of Science ID 000440377500025

  View details for PubMedID 30067990

• Deep Profiling of Mouse Splenic Architecture with CODEX Multiplexed Imaging. Cell Goltsev, Y., Samusik, N., Kennedy-Darling, J., Bhate, S., Hale, M., Vazquez, G., Black, S., Nolan, G. P. 2018

  Abstract

  A highly multiplexed cytometric imaging approach, termed co-detection by indexing (CODEX), is used here to create multiplexed datasets of normal and lupus (MRL/lpr) murine spleens. CODEX iteratively visualizes antibody binding events using DNA barcodes, fluorescent dNTP analogs, and an in situ polymerization-based indexing procedure. An algorithmic pipeline for single-cell antigen quantification in tightly packed tissues was developed and used to overlay well-known morphological features with de novo characterization of lymphoid tissue architecture at a single-cell and cellular neighborhood levels. We observed an unexpected, profound impact of the cellular neighborhood on the expression of protein receptors on immune cells. By comparing normal murine spleen to spleens from animals with systemicautoimmune disease (MRL/lpr), extensive and previously uncharacterized splenic cell-interaction dynamics in the healthy versus diseased state was observed. The fidelity of multiplexed spatial cytometry demonstrated here allows for quantitative systemic characterization of tissue architecture in normal and clinically aberrant samples.

  View details for DOI 10.1016/j.cell.2018.07.010

  View details for PubMedID 30078711

• Three-dimensional intact-tissue sequencing of single-cell transcriptional states SCIENCE Wang, X., Allen, W. E., Wright, M. A., Sylwestrak, E. L., Samusik, N., Vesuna, S., Evans, K., Liu, C., Ramakrishnan, C., Liu, J., Nolan, G. P., Bava, F., Deisseroth, K. 2018; 361 (6400): 380-+

  View details for DOI 10.1126/science.aat5691;eaat5691

  View details for Web of Science ID 000439923700038

• The Atacama skeleton. Genome research Nolan, G., Butte, A. 2018; 28 (5): 607–8

  View details for DOI 10.1101/gr.237834.118

  View details for PubMedID 29602903

• Single-cell developmental classification of B cell precursor acute lymphoblastic leukemia at diagnosis reveals predictors of relapse. Nature medicine Good, Z., Sarno, J., Jager, A., Samusik, N., Aghaeepour, N., Simonds, E. F., White, L., Lacayo, N. J., Fantl, W. J., Fazio, G., Gaipa, G., Biondi, A., Tibshirani, R., Bendall, S. C., Nolan, G. P., Davis, K. L. 2018; 24 (4): 474–83

  Abstract

  Insight into the cancer cell populations that are responsible for relapsed disease is needed to improve outcomes. Here we report a single-cell-based study of B cell precursor acute lymphoblastic leukemia at diagnosis that reveals hidden developmentally dependent cell signaling states that are uniquely associated with relapse. By using mass cytometry we simultaneously quantified 35 proteins involved in B cell development in 60 primary diagnostic samples. Each leukemia cell was then matched to its nearest healthy B cell population by a developmental classifier that operated at the single-cell level. Machine learning identified six features of expanded leukemic populations that were sufficient to predict patient relapse at diagnosis. These features implicated the pro-BII subpopulation of B cells with activated mTOR signaling, and the pre-BI subpopulation of B cells with activated and unresponsive pre-B cell receptor signaling, to be associated with relapse. This model, termed 'developmentally dependent predictor of relapse' (DDPR), significantly improves currently established risk stratification methods. DDPR features exist at diagnosis and persist at relapse. By leveraging a data-driven approach, we demonstrate the predictive value of single-cell 'omics' for patient stratification in a translational setting and provide a framework for its application to human cancer.

  View details for DOI 10.1038/nm.4505

  View details for PubMedID 29505032

• SRC/ABL inhibition disrupts CRLF2-driven signaling to induce cell death in B-cell acute lymphoblastic leukemia. Oncotarget Sarno, J., Savino, A. M., Buracchi, C., Palmi, C., Pinto, S., Bugarin, C., Jager, A., Bresolin, S., Barber, R. C., Silvestri, D., Israeli, S., Dyer, M. J., Cazzaniga, G., Nolan, G. P., Biondi, A., Davis, K. L., Gaipa, G. 2018; 9 (33): 22872–85

  Abstract

  Children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) overexpressing the CRLF2 gene (hiCRLF2) have poor prognosis. CRLF2 protein overexpression leads to activated JAK/STAT signaling and trials are underway using JAK inhibitors to overcome treatment failure. Pre-clinical studies indicated limited efficacy of single JAK inhibitors, thus additional pathways must be targeted in hiCRLF2 cells. To identify additional activated networks, we used single-cell mass cytometry to examine 15 BCP-ALL primary patient samples. We uncovered a coordinated signaling network downstream of CRLF2 characterized by co-activation of JAK/STAT, PI3K, and CREB pathways. This CRLF2-driven network could be more effectively disrupted by SRC/ABL inhibition than single-agent JAK or PI3K inhibition, and this could be demonstrated even in primary minimal residual disease (MRD) cells. Our study suggests SCR/ABL inhibition as effective in disrupting the cooperative functional networks present in hiCRLF2 BCP-ALL patients, supporting further investigation of this strategy in pre-clinical studies.

  View details for DOI 10.18632/oncotarget.25089

  View details for PubMedID 29796158

• Whole-genome sequencing of Atacama skeleton shows novel mutations linked with dysplasia GENOME RESEARCH Bhattacharya, S., Li, J., Sockell, A., Kan, M. J., Bava, F. A., Chen, S., Avila-Arcos, M. C., Ji, X., Smith, E., Asadi, N. B., Lachman, R. S., Lam, H. K., Bustamante, C. D., Butte, A. J., Nolan, G. P. 2018; 28 (4): 423–31

  Abstract

  Over a decade ago, the Atacama humanoid skeleton (Ata) was discovered in the Atacama region of Chile. The Ata specimen carried a strange phenotype-6-in stature, fewer than expected ribs, elongated cranium, and accelerated bone age-leading to speculation that this was a preserved nonhuman primate, human fetus harboring genetic mutations, or even an extraterrestrial. We previously reported that it was human by DNA analysis with an estimated bone age of about 6-8 yr at the time of demise. To determine the possible genetic drivers of the observed morphology, DNA from the specimen was subjected to whole-genome sequencing using the Illumina HiSeq platform with an average 11.5× coverage of 101-bp, paired-end reads. In total, 3,356,569 single nucleotide variations (SNVs) were found as compared to the human reference genome, 518,365 insertions and deletions (indels), and 1047 structural variations (SVs) were detected. Here, we present the detailed whole-genome analysis showing that Ata is a female of human origin, likely of Chilean descent, and its genome harbors mutations in genes (COL1A1, COL2A1, KMT2D, FLNB, ATR, TRIP11, PCNT) previously linked with diseases of small stature, rib anomalies, cranial malformations, premature joint fusion, and osteochondrodysplasia (also known as skeletal dysplasia). Together, these findings provide a molecular characterization of Ata's peculiar phenotype, which likely results from multiple known and novel putative gene mutations affecting bone development and ossification.

  View details for DOI 10.1101/gr.223693.117

  View details for Web of Science ID 000428993500001

  View details for PubMedID 29567674

  View details for PubMedCentralID PMC5880234

• Single-cell mass cytometry reveals distinct populations of brain myeloid cells in mouse neuroinflammation and neurodegeneration models. Nature neuroscience Ajami, B., Samusik, N., Wieghofer, P., Ho, P. P., Crotti, A., Bjornson, Z., Prinz, M., Fantl, W. J., Nolan, G. P., Steinman, L. 2018; 21 (4): 541–51

  Abstract

  Neuroinflammation and neurodegeneration may represent two poles of brain pathology. Brain myeloid cells, particularly microglia, play key roles in these conditions. We employed single-cell mass cytometry (CyTOF) to compare myeloid cell populations in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, the R6/2 model of Huntington's disease (HD) and the mutant superoxide dismutase 1 (mSOD1) model of amyotrophic lateral sclerosis (ALS). We identified three myeloid cell populations exclusive to the CNS and present in each disease model. Blood-derived monocytes comprised five populations and migrated to the brain in EAE, but not in HD and ALS models. Single-cell analysis resolved differences in signaling and cytokine production within similar myeloid populations in EAE compared to HD and ALS models. Moreover, these analyses highlighted alpha5 integrin on myeloid cells as a potential therapeutic target for neuroinflammation. Together, these findings illustrate how neuropathology may differ between inflammatory and degenerative brain disease.

  View details for DOI 10.1038/s41593-018-0100-x

  View details for PubMedID 29507414

• Publisher Correction: High-resolution myogenic lineage mapping by single-cell mass cytometry. Nature cell biology Porpiglia, E., Samusik, N., Van Ho, A. T., Cosgrove, B. D., Mai, T., Davis, K. L., Jager, A., Nolan, G. P., Bendall, S. C., Fantl, W. J., Blau, H. M. 2018

  Abstract

  In the version of this Article originally published, the name of author Andrew Tri Van Ho was coded wrongly, resulting in it being incorrect when exported to citation databases. This has been corrected, though no visible changes will be apparent.

  View details for DOI 10.1038/s41556-018-0043-1

  View details for PubMedID 29507406

• 50-dimensional microenvironment analysis of human and mouse bone marrow during malignant transformation Schuerch, C. M., Barlow, G. L., Bhate, S. S., Samusik, N., Nolan, G., Goltsev, Y. NATURE PUBLISHING GROUP. 2018: 550

  View details for Web of Science ID 000429308603413

• Commonly Occurring Cell Subsets in High-Grade Serous Ovarian Tumors Identified by Single-Cell Mass Cytometry CELL REPORTS Gonzalez, V. D., Samusik, N., Chen, T. J., Savig, E. S., Aghaeepour, N., Quigley, D. A., Huang, Y., Giangarra, V., Borowsky, A. D., Hubbard, N. E., Chen, S., Han, G., Ashworth, A., Kipps, T. J., Berek, J. S., Nolan, G. P., Fantl, W. J. 2018; 22 (7): 1875–88

  Abstract

  We have performed an in-depth single-cell phenotypic characterization of high-grade serous ovarian cancer (HGSOC) by multiparametric mass cytometry (CyTOF). Using a CyTOF antibody panel to interrogate features of HGSOC biology, combined with unsupervised computational analysis, we identified noteworthy cell types co-occurring across the tumors. In addition to a dominant cell subset, each tumor harbored rarer cell phenotypes. One such group co-expressed E-cadherin and vimentin (EV), suggesting their potential role in epithelial mesenchymal transition, which was substantiated by pairwise correlation analyses. Furthermore, tumors from patients with poorer outcome had an increased frequency of another rare cell type that co-expressed vimentin, HE4, and cMyc. These poorer-outcome tumors also populated more cell phenotypes, as quantified by Simpson's diversity index. Thus, despite the recognized genomic complexity of the disease, the specific cell phenotypes uncovered here offer a focus for therapeutic intervention and disease monitoring.

  View details for DOI 10.1016/j.celrep.2018.01.053

  View details for Web of Science ID 000424981600021

  View details for PubMedID 29444438

• DRUG-NEM: Optimizing drug combinations using single-cell perturbation response to account for intratumoral heterogeneity. Proceedings of the National Academy of Sciences of the United States of America Anchang, B., Davis, K. L., Fienberg, H. G., Williamson, B. D., Bendall, S. C., Karacosta, L. G., Tibshirani, R., Nolan, G. P., Plevritis, S. K. 2018; 115 (18): E4294–E4303

  Abstract

  An individual malignant tumor is composed of a heterogeneous collection of single cells with distinct molecular and phenotypic features, a phenomenon termed intratumoral heterogeneity. Intratumoral heterogeneity poses challenges for cancer treatment, motivating the need for combination therapies. Single-cell technologies are now available to guide effective drug combinations by accounting for intratumoral heterogeneity through the analysis of the signaling perturbations of an individual tumor sample screened by a drug panel. In particular, Mass Cytometry Time-of-Flight (CyTOF) is a high-throughput single-cell technology that enables the simultaneous measurements of multiple ([Formula: see text]40) intracellular and surface markers at the level of single cells for hundreds of thousands of cells in a sample. We developed a computational framework, entitled Drug Nested Effects Models (DRUG-NEM), to analyze CyTOF single-drug perturbation data for the purpose of individualizing drug combinations. DRUG-NEM optimizes drug combinations by choosing the minimum number of drugs that produce the maximal desired intracellular effects based on nested effects modeling. We demonstrate the performance of DRUG-NEM using single-cell drug perturbation data from tumor cell lines and primary leukemia samples.

  View details for DOI 10.1073/pnas.1711365115

  View details for PubMedID 29654148

• GateFinder: Projection-based Gating Strategy Optimization for Flow and Mass Cytometry. Bioinformatics (Oxford, England) Aghaeepour, N., Simonds, E. F., Knapp, D. J., Bruggner, R., Sachs, K., Culos, A., Gherardini, P. F., Samusik, N., Fragiadakis, G., Bendall, S., Gaudilliere, B., Angst, M. S., Eaves, C. J., Weiss, W. A., Fantl, W., Nolan, G. 2018

  Abstract

  High-parameter single-cell technologies can reveal novel cell populations of interest, but studying or validating these populations using lower-parameter methods remains challenging.Here we present GateFinder, an algorithm that enriches high-dimensional cell types with simple, stepwise polygon gates requiring only two markers at a time. A series of case studies of complex cell types illustrates how simplified enrichment strategies can enable more efficient assays, reveal novel biomarkers, and clarify underlying biology.The GateFinder algorithm is implemented as a free and open-source package for BioConductor: https://nalab.stanford.edu/gatefinder.gnolan@stanford.edu or naghaeep@stanford.edu.Supplementary data are available at Bioinformatics online.

  View details for DOI 10.1093/bioinformatics/bty430

  View details for PubMedID 29850785

• The Human Cell Atlas ELIFE Regev, A., Teichmann, S. A., Lander, E. S., Amt, I., Benoist, C., Birney, E., Bodenmiller, B., Campbell, P., Carninci, P., Clatworthy, M., Clevers, H., Deplancke, B., Dunham, I., Eberwine, J., Elis, R., Enard, W., Farmer, A., Fugger, L., Gottgens, B., Hacohen, N., Haniffa, M., Hemberg, M., Kim, S., Klenerman, P., Kriegstein, A., Lein, E. D., Linnarsson, S., Lundberg, E., Lundeberg, J., Majumder, P., Marioni, J. C., Merad, M., Mhlanga, M., Nawijin, M., Netea, M., Nolan, G., Pe'er, D., Phillipakis, A., Ponting, C. P., Quake, S., Reik, W., Rozenblatt-Rosen, O., Sanes, J., Satija, R., Schumacher, T. N., Shalek, A., Shapiro, E., Sharma, P., Shin, J. W., Stegle, O., Stratton, M., Stubbington, M. T., Theis, F. J., Uhlen, M., Van Oudenaarden, A., Wagner, A., Watt, F., Weissman, J., Wold, B., Xavier, R., Yosef, N., HUMAN CELL ATLAS MEETING 2017; 6

  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.

  View details for DOI 10.7554/eLife.27041

  View details for Web of Science ID 000419858100001

  View details for PubMedID 29206104

  View details for PubMedCentralID PMC5762154

• Atomic mass tag of bismuth-209 for increasing the immunoassay multiplexing capacity of mass cytometry CYTOMETRY PART A Han, G., Chen, S., Gonzalez, V. D., Zunder, E. R., Fantl, W. J., Nolan, G. P. 2017; 91A (12): 1150–63

  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.

  View details for DOI 10.1002/cyto.a.23283

  View details for Web of Science ID 000418422200004

  View details for PubMedID 29205767

  View details for PubMedCentralID PMC5802970

• Jak1 Integrates Cytokine Sensing to Regulate Hematopoietic Stem Cell Function and Stress Hematopoiesis CELL STEM CELL Kleppe, M., Spitzer, M. H., Li, S., Hill, C. E., Dong, L., Papalexi, E., De Groote, S., Bowman, R. L., Keller, M., Koppikar, P., Rapaport, F. T., Teruya-Feldstein, J., Gandara, J., Mason, C. E., Nolan, G. P., Levine, R. L. 2017; 21 (4): 489-+

  Abstract

  JAK1 is a critical effector of pro-inflammatory cytokine signaling and plays important roles in immune function, while abnormal JAK1 activity has been linked to immunological and neoplastic diseases. Specific functions of JAK1 in the context of hematopoiesis, and specifically within hematopoietic stem cells (HSCs), have not clearly been delineated. Here, we show that conditional Jak1 loss in HSCs reduces their self-renewal and markedly alters lymphoid/myeloid differentiation in vivo. Jak1-deficient HSCs exhibit decreased competitiveness in vivo and are unable to rescue hematopoiesis in the setting of myelosuppression. They exhibit increased quiescence, an inability to enter the cell cycle in response to hematopoietic stress, and a marked reduction in cytokine sensing, including in response to type I interferons and IL-3. Moreover, Jak1 loss is not fully rescued by expression of a constitutively active Jak2 allele. Together, these data highlight an essential role for Jak1 in HSC homeostasis and stress responses.

  View details for DOI 10.1016/j.stem.2017.08.011

  View details for Web of Science ID 000412344700012

  View details for PubMedID 28965767

  View details for PubMedCentralID PMC5847260

• NKG2D ligand expression in Crohn's disease and NKG2D-dependent stimulation of CD8(+) T cell migration EXPERIMENTAL AND MOLECULAR PATHOLOGY Vadstrup, K., Galsgaard, E., Jensen, H., Lanier, L. L., Ryan, J. C., Chen, S., Nolan, G. P., Vester-Andersen, M., Pedersen, J., Gerwien, J., Jensen, T., Bendtsen, F. 2017; 103 (1): 56–70

  Abstract

  Interaction between the activating NKG2D receptor on lymphocytes and its ligands MICA, MICB, and ULBP1-6 modulate T and NK cell activity and may contribute to the pathogenesis of Crohn's disease (CD). NKG2D ligands are generally not expressed on the cell surface of normal, non-stressed cells, but expression of MICA and MICB in CD intestine has been reported. In this exploratory study, we further characterize the expression of NKG2D and its ligands, including the less well-described ULBP4-6, in CD, and test if NKG2D ligand interactions are involved in the migration of activated T cells into the affected mucosal compartments. Intestinal tissue from CD patients and healthy controls were analyzed by flow cytometry, mass cytometry, and immunohistochemistry for expression of NKG2D and ligands, and for cytokine release. Furthermore, NKG2D-dependent chemotaxis of activated CD8+ T cells across a monolayer of ligand-expressing human intestinal endothelial cells was examined. Activated lymphocytes down-regulated NKG2D expression upon accumulation in inflamed CD intestine. NKG2D expression on CD56+ T and γδ T cells from inflamed tissue seemed inversely correlated with CRP levels and cytokine release. B cells, monocytes, mucosal epithelium, and vascular endothelium expressed NKG2D ligands in inflamed CD intestine. The expression of NKG2D ligands was correlated with cytokine release, but was highly variable between patients. Stimulation of vascular intestinal endothelial cells in vitro induced expression of NKG2D ligands, including MICA/B and ULBP2/6. Blockade of NKG2D on CD8+ T cells inhibited the migration over ligand-expressing endothelial cells. Intestinal induction of NKG2D ligands and ligand-induced down-regulation of NKG2D in CD suggest that the NKG2D-ligand interaction may be involved in both the activation and recruitment of NKG2D+ lymphocytes into the inflamed CD intestine.

  View details for DOI 10.1016/j.yexmp.2017.06.010

  View details for Web of Science ID 000408182800009

  View details for PubMedID 28684217

  View details for PubMedCentralID PMC5560431

• In silico modeling identifies CD45 as a regulator of IL-2 synergy in the NKG2D-mediated activation of immature human NK cells SCIENCE SIGNALING Mukherjee, S., Jensen, H., Stewart, W., Stewart, D., Ray, W. C., Chen, S., Nolan, G. P., Lanier, L. L., Das, J. 2017; 10 (485)

  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.

  View details for DOI 10.1126/scisignal.aai9062

  View details for Web of Science ID 000404110100001

  View details for PubMedID 28655861

  View details for PubMedCentralID PMC5556952

• Unifying mechanism for different fibrotic diseases PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wernig, G., Chen, S., Cui, L., Van Neste, C., Tsai, J. M., Kambham, N., Vogel, H., Natkunam, Y., Gilliland, D. G., Nolan, G., Weissman, I. L. 2017; 114 (18): 4757-4762

  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.

  View details for DOI 10.1073/pnas.1621375114

  View details for Web of Science ID 000400358000057

  View details for PubMedID 28424250

  View details for PubMedCentralID PMC5422830

• High-resolution myogenic lineage mapping by single-cell mass cytometry NATURE CELL BIOLOGY Porpiglia, E., Samusik, N., Van Ho, A. T., Cosgrove, B. D., Mai, T., Davis, K. L., Jager, A., Nolan, G. P., Bendall, S. C., Fantl, W. J., Blau, H. M. 2017; 19 (5): 558-?

  Abstract

  Muscle regeneration is a dynamic process during which cell state and identity change over time. A major roadblock has been a lack of tools to resolve a myogenic progression in vivo. Here we capitalize on a transformative technology, single-cell mass cytometry (CyTOF), to identify in vivo skeletal muscle stem cell and previously unrecognized progenitor populations that precede differentiation. We discovered two cell surface markers, CD9 and CD104, whose combined expression enabled in vivo identification and prospective isolation of stem and progenitor cells. Data analysis using the X-shift algorithm paired with single-cell force-directed layout visualization defined a molecular signature of the activated stem cell state (CD44(+)/CD98(+)/MyoD(+)) and delineated a myogenic trajectory during recovery from acute muscle injury. Our studies uncover the dynamics of skeletal muscle regeneration in vivo and pave the way for the elucidation of the regulatory networks that underlie cell-state transitions in muscle diseases and ageing.

  View details for DOI 10.1038/ncb3507

  View details for Web of Science ID 000400376100019

  View details for PubMedID 28414312

• Mass cytometry identifies a distinct monocyte cytokine signature shared by clinically heterogeneous pediatric SLE patients. Journal of autoimmunity O'Gorman, W. E., Kong, D. S., Balboni, I. M., Rudra, P., Bolen, C. R., Ghosh, D., DAVIS, M. M., Nolan, G. P., Hsieh, E. W. 2017

  Abstract

  Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease with heightened disease severity in children. The incomplete understanding of the precise cellular and molecular events that drive disease activity pose a significant hurdle to the development of targeted therapeutic agents. Here, we performed single-cell phenotypic and functional characterization of pediatric SLE patients and healthy controls blood via mass cytometry. We identified a distinct CD14(hi) monocyte cytokine signature, with increased levels of monocyte chemoattractant protein-1 (MCP1), macrophage inflammatory protein-1β (Mip1β), and interleukin-1 receptor antagonist (IL-1RA). This signature was shared by every clinically heterogeneous patient, and reproduced in healthy donors' blood upon ex-vivo exposure to plasma from clinically active patients only. This SLE-plasma induced signature was abrogated by JAK1/JAK2 selective inhibition. This study demonstrates the utility of mass cytometry to evaluate immune dysregulation in pediatric autoimmunity, by identification of a multi-parametric immune signature that can be further dissected to delineate the events that drive disease pathogenesis.

  View details for DOI 10.1016/j.jaut.2017.03.010

  View details for PubMedID 28389038

• Microsphere cytometry to interrogate microenvironment-dependent cell signaling. Integrative biology Ertsås, H. C., Nolan, G. P., LaBarge, M. A., Lorens, J. B. 2017; 9 (2): 123-134

  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.

  View details for DOI 10.1039/c6ib00207b

  View details for PubMedID 28102399

• EBI3 regulates the NK cell response to mouse cytomegalovirus infection. Proceedings of the National Academy of Sciences of the United States of America Jensen, H., Chen, S., Folkersen, L., Nolan, G. P., Lanier, L. L. 2017; 114 (7): 1625-1630

  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.

  View details for DOI 10.1073/pnas.1700231114

  View details for PubMedID 28143936

• Mass cytometry analysis reveals hyperactive NF Kappa B signaling in myelofibrosis and secondary acute myeloid leukemia. Leukemia Fisher, D. A., Malkova, O., Engle, E. K., Miner, C. A., Fulbright, M. C., Behbehani, G. K., COLLINS, T. B., Bandyopadhyay, S., Zhou, A., Nolan, G. P., Oh, S. T. 2017

  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.Leukemia advance online publication, 3 February 2017; doi:10.1038/leu.2016.377.

  View details for DOI 10.1038/leu.2016.377

  View details for PubMedID 28008177

• Expression of specific inflammasome gene modules stratifies older individuals into two extreme clinical and immunological states NATURE MEDICINE Furman, D., Chang, J., Lartigue, L., Bolen, C. R., Haddad, F., Gaudilliere, B., Ganio, E. A., Fragiadakis, G. K., Spitzer, M. H., Douchet, I., Daburon, S., Moreau, J., Nolan, G. P., Blanco, P., Dechanet-Merville, J., Dekker, C. L., Jojic, V., Kuo, C. J., Davis, M. M., Faustin, B. 2017; 23 (2): 174-184

  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 N(4)-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.

  View details for DOI 10.1038/nm.4267

  View details for Web of Science ID 000393729000009

  View details for PubMedID 28092664

• High-throughput precision measurement of subcellular localization in single cells. Cytometry. Part A : the journal of the International Society for Analytical Cytology Burns, T. J., Frei, A. P., Gherardini, P. F., Bava, F. A., Batchelder, J. E., Yoshiyasu, Y., Yu, J. M., Groziak, A. R., Kimmey, S. C., Gonzalez, V. D., Fantl, W. J., Nolan, G. P. 2017; 91 (2): 180-189

  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.

  View details for DOI 10.1002/cyto.a.23054

  View details for PubMedID 28094900

• Systemic Immunity Is Required for Effective Cancer Immunotherapy. Cell Spitzer, M. H., Carmi, Y., Reticker-Flynn, N. E., Kwek, S. S., Madhireddy, D., Martins, M. M., Gherardini, P. F., Prestwood, T. R., Chabon, J., Bendall, S. C., Fong, L., Nolan, G. P., Engleman, E. G. 2017; 168 (3): 487-502 e15

  Abstract

  Immune responses involve coordination across cell types and tissues. However, studies in cancer immunotherapy have focused heavily on local immune responses in the tumor microenvironment. To investigate immune activity more broadly, we performed an organism-wide study in genetically engineered cancer models using mass cytometry. We analyzed immune responses in several tissues after immunotherapy by developing intuitive models for visualizing single-cell data with statistical inference. Immune activation was evident in the tumor and systemically shortly after effective therapy was administered. However, during tumor rejection, only peripheral immune cells sustained their proliferation. This systemic response was coordinated across tissues and required for tumor eradication in several immunotherapy models. An emergent population of peripheral CD4 T cells conferred protection against new tumors and was significantly expanded in patients responding to immunotherapy. These studies demonstrate the critical impact of systemic immune responses that drive tumor rejection.

  View details for DOI 10.1016/j.cell.2016.12.022

  View details for PubMedID 28111070

  View details for PubMedCentralID PMC5312823

• Distinct signaling programs control human hematopoietic stem cell survival and proliferation. Blood Knapp, D. J., Hammond, C. A., Aghaeepour, N., Miller, P. H., Pellacani, D., Beer, P. A., Sachs, K., Qiao, W., Wang, W., Humphries, R. K., Sauvageau, G., Zandstra, P. W., Bendall, S. C., Nolan, G. P., Hansen, C., Eaves, C. J. 2017; 129 (3): 307-318

  Abstract

  Several growth factors (GFs) that together promote quiescent human hematopoietic stem cell (HSC) expansion ex vivo have been identified; however, the molecular mechanisms by which these GFs regulate the survival, proliferation. and differentiation of human HSCs remain poorly understood. We now describe experiments in which we used mass cytometry to simultaneously measure multiple surface markers, transcription factors, active signaling intermediates, viability, and cell-cycle indicators in single CD34(+) cord blood cells before and up to 2 hours after their stimulation with stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor (5 GFs) either alone or combined. Cells with a CD34(+)CD38(-)CD45RA(-)CD90(+)CD49f(+) (CD49f(+)) phenotype (∼10% HSCs with >6-month repopulating activity in immunodeficient mice) displayed rapid increases in activated STAT1/3/5, extracellular signal-regulated kinase 1/2, AKT, CREB, and S6 by 1 or more of these GFs, and β-catenin only when the 5 GFs were combined. Certain minority subsets within the CD49f(+) compartment were poorly GF-responsive and, among the more GF-responsive subsets of CD49f(+) cells, different signaling intermediates correlated with the levels of the myeloid- and lymphoid-associated transcription factors measured. Phenotypically similar, but CD90(-)CD49f(-) cells (MPPs) contained lower baseline levels of multiple signaling intermediates than the CD90(+)CD49f(+) cells, but showed similar response amplitudes to the same GFs. Importantly, we found activation or inhibition of AKT and β-catenin directly altered immediate CD49f(+) cell survival and proliferation. These findings identify rapid signaling events that 5 GFs elicit directly in the most primitive human hematopoietic cell types to promote their survival and proliferation.

  View details for DOI 10.1182/blood-2016-09-740654

  View details for PubMedID 27827829

  View details for PubMedCentralID PMC5271174

• High-throughput precision measurement of subcellular localization in single cells CYTOMETRY PART A Burns, T. J., Frei, A. P., Gherardini, P. F., Bava, F. A., Batchelder, J. E., Yoshiyasu, Y., Yu, J. M., Groziak, A. R., Kimmey, S. C., Gonzalez, V. D., Fantl, W. J., Nolan, G. P. 2017; 91A (2): 180-189

  View details for DOI 10.1002/cyto.a.23054

  View details for Web of Science ID 000395651800011

• Scalable multi-sample single-cell data analysis by Partition-Assisted Clustering and Multiple Alignments of Networks. PLoS computational biology Li, Y. H., Li, D., Samusik, N., Wang, X., Guan, L., Nolan, G. P., Wong, W. H. 2017; 13 (12): e1005875

  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.

  View details for DOI 10.1371/journal.pcbi.1005875

  View details for PubMedID 29281633

• High-Dimensional Phenotypic Mapping of Human Dendritic Cells Reveals Interindividual Variation and Tissue Specialization. Immunity Alcántara-Hernández, M., Leylek, R., Wagar, L. E., Engleman, E. G., Keler, T., Marinkovich, M. P., Davis, M. M., Nolan, G. P., Idoyaga, J. 2017

  Abstract

  Given the limited efficacy of clinical approaches that rely on ex vivo generated dendritic cells (DCs), it is imperative to design strategies that harness specialized DC subsets in situ. This requires delineating the expression of surface markers by DC subsets among individuals and tissues. Here, we performed a multiparametric phenotypic characterization and unbiased analysis of human DC subsets in blood, tonsil, spleen, and skin. We uncovered previously unreported phenotypic heterogeneity of human cDC2s among individuals, including variable expression of functional receptors such as CD172a. We found marked differences in DC subsets localized in blood and lymphoid tissues versus skin, and a striking absence of the newly discovered Axl+ DCs in the skin. Finally, we evaluated the capacity of anti-receptor monoclonal antibodies to deliver vaccine components to skin DC subsets. These results offer a promising path for developing DC subset-specific immunotherapies that cannot be provided by transcriptomic analysis alone.

  View details for DOI 10.1016/j.immuni.2017.11.001

  View details for PubMedID 29221729

• A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites. Nature Dodd, D., Spitzer, M. H., Van Treuren, W., Merrill, B. D., Hryckowian, A. J., Higginbottom, S. K., Le, A., Cowan, T. M., Nolan, G. P., Fischbach, M. A., Sonnenburg, J. L. 2017; 551 (7682): 648–52

  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.

  View details for DOI 10.1038/nature24661

  View details for PubMedID 29168502

• Upregulation of HERV-K is Linked to Immunity and Inflammation in Pulmonary Arterial Hypertension. Circulation Saito, T., Miyagawa, K., Chen, S. Y., Tamosiuniene, R., Wang, L., Sharp, O., Samayoa, E., Harada, D., Moonen, J. A., Cao, A., Chen, P. I., Hennigs, J. K., Gu, M., Li, C. G., Leib, R. D., Li, D., Adams, C. M., Del Rosario, P. A., Bill, M. A., Haddad, F., Montoya, J. G., Robinson, W., Fantl, W. J., Nolan, G. P., Zamanian, R. T., Nicolls, M. R., Chiu, C. Y., Ariza, M. E., Rabinovitch, M. 2017

  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. Circulating autoantibodies, lung perivascular lymphoid tissue and elevated cytokines have been related to PAH pathogenesis but without clear understanding of how these abnormalities are initiated, perpetuated and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies. Methods -Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry (LCMS), confirmed by ELISA, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next generation sequencing, functional studies in cultured monocytes and endothelial cells (EC) and hemodynamic and lung studies in a rat. Results -SAM domain and HD1 domain-containing protein (SAMHD1), an innate immune factor that suppresses HIV replication was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH vs. 12 control lungs. Elevated SAMHD1 was localized to endothelial cells (EC), perivascular dendritic cells and macrophages and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH vs. control lungs (n=4 each). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase (dUTPase) mRNAs were elevated in PAH vs. control lungs (n=10) and proteins were localized to macrophages. HERV-K dUTPase induced SAMHD1 and pro-inflammatory cytokines (e.g., IL6, IL1β and TNFα) in circulating monocytes and pulmonary arterial (PA) EC, and activated B cells. Vulnerability of PAEC to apoptosis was increased by HERV-K dUTPase in an IL6 independent manner. Furthermore, three weekly injections of HERV-K dUTPase induced hemodynamic and vascular changes of pulmonary hypertension in rats (n=8), and elevated IL6. Conclusions -Our study reveals that upregulation of the endogenous retrovirus HERV-K could both initiate and sustain activation of the immune system and cause vascular changes associated with PAH.

  View details for DOI 10.1161/CIRCULATIONAHA.117.027589

  View details for PubMedID 28935667

• An immune clock of human pregnancy. Science immunology Aghaeepour, N., Ganio, E. A., Mcilwain, D., Tsai, A. S., Tingle, M., Van Gassen, S., Gaudilliere, D. K., Baca, Q., McNeil, L., Okada, R., Ghaemi, M. S., Furman, D., Wong, R. J., Winn, V. D., Druzin, M. L., El-Sayed, Y. Y., Quaintance, C., Gibbs, R., Darmstadt, G. L., Shaw, G. M., Stevenson, D. K., Tibshirani, R., Nolan, G. P., Lewis, D. B., Angst, M. S., Gaudilliere, B. 2017; 2 (15)

  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.

  View details for DOI 10.1126/sciimmunol.aan2946

  View details for PubMedID 28864494

• Mapping the Fetomaternal Peripheral Immune System at Term Pregnancy. Journal of immunology Fragiadakis, G. K., Baca, Q. J., Gherardini, P. F., Ganio, E. A., Gaudilliere, D. K., Tingle, M., Lancero, H. L., McNeil, L. S., Spitzer, M. H., Wong, R. J., Shaw, G. M., Darmstadt, G. L., Sylvester, K. G., Winn, V. D., Carvalho, B., Lewis, D. B., Stevenson, D. K., Nolan, G. P., Aghaeepour, N., Angst, M. S., Gaudilliere, B. L. 2016

  Abstract

  Preterm labor and infections are the leading causes of neonatal deaths worldwide. During pregnancy, immunological cross talk between the mother and her fetus is critical for the maintenance of pregnancy and the delivery of an immunocompetent neonate. A precise understanding of healthy fetomaternal immunity is the important first step to identifying dysregulated immune mechanisms driving adverse maternal or neonatal outcomes. This study combined single-cell mass cytometry of paired peripheral and umbilical cord blood samples from mothers and their neonates with a graphical approach developed for the visualization of high-dimensional data to provide a high-resolution reference map of the cellular composition and functional organization of the healthy fetal and maternal immune systems at birth. The approach enabled mapping of known phenotypical and functional characteristics of fetal immunity (including the functional hyperresponsiveness of CD4(+) and CD8(+) T cells and the global blunting of innate immune responses). It also allowed discovery of new properties that distinguish the fetal and maternal immune systems. For example, examination of paired samples revealed differences in endogenous signaling tone that are unique to a mother and her offspring, including increased ERK1/2, MAPK-activated protein kinase 2, rpS6, and CREB phosphorylation in fetal Tbet(+)CD4(+) T cells, CD8(+) T cells, B cells, and CD56(lo)CD16(+) NK cells and decreased ERK1/2, MAPK-activated protein kinase 2, and STAT1 phosphorylation in fetal intermediate and nonclassical monocytes. This highly interactive functional map of healthy fetomaternal immunity builds the core reference for a growing data repository that will allow inferring deviations from normal associated with adverse maternal and neonatal outcomes.

  View details for PubMedID 27793998

  View details for PubMedCentralID PMC5125527

• Mutant IDH1 Downregulates ATM and Alters DNA Repair and Sensitivity to DNA Damage Independent of TET2. Cancer cell Inoue, S., Li, W. Y., Tseng, A., Beerman, I., Elia, A. J., Bendall, S. C., Lemonnier, F., Kron, K. J., Cescon, D. W., Hao, Z., Lind, E. F., Takayama, N., Planello, A. C., Shen, S. Y., Shih, A. H., Larsen, D. M., Li, Q., Snow, B. E., Wakeham, A., Haight, J., Gorrini, C., Bassi, C., Thu, K. L., Murakami, K., Elford, A. R., Ueda, T., Straley, K., Yen, K. E., Melino, G., Cimmino, L., Aifantis, I., Levine, R. L., De Carvalho, D. D., Lupien, M., Rossi, D. J., Nolan, G. P., Cairns, R. A., Mak, T. W. 2016; 30 (2): 337-348

  Abstract

  Mutations in the isocitrate dehydrogenase-1 gene (IDH1) are common drivers of acute myeloid leukemia (AML) but their mechanism is not fully understood. It is thought that IDH1 mutants act by inhibiting TET2 to alter DNA methylation, but there are significant unexplained clinical differences between IDH1- and TET2-mutant diseases. We have discovered that mice expressing endogenous mutant IDH1 have reduced numbers of hematopoietic stem cells (HSCs), in contrast to Tet2 knockout (TET2-KO) mice. Mutant IDH1 downregulates the DNA damage (DD) sensor ATM by altering histone methylation, leading to impaired DNA repair, increased sensitivity to DD, and reduced HSC self-renewal, independent of TET2. ATM expression is also decreased in human IDH1-mutated AML. These findings may have implications for treatment of IDH-mutant leukemia.

  View details for DOI 10.1016/j.ccell.2016.05.018

  View details for PubMedID 27424808

  View details for PubMedCentralID PMC5022794

• Visualization and cellular hierarchy inference of single-cell data using SPADE. Nature protocols Anchang, B., Hart, T. D., Bendall, S. C., Qiu, P., Bjornson, Z., Linderman, M., Nolan, G. P., Plevritis, S. K. 2016; 11 (7): 1264-1279

  Abstract

  High-throughput single-cell technologies provide an unprecedented view into cellular heterogeneity, yet they pose new challenges in data analysis and interpretation. In this protocol, we describe the use of Spanning-tree Progression Analysis of Density-normalized Events (SPADE), a density-based algorithm for visualizing single-cell data and enabling cellular hierarchy inference among subpopulations of similar cells. It was initially developed for flow and mass cytometry single-cell data. We describe SPADE's implementation and application using an open-source R package that runs on Mac OS X, Linux and Windows systems. A typical SPADE analysis on a 2.27-GHz processor laptop takes ∼5 min. We demonstrate the applicability of SPADE to single-cell RNA-seq data. We compare SPADE with recently developed single-cell visualization approaches based on the t-distribution stochastic neighborhood embedding (t-SNE) algorithm. We contrast the implementation and outputs of these methods for normal and malignant hematopoietic cells analyzed by mass cytometry and provide recommendations for appropriate use. Finally, we provide an integrative strategy that combines the strengths of t-SNE and SPADE to infer cellular hierarchy from high-dimensional single-cell data.

  View details for DOI 10.1038/nprot.2016.066

  View details for PubMedID 27310265

• Small molecule inhibition of cAMP response element binding protein in human acute myeloid leukemia cells. Leukemia Mitton, B., Chae, H., Hsu, K., Dutta, R., Aldana-Masangkay, G., FERRARI, R., Davis, K., Tiu, B. C., Kaul, A., Lacayo, N., Dahl, G., Xie, F., Li, B. X., Breese, M. R., Landaw, E. M., Nolan, G., Pellegrini, M., Romanov, S., Xiao, X., Sakamoto, K. M. 2016

  Abstract

  The transcription factor CREB (cAMP Response-Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell-cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell-cycle and survival pathways, which may represent a novel approach for AML therapy.

  View details for DOI 10.1038/leu.2016.139

  View details for PubMedID 27211267

  View details for PubMedCentralID PMC5143163

• In Reply. Anesthesiology Angst, M. S., Fragiadakis, G. K., Gaudillière, B., Aghaeepour, N., Nolan, G. P. 2016; 124 (6): 1414-1415

  View details for DOI 10.1097/ALN.0000000000001091

  View details for PubMedID 27187126

  View details for PubMedCentralID PMC4914362

• Automated mapping of phenotype space with single-cell data NATURE METHODS Samusik, N., Good, Z., Spitzer, M. H., Davis, K. L., Nolan, G. P. 2016; 13 (6): 493-?

  Abstract

  Accurate identification of cell subsets in complex populations is key to discovering novelty in multidimensional single-cell experiments. We present X-shift (http://web.stanford.edu/~samusik/vortex/), an algorithm that processes data sets using fast k-nearest-neighbor estimation of cell event density and arranges populations by marker-based classification. X-shift enables automated cell-subset clustering and access to biological insights that 'prior knowledge' might prevent the researcher from discovering.

  View details for DOI 10.1038/NMETH.3863

  View details for Web of Science ID 000377480100015

  View details for PubMedID 27183440

  View details for PubMedCentralID PMC4896314

• Mass Cytometry: Single Cells, Many Features CELL Spitzer, M. H., Nolan, G. P. 2016; 165 (4): 780-791

  Abstract

  Technology development in biological research often aims to either increase the number of cellular features that can be surveyed simultaneously or enhance the resolution at which such observations are possible. For decades, flow cytometry has balanced these goals to fill a critical need by enabling the measurement of multiple features in single cells, commonly to examine complex or hierarchical cellular systems. Recently, a format for flow cytometry has been developed that leverages the precision of mass spectrometry. This fusion of the two technologies, termed mass cytometry, provides measurement of over 40 simultaneous cellular parameters at single-cell resolution, significantly augmenting the ability of cytometry to evaluate complex cellular systems and processes. In this Primer, we review the current state of mass cytometry, providing an overview of the instrumentation, its present capabilities, and methods of data analysis, as well as thoughts on future developments and applications.

  View details for DOI 10.1016/j.cell.2016.04.019

  View details for Web of Science ID 000375800300008

  View details for PubMedID 27153492

• Highly multiplexed simultaneous detection of RNAs and proteins in single cells. Nature methods Frei, A. P., Bava, F., Zunder, E. R., Hsieh, E. W., Chen, S., Nolan, G. P., Gherardini, P. F. 2016; 13 (3): 269-275

  Abstract

  To enable the detection of expression signatures specific to individual cells, we developed PLAYR (proximity ligation assay for RNA), a method for highly multiplexed transcript quantification by flow and mass cytometry that is compatible with standard antibody staining. When used with mass cytometry, PLAYR allowed for the simultaneous quantification of more than 40 different mRNAs and proteins. In primary cells, we quantified multiple transcripts, with the identity and functional state of each analyzed cell defined on the basis of the expression of a separate set of transcripts or proteins. By expanding high-throughput deep phenotyping of cells beyond protein epitopes to include RNA expression, PLAYR opens a new avenue for the characterization of cellular metabolism.

  View details for DOI 10.1038/nmeth.3742

  View details for PubMedID 26808670

• Coordinate actions of innate immune responses oppose those of the adaptive immune system during Salmonella infection of mice SCIENCE SIGNALING Hotson, A. N., Gopinath, S., Nicolau, M., Khasanova, A., Finck, R., Monack, D., Nolan, G. P. 2016; 9 (410)

  View details for DOI 10.1126/scisignal.aaa9303

  View details for Web of Science ID 000368662000001

• A benchmark for evaluation of algorithms for identification of cellular correlates of clinical outcomes. Cytometry. Part A : the journal of the International Society for Analytical Cytology Aghaeepour, N., Chattopadhyay, P., Chikina, M., Dhaene, T., Van Gassen, S., Kursa, M., Lambrecht, B. N., Malek, M., McLachlan, G. J., Qian, Y., Qiu, P., Saeys, Y., Stanton, R., Tong, D., Vens, C., Walkowiak, S., Wang, K., Finak, G., Gottardo, R., Mosmann, T., Nolan, G. P., Scheuermann, R. H., Brinkman, R. R. 2016; 89 (1): 16-21

  Abstract

  The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of computational methods for identifying cell populations in multidimensional flow cytometry data. Here we report the results of FlowCAP-IV where algorithms from seven different research groups predicted the time to progression to AIDS among a cohort of 384 HIV+ subjects, using antigen-stimulated peripheral blood mononuclear cell (PBMC) samples analyzed with a 14-color staining panel. Two approaches (FlowReMi.1 and flowDensity-flowType-RchyOptimyx) provided statistically significant predictive value in the blinded test set. Manual validation of submitted results indicated that unbiased analysis of single cell phenotypes could reveal unexpected cell types that correlated with outcomes of interest in high dimensional flow cytometry datasets.

  View details for DOI 10.1002/cyto.a.22732

  View details for PubMedID 26447924

• A benchmark for evaluation of algorithms for identification of cellular correlates of clinical outcomes CYTOMETRY PART A Aghaeepour, N., Chattopadhyay, P., Chikina, M., Dhaene, T., Van Gassen, S., Kursa, M., Lambrecht, B. N., Malek, M., McLachlan, G. J., Qian, Y., Qiu, P., Saeys, Y., Stanton, R., Tong, D., Vens, C., Walkowiak, S., Wang, K., Finak, G., Gottardo, R., Mosmann, T., Nolan, G. P., Scheuermann, R. H., Brinkman, R. R. 2016; 89A (1): 16-21

  View details for DOI 10.1002/cyto.a.22732

  View details for Web of Science ID 000369061600004

• AN UPDATED DEBARCODING TOOL FOR MASS CYTOMETRY WITH CELL TYPE-SPECIFIC AND CELL SAMPLE-SPECIFIC STRINGENCY ADJUSTMENT. Pacific Symposium on Biocomputing. Pacific Symposium on Biocomputing Fread, K. I., Strickland, W. D., Nolan, G. P., Zunder, E. R. 2016; 22: 588-598

  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.

  View details for PubMedID 27897009

• Patient-specific Immune States before Surgery Are Strong Correlates of Surgical Recovery ANESTHESIOLOGY Fragiadakis, G. K., Gaudilliere, B., Ganio, E. A., Aghaeepour, N., Tingle, M., Nolan, G. P., Angst, M. S. 2015; 123 (6): 1241-1255

  Abstract

  Recovery after surgery is highly variable. Risk-stratifying patients based on their predicted recovery profile will afford individualized perioperative management strategies. Recently, application of mass cytometry in patients undergoing hip arthroplasty revealed strong immune correlates of surgical recovery in blood samples collected shortly after surgery. However, the ability to interrogate a patient's immune state before surgery and predict recovery is highly desirable in perioperative medicine.To evaluate a patient's presurgical immune state, cell-type-specific intracellular signaling responses to ex vivo ligands (lipopolysaccharide, interleukin [IL]-6, IL-10, and IL-2/granulocyte macrophage colony-stimulating factor) were quantified by mass cytometry in presurgical blood samples. Selected ligands modulate signaling processes perturbed by surgery. Twenty-three cell surface and 11 intracellular markers were used for the phenotypic and functional characterization of major immune cell subsets. Evoked immune responses were regressed against patient-centered outcomes, contributing to protracted recovery including functional impairment, postoperative pain, and fatigue.Evoked signaling responses varied significantly and defined patient-specific presurgical immune states. Eighteen signaling responses correlated significantly with surgical recovery parameters (|R| = 0.37 to 0.70; false discovery rate < 0.01). Signaling responses downstream of the toll-like receptor 4 in cluster of differentiation (CD) 14 monocytes were particularly strong correlates, accounting for 50% of observed variance. Immune correlates identified in presurgical blood samples mirrored correlates identified in postsurgical blood samples.Convergent findings in pre- and postsurgical analyses provide validation of reported immune correlates and suggest a critical role of the toll-like receptor 4 signaling pathway in monocytes for the clinical recovery process. The comprehensive assessment of patients' preoperative immune state is promising for predicting important recovery parameters and may lead to clinical tests using standard flow cytometry.

  View details for DOI 10.1097/ALN.0000000000000887

  View details for Web of Science ID 000365483600009

  View details for PubMedID 26655308

  View details for PubMedCentralID PMC4681408

• Deletions in the cytoplasmic domain of iRhom1 and iRhom2 promote shedding of the TNF receptor by the protease ADAM17 SCIENCE SIGNALING Maney, S. K., Mcllwain, D. R., Polz, R., Pandyra, A. A., Sundaram, B., Wolff, D., Ohishi, K., Maretzky, T., Brooke, M. A., Evers, A., Vasudevan, A. A., Aghaeepour, N., Scheller, J., Muenk, C., Haeussinger, D., Mak, T. W., Nolan, G. P., Kelsell, D. P., Blobel, C. P., Lang, K. S., Lang, P. A. 2015; 8 (401)

  View details for DOI 10.1126/scisignal.aac5356

  View details for Web of Science ID 000365866400003

• Single-cell systems-level analysis of human Toll-like receptor activation defines a chemokine signature in patients with systemic lupus erythematosus JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY O'Gorman, W. E., Hsieh, E. W., Savig, E. S., Gherardini, P. F., Hernandez, J. D., Hansmann, L., Balboni, I. M., Utz, P. J., Bendall, S. C., Fantl, W. J., Lewis, D. B., Nolan, G. P., Davis, M. M. 2015; 136 (5): 1326-1336

  Abstract

  Activation of Toll-like receptors (TLRs) induces inflammatory responses involved in immunity to pathogens and autoimmune pathogenesis, such as in patients with systemic lupus erythematosus (SLE). Although TLRs are differentially expressed across the immune system, a comprehensive analysis of how multiple immune cell subsets respond in a system-wide manner has not been described.We sought to characterize TLR activation across multiple immune cell subsets and subjects, with the goal of establishing a reference framework against which to compare pathologic processes.Peripheral whole-blood samples were stimulated with TLR ligands and analyzed by means of mass cytometry simultaneously for surface marker expression, activation states of intracellular signaling proteins, and cytokine production. We developed a novel data visualization tool to provide an integrated view of TLR signaling networks with single-cell resolution. We studied 17 healthy volunteer donors and 8 patients with newly diagnosed and untreated SLE.Our data revealed the diversity of TLR-induced responses within cell types, with TLR ligand specificity. Subsets of natural killer cells and T cells selectively induced nuclear factor κ light chain enhancer of activated B cells in response to TLR2 ligands. CD14(hi) monocytes exhibited the most polyfunctional cytokine expression patterns, with more than 80 distinct cytokine combinations. Monocytic TLR-induced cytokine patterns were shared among a group of healthy donors, with minimal intraindividual and interindividual variability. Furthermore, autoimmune disease altered baseline cytokine production; newly diagnosed untreated SLE patients shared a distinct monocytic chemokine signature, despite clinical heterogeneity.Mass cytometry defined a systems-level reference framework for human TLR activation, which can be applied to study perturbations in patients with inflammatory diseases, such as SLE.

  View details for DOI 10.1016/j.jaci.2015.04.008

  View details for Web of Science ID 000364787200023

  View details for PubMedID 26037552

• Implementing Mass Cytometry at the Bedside to Study the Immunological Basis of Human Diseases: Distinctive Immune Features in Patients with a History of Term or Preterm Birth CYTOMETRY PART A Gaudilliere, B., Ganio, E. A., Tingle, M., Lancero, H. L., Fragiadakis, G. K., Baca, Q. J., Aghaeepour, N., Wong, R. J., Quaintance, C., El-Sayed, Y. Y., Shaw, G. M., Lewis, D. B., Stevenson, D. K., Nolan, G. P., Angst, M. S. 2015; 87A (9): 817-829

  Abstract

  Single-cell technologies have immense potential to shed light on molecular and biological processes that drive human diseases. Mass cytometry (or Cytometry by Time Of Flight mass spectrometry, CyTOF) has already been employed in clinical studies to comprehensively survey patients' circulating immune system. As interest in the "bedside" application of mass cytometry is growing, the delineation of relevant methodological issues is called for. This report uses a newly generated dataset to discuss important methodological considerations when mass cytometry is implemented in a clinical study. Specifically, the use of whole blood samples versus peripheral blood mononuclear cells (PBMCs), design of mass-tagged antibody panels, technical and analytical implications of sample barcoding, and application of traditional and unsupervised approaches to analyze high-dimensional mass cytometry datasets are discussed. A mass cytometry assay was implemented in a cross-sectional study of 19 women with a history of term or preterm birth to determine whether immune traits in peripheral blood differentiate the two groups in the absence of pregnancy. Twenty-seven phenotypic and 11 intracellular markers were simultaneously analyzed in whole blood samples stimulated with lipopolysaccharide (LPS at 0, 0.1, 1, 10, and 100 ng mL(-1) ) to examine dose-dependent signaling responses within the toll-like receptor 4 (TLR4) pathway. Complementary analyses, grounded in traditional or unsupervised gating strategies of immune cell subsets, indicated that the prpS6 and pMAPKAPK2 responses in classical monocytes are accentuated in women with a history of preterm birth (FDR<1%). The results suggest that women predisposed to preterm birth may be prone to mount an exacerbated TLR4 response during the course of pregnancy. This important hypothesis-generating finding points to the power of single-cell mass cytometry to detect biologically important differences in a relatively small patient cohort. © 2015 International Society for Advancement of Cytometry.

  View details for DOI 10.1002/cyto.a.22720

  View details for Web of Science ID 000360590500009

• Implementing Mass Cytometry at the Bedside to Study the Immunological Basis of Human Diseases: Distinctive Immune Features in Patients with a History of Term or Preterm Birth. Cytometry. Part A : the journal of the International Society for Analytical Cytology Gaudillière, B., Ganio, E. A., Tingle, M., Lancero, H. L., Fragiadakis, G. K., Baca, Q. J., Aghaeepour, N., Wong, R. J., Quaintance, C., El-Sayed, Y. Y., Shaw, G. M., Lewis, D. B., Stevenson, D. K., Nolan, G. P., Angst, M. S. 2015; 87 (9): 817-829

  Abstract

  Single-cell technologies have immense potential to shed light on molecular and biological processes that drive human diseases. Mass cytometry (or Cytometry by Time Of Flight mass spectrometry, CyTOF) has already been employed in clinical studies to comprehensively survey patients' circulating immune system. As interest in the "bedside" application of mass cytometry is growing, the delineation of relevant methodological issues is called for. This report uses a newly generated dataset to discuss important methodological considerations when mass cytometry is implemented in a clinical study. Specifically, the use of whole blood samples versus peripheral blood mononuclear cells (PBMCs), design of mass-tagged antibody panels, technical and analytical implications of sample barcoding, and application of traditional and unsupervised approaches to analyze high-dimensional mass cytometry datasets are discussed. A mass cytometry assay was implemented in a cross-sectional study of 19 women with a history of term or preterm birth to determine whether immune traits in peripheral blood differentiate the two groups in the absence of pregnancy. Twenty-seven phenotypic and 11 intracellular markers were simultaneously analyzed in whole blood samples stimulated with lipopolysaccharide (LPS at 0, 0.1, 1, 10, and 100 ng mL(-1) ) to examine dose-dependent signaling responses within the toll-like receptor 4 (TLR4) pathway. Complementary analyses, grounded in traditional or unsupervised gating strategies of immune cell subsets, indicated that the prpS6 and pMAPKAPK2 responses in classical monocytes are accentuated in women with a history of preterm birth (FDR<1%). The results suggest that women predisposed to preterm birth may be prone to mount an exacerbated TLR4 response during the course of pregnancy. This important hypothesis-generating finding points to the power of single-cell mass cytometry to detect biologically important differences in a relatively small patient cohort. © 2015 International Society for Advancement of Cytometry.

  View details for DOI 10.1002/cyto.a.22720

  View details for PubMedID 26190063

• Mass Cytometric Functional Profiling of Acute Myeloid Leukemia Defines Cell-Cycle and Immunophenotypic Properties That Correlate with Known Responses to Therapy. Cancer discovery Behbehani, G. K., Samusik, N., Bjornson, Z. B., Fantl, W. J., Medeiros, B. C., Nolan, G. P. 2015; 5 (9): 988-1003

  Abstract

  Acute myeloid leukemia (AML) is characterized by a high relapse rate that has been attributed to the quiescence of leukemia stem cells (LSCs), which renders them resistant to chemotherapy. However, this hypothesis is largely supported by indirect evidence and fails to explain the large differences in relapse rates across AML subtypes. To address this, bone marrow aspirates from 41 AML patients and five healthy donors were analyzed by high-dimensional mass cytometry. All patients displayed immunophenotypic and intracellular signaling abnormalities within CD34+CD38low populations and several karyotype and genotype-specific surface marker patterns were identified. The immunophenotypic stem and early progenitor cell populations from patients with clinically favorable core-binding factor AML demonstrated a five-fold higher fraction of cells in S-phase compared to other AML samples. Conversely, LSCs in less clinically favorable FLT3-ITD AML exhibited dramatic reductions in S-phase fraction. Mass cytometry also allowed direct observation of the in vivo effects of cytotoxic chemotherapy.

  View details for DOI 10.1158/2159-8290.CD-15-0298

  View details for PubMedID 26091827

• Synthetically Modified Viral Capsids as Versatile Carriers for Use in Antibody-Based Cell Targeting. Bioconjugate chemistry ElSohly, A. M., Netirojjanakul, C., Aanei, I. L., Jager, A., Bendall, S. C., Farkas, M. E., Nolan, G. P., Francis, M. B. 2015; 26 (8): 1590-1596

  Abstract

  The present study describes an efficient and reliable method for the preparation of MS2 viral capsids that are synthetically modified with antibodies using a rapid oxidative coupling strategy. The overall protocol delivers conjugates in high yields and recoveries, requires a minimal excess of antibody to achieve modification of more than 95% of capsids, and can be completed in a short period of time. Antibody-capsid conjugates targeting extracellular receptors on human breast cancer cell lines were prepared and characterized. Notably, conjugation to the capsid did not significantly perturb the binding of the antibodies, as indicated by binding affinities similar to those obtained for the parent antibodies. An array of conjugates was synthesized with various reporters on the interior surface of the capsids to be used in cell studies, including fluorescence-based flow cytometry, confocal microscopy, and mass cytometry. The results of these studies lay the foundation for further exploration of these constructs in the context of clinically relevant applications, including drug delivery and in vivo diagnostics.

  View details for DOI 10.1021/acs.bioconjchem.5b00226

  View details for PubMedID 26076186

• mir-181a-1/b-1 Modulates Tolerance through Opposing Activities in Selection and Peripheral T Cell Function JOURNAL OF IMMUNOLOGY Schaffert, S. A., Loh, C., Wang, S., Arnold, C. P., Axtell, R. C., Newell, E. W., Nolan, G., Ansel, K. M., Davis, M. M., Steinman, L., Chen, C. 2015; 195 (4): 1470-1479

  Abstract

  Understanding the consequences of tuning TCR signaling on selection, peripheral T cell function, and tolerance in the context of native TCR repertoires may provide insight into the physiological control of tolerance. In this study, we show that genetic ablation of a natural tuner of TCR signaling, mir-181a-1/b-1, in double-positive thymocytes dampened TCR and Erk signaling and increased the threshold of positive selection. Whereas mir-181a-1/b-1 deletion in mice resulted in an increase in the intrinsic reactivity of naive T cells to self-antigens, it did not cause spontaneous autoimmunity. Loss of mir-181a-1/b-1 dampened the induction of experimental autoimmune encephalomyelitis and reduced basal TCR signaling in peripheral T cells and their migration from lymph nodes to pathogenic sites. Taken together, these results demonstrate that tolerance can be modulated by microRNA gene products through the control of opposing activities in T cell selection and peripheral T cell function.

  View details for DOI 10.4049/jimmunol.1401587

  View details for Web of Science ID 000360013200017

  View details for PubMedID 26163591

• Synthetically Modified Viral Capsids as Versatile Carriers for Use in Antibody-Based Cell Targeting BIOCONJUGATE CHEMISTRY ElSohly, A. M., Netirojjanakul, C., Aanei, I. L., Jager, A., Bendall, S. C., Farkas, M. E., Nolan, G. P., Francis, M. B. 2015; 26 (8): 1590-1596

  View details for DOI 10.1021/acs.bioconjchem.5b00226

  View details for Web of Science ID 000359962900024

• An interactive reference framework for modeling a dynamic immune system SCIENCE Spitzer, M. H., Gherardini, P. F., Fragiadakis, G. K., Bhattacharya, N., Yuan, R. T., Hotson, A. N., Finck, R., Carmi, Y., Zunder, E. R., Fantl, W. J., Bendall, S. C., Engleman, E. G., Nolan, G. P. 2015; 349 (6244): 155-?

  View details for DOI 10.1126/science.1259425

  View details for Web of Science ID 000357664300034

• IMMUNOLOGY. An interactive reference framework for modeling a dynamic immune system. Science Spitzer, M. H., Gherardini, P. F., Fragiadakis, G. K., Bhattacharya, N., Yuan, R. T., Hotson, A. N., Finck, R., Carmi, Y., Zunder, E. R., Fantl, W. J., Bendall, S. C., Engleman, E. G., Nolan, G. P. 2015; 349 (6244)

  Abstract

  Immune cells function in an interacting hierarchy that coordinates the activities of various cell types according to genetic and environmental contexts. We developed graphical approaches to construct an extensible immune reference map from mass cytometry data of cells from different organs, incorporating landmark cell populations as flags on the map to compare cells from distinct samples. The maps recapitulated canonical cellular phenotypes and revealed reproducible, tissue-specific deviations. The approach revealed influences of genetic variation and circadian rhythms on immune system structure, enabled direct comparisons of murine and human blood cell phenotypes, and even enabled archival fluorescence-based flow cytometry data to be mapped onto the reference framework. This foundational reference map provides a working definition of systemic immune organization to which new data can be integrated to reveal deviations driven by genetics, environment, or pathology.

  View details for DOI 10.1126/science.1259425

  View details for PubMedID 26160952

  View details for PubMedCentralID PMC4537647

• Reversibility of Defective Hematopoiesis Caused by Telomere Shortening in Telomerase Knockout Mice PLOS ONE Raval, A., Behbehani, G. K., Le Xuan Truong Nguyen, L. X., Thomas, D., Kusler, B., Garbuzov, A., Ramunas, J., Holbrook, C., Park, C. Y., Blau, H., Nolan, G. P., Artandi, S. E., Mitchell, B. S. 2015; 10 (7)

  View details for DOI 10.1371/journal.pone.0131722

  View details for Web of Science ID 000358154400065

• Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis CELL Levine, J. H., Simonds, E. F., Bendall, S. C., Davis, K. L., Amir, E. D., Tadmor, M. D., Litvin, O., Fienberg, H. G., Jager, A., Zunder, E. R., Finck, R., Gedman, A. L., Radtke, I., Downing, J. R., Pe'er, D., Nolan, G. P. 2015; 162 (1): 184-197

  Abstract

  Acute myeloid leukemia (AML) manifests as phenotypically and functionally diverse cells, often within the same patient. Intratumor phenotypic and functional heterogeneity have been linked primarily by physical sorting experiments, which assume that functionally distinct subpopulations can be prospectively isolated by surface phenotypes. This assumption has proven problematic, and we therefore developed a data-driven approach. Using mass cytometry, we profiled surface and intracellular signaling proteins simultaneously in millions of healthy and leukemic cells. We developed PhenoGraph, which algorithmically defines phenotypes in high-dimensional single-cell data. PhenoGraph revealed that the surface phenotypes of leukemic blasts do not necessarily reflect their intracellular state. Using hematopoietic progenitors, we defined a signaling-based measure of cellular phenotype, which led to isolation of a gene expression signature that was predictive of survival in independent cohorts. This study presents new methods for large-scale analysis of single-cell heterogeneity and demonstrates their utility, yielding insights into AML pathophysiology.

  View details for DOI 10.1016/j.cell.2015.05.047

  View details for Web of Science ID 000357542300019

  View details for PubMedID 26095251

  View details for PubMedCentralID PMC4508757

• Unipotent Megakaryopoietic Pathway Bridging Hematopoietic Stem Cells and Mature Megakaryocytes STEM CELLS Nishikii, H., Kanazawa, Y., Umemoto, T., Goltsev, Y., Matsuzaki, Y., Matsushita, K., Yamato, M., Nolan, G. P., Negrin, R., Chiba, S. 2015; 33 (7): 2196-2207

  Abstract

  Recent identification of platelet/megakaryocyte-biased hematopoietic stem/repopulating cells requires revision of the intermediate pathway for megakaryopoiesis. Here, we show a unipotent megakaryopoietic pathway bypassing the bipotent megakaryocyte/erythroid progenitors (biEMPs). Cells purified from mouse bone marrow by CD42b (GPIbα) marking were demonstrated to be unipotent megakaryocytic progenitors (MKPs) by culture and transplantation. A subpopulation of freshly isolated CD41(+) cells in the lineage Sca1(+) cKit(+) (LSK) fraction (subCD41(+) LSK) differentiated only into MKP and mature megakaryocytes in culture. Although CD41(+) LSK cells as a whole were capable of differentiating into all myeloid and lymphoid cells in vivo, they produced unipotent MKP, mature megakaryocytes, and platelets in vitro and in vivo much more efficiently than Flt3(+) CD41(-) LSK cells, especially at the early phase after transplantation. In single cell polymerase chain reaction and thrombopoietin (TPO) signaling analyses, the MKP and a fraction of CD41(+) LSK, but not the biEMP, showed the similarities in mRNA expression profile and visible TPO-mediated phosphorylation. On increased demand of platelet production after 5-FU treatment, a part of CD41(+) LSK population expressed CD42b on the surface, and 90% of them showed unipotent megakaryopoietic capacity in single cell culture and predominantly produced platelets in vivo at the early phase after transplantation. These results suggest that the CD41(+) CD42b(+) LSK are straightforward progenies of megakaryocytes/platelet-biased stem/repopulating cells, but not progenies of biEMP. Consequently, we show a unipotent/highly biased megakaryopoietic pathway interconnecting stem/repopulating cells and mature megakaryocytes, the one that may play physiologic roles especially in emergency megakaryopoiesis. Stem Cells 2015;33:2196-2207.

  View details for DOI 10.1002/stem.1985

  View details for Web of Science ID 000356668200011

  View details for PubMedID 25753067

• Early reprogramming regulators identified by prospective isolation and mass cytometry NATURE Lujan, E., Zunder, E. R., Ng, Y. H., Goronzy, I. N., Nolan, G. P., Wernig, M. 2015; 521 (7552): 352-?

  Abstract

  In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of non-productive and staggered productive intermediates arise at different reprogramming time points. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells, prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that, during reprogramming, cells progressively lose donor cell identity and gradually acquire iPS cell properties. Here we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen, we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200, that are absent in both fibroblasts and iPS cells. Single-cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic, reprogramming phase. Expression profiling reveals early upregulation of the transcriptional regulators Nr0b1 and Etv5 in this reprogramming state, preceding activation of key pluripotency regulators such as Rex1 (also known as Zfp42), Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus represent some of the earliest known regulators of iPS cell induction. Our study deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition.

  View details for DOI 10.1038/nature14274

  View details for Web of Science ID 000354816500056

  View details for PubMedID 25830878

• Mass cytometry as a platform for the discovery of cellular biomarkers to guide effective rheumatic disease therapy ARTHRITIS RESEARCH & THERAPY Nair, N., Mei, H. E., Chen, S., Hale, M., Nolan, G. P., Maecker, H. T., Genovese, M., Fathman, C. G., Whiting, C. C. 2015; 17

  Abstract

  The development of biomarkers for autoimmune diseases has been hampered by a lack of understanding of disease etiopathogenesis and of the mechanisms underlying the induction and maintenance of inflammation, which involves complex activation dynamics of diverse cell types. The heterogeneous nature and suboptimal clinical response to treatment observed in many autoimmune syndromes highlight the need to develop improved strategies to predict patient outcome to therapy and personalize patient care. Mass cytometry, using CyTOF®, is an advanced technology that facilitates multiparametric, phenotypic analysis of immune cells at single-cell resolution. In this review, we outline the capabilities of mass cytometry and illustrate the potential of this technology to enhance the discovery of cellular biomarkers for rheumatoid arthritis, a prototypical autoimmune disease.

  View details for DOI 10.1186/s13075-015-0644-z

  View details for Web of Science ID 000354850500001

  View details for PubMedID 25981462

  View details for PubMedCentralID PMC4436107

• Nomenclature of Toso, Fas Apoptosis Inhibitory Molecule 3, and IgM FcR JOURNAL OF IMMUNOLOGY Kubagawa, H., Carroll, M. C., Jacob, C. O., Lang, K. S., Lee, K., Mak, T., McAndrews, M., Morse, H. C., Nolan, G. P., Ohno, H., Richter, G. H., Seal, R., Wang, J., Wiestner, A., Coligan, J. E. 2015; 194 (9): 4055-4057

  Abstract

  Hiromi Kubagawa and John E. Coligan coordinated an online meeting to define an appropriate nomenclature for the cell surface glycoprotein presently designated by different names: Toso, Fas apoptosis inhibitory molecule 3 (FAIM3), and IgM FcR (FcμR). FAIM3 and Faim3 are the currently approved symbols for the human and mouse genes, respectively, in the National Center for Biotechnology Information, Ensembl, and other databases. However, recent functional results reported by several groups of investigators strongly support a recommendation for renaming FAIM3/Faim3 as FCMR/Fcmr, a name better reflecting its physiological function as the FcR for IgM. Participants included 12 investigators involved in studying Toso/FAIM3(Faim3)/FμR, representatives from the Human Genome Nomenclature Committee (Ruth Seal) and the Mouse Genome Nomenclature Committee (Monica McAndrews), and an observer from the IgM research field (Michael Carroll). In this article, we provide a brief background of the key research on the Toso/FAIM3(Faim3)/FcμR proteins, focusing on the ligand specificity and functional activity, followed by a brief summary of discussion about adopting a single name for this molecule and its gene and a resulting recommendation for genome nomenclature committees.

  View details for DOI 10.4049/jimmunol.1500222

  View details for Web of Science ID 000353727400001

  View details for PubMedID 25888699

• A Continuous Molecular Roadmap to iPSC Reprogramming through Progression Analysis of Single-Cell Mass Cytometry. Cell stem cell Zunder, E. R., Lujan, E., Goltsev, Y., Wernig, M., Nolan, G. P. 2015; 16 (3): 323-337

  Abstract

  To analyze cellular reprogramming at the single-cell level, mass cytometry was used to simultaneously measure markers of pluripotency, differentiation, cell-cycle status, and cellular signaling throughout the reprogramming process. Time-resolved progression analysis of the resulting data sets was used to construct a continuous molecular roadmap for three independent reprogramming systems. Although these systems varied substantially in Oct4, Sox2, Klf4, and c-Myc stoichiometry, they presented a common set of reprogramming landmarks. Early in the reprogramming process, Oct4(high)Klf4(high) cells transitioned to a CD73(high)CD104(high)CD54(low) partially reprogrammed state. Ki67(low) cells from this intermediate population reverted to a MEF-like phenotype, but Ki67(high) cells advanced through the M-E-T and then bifurcated into two distinct populations: an ESC-like Nanog(high)Sox2(high)CD54(high) population and a mesendoderm-like Nanog(low)Sox2(low)Lin28(high)CD24(high)PDGFR-α(high) population. The methods developed here for time-resolved, single-cell progression analysis may be used for the study of additional complex and dynamic systems, such as cancer progression and embryonic development.

  View details for DOI 10.1016/j.stem.2015.01.015

  View details for PubMedID 25748935

• T-cell STAT3 is required for the maintenance of humoral immunity to LCMV. European journal of immunology McIlwain, D. R., Grusdat, M., Pozdeev, V. I., Xu, H. C., Shinde, P., Reardon, C., Hao, Z., Beyer, M., Bergthaler, A., Häussinger, D., Nolan, G. P., Lang, K. S., Lang, P. A. 2015; 45 (2): 418-427

  View details for DOI 10.1002/eji.201445060

  View details for PubMedID 25393615

• Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm. Nature protocols Zunder, E. R., Finck, R., Behbehani, G. K., Amir, E. D., Krishnaswamy, S., Gonzalez, V. D., Lorang, C. G., Bjornson, Z., Spitzer, M. H., Bodenmiller, B., Fantl, W. J., Pe'er, D., Nolan, G. P. 2015; 10 (2): 316-333

  View details for DOI 10.1038/nprot.2015.020

  View details for PubMedID 25612231

• T-cell STAT3 is required for the maintenance of humoral immunity to LCMV. European journal of immunology McIlwain, D. R., Grusdat, M., Pozdeev, V. I., Xu, H. C., Shinde, P., Reardon, C., Hao, Z., Beyer, M., Bergthaler, A., Häussinger, D., Nolan, G. P., Lang, K. S., Lang, P. A. 2015; 45 (2): 418-427

  Abstract

  STAT3 is a critical transcription factor activated downstream of cytokine signaling and is integral for the function of multiple immune cell types. Human mutations in STAT3 cause primary immunodeficiency resulting in impaired control of a variety of infections, including reactivation of latent viruses. In this study, we investigate how T-cell functions of STAT3 contribute to responses to viral infection by inducing chronic lymphocytic choriomeningitis virus (LCMV) infection in mice lacking STAT3 specifically in T cells. Although mice with conditional disruption of STAT3 in T cells were able to mount early responses to viral infection similar to control animals, including expansion of effector T cells, we found generation of T-follicular helper (Tfh) cells to be impaired. As a result, STAT3 T cell deficient mice produced attenuated germinal center reactions, and did not accumulate bone marrow virus specific IgG-secreting cells, resulting in failure to maintain levels of virus-specific IgG or mount neutralizing responses to LCMV in the serum. These effects were associated with reduced control of viral replication and prolonged infection. Our results demonstrate the importance of STAT3 in T cells for the generation of functional long-term humoral immunity to viral infections.

  View details for DOI 10.1002/eji.201445060

  View details for PubMedID 25393615

• Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm. Nature protocols Zunder, E. R., Finck, R., Behbehani, G. K., Amir, E. D., Krishnaswamy, S., Gonzalez, V. D., Lorang, C. G., Bjornson, Z., Spitzer, M. H., Bodenmiller, B., Fantl, W. J., Pe'er, D., Nolan, G. P. 2015; 10 (2): 316-333

  Abstract

  Mass-tag cell barcoding (MCB) labels individual cell samples with unique combinatorial barcodes, after which they are pooled for processing and measurement as a single multiplexed sample. The MCB method eliminates variability between samples in antibody staining and instrument sensitivity, reduces antibody consumption and shortens instrument measurement time. Here we present an optimized MCB protocol. The use of palladium-based labeling reagents expands the number of measurement channels available for mass cytometry and reduces interference with lanthanide-based antibody measurement. An error-detecting combinatorial barcoding scheme allows cell doublets to be identified and removed from the analysis. A debarcoding algorithm that is single cell-based rather than population-based improves the accuracy and efficiency of sample deconvolution. This debarcoding algorithm has been packaged into software that allows rapid and unbiased sample deconvolution. The MCB procedure takes 3-4 h, not including sample acquisition time of ∼1 h per million cells.

  View details for DOI 10.1038/nprot.2015.020

  View details for PubMedID 25612231

• Reversibility of Defective Hematopoiesis Caused by Telomere Shortening in Telomerase Knockout Mice. PloS one Raval, A., Behbehani, G. K., Nguyen, L. X., Thomas, D., Kusler, B., Garbuzov, A., Ramunas, J., Holbrook, C., Park, C. Y., Blau, H., Nolan, G. P., Artandi, S. E., Mitchell, B. S. 2015; 10 (7)

  Abstract

  Telomere shortening is common in bone marrow failure syndromes such as dyskeratosis congenita (DC), aplastic anemia (AA) and myelodysplastic syndromes (MDS). However, improved knowledge of the lineage-specific consequences of telomere erosion and restoration of telomere length in hematopoietic progenitors is required to advance therapeutic approaches. We have employed a reversible murine model of telomerase deficiency to compare the dependence of erythroid and myeloid lineage differentiation on telomerase activity. Fifth generation Tert-/- (G5 Tert-/-) mice with shortened telomeres have significant anemia, decreased erythroblasts and reduced hematopoietic stem cell (HSC) populations associated with neutrophilia and increased myelopoiesis. Intracellular multiparameter analysis by mass cytometry showed significantly reduced cell proliferation and increased sensitivity to activation of DNA damage checkpoints in erythroid progenitors and in erythroid-biased CD150hi HSC, but not in myeloid progenitors. Strikingly, Cre-inducible reactivation of telomerase activity restored hematopoietic stem and progenitor cell (HSPC) proliferation, normalized the DNA damage response, and improved red cell production and hemoglobin levels. These data establish a direct link between the loss of TERT activity, telomere shortening and defective erythropoiesis and suggest that novel strategies to restore telomerase function may have an important role in the treatment of the resulting anemia.

  View details for DOI 10.1371/journal.pone.0131722

  View details for PubMedID 26133370

  View details for PubMedCentralID PMC4489842

• Deletions in the cytoplasmic domain of iRhom1 and iRhom2 promote shedding of the TNF receptor by the protease ADAM17. Science signaling Maney, S. K., McIlwain, D. R., Polz, R., Pandyra, A. A., Sundaram, B., Wolff, D., Ohishi, K., Maretzky, T., Brooke, M. A., Evers, A., Vasudevan, A. A., Aghaeepour, N., Scheller, J., Münk, C., Häussinger, D., Mak, T. W., Nolan, G. P., Kelsell, D. P., Blobel, C. P., Lang, K. S., Lang, P. A. 2015; 8 (401): ra109-?

  Abstract

  The protease ADAM17 (a disintegrin and metalloproteinase 17) catalyzes the shedding of various transmembrane proteins from the surface of cells, including tumor necrosis factor (TNF) and its receptors. Liberation of TNF receptors (TNFRs) from cell surfaces can dampen the cellular response to TNF, a cytokine that is critical in the innate immune response and promotes programmed cell death but can also promote sepsis. Catalytically inactive members of the rhomboid family of proteases, iRhom1 and iRhom2, mediate the intracellular transport and maturation of ADAM17. Using a genetic screen, we found that the presence of either iRhom1 or iRhom2 lacking part of their extended amino-terminal cytoplasmic domain (herein referred to as ΔN) increases ADAM17 activity, TNFR shedding, and resistance to TNF-induced cell death in fibrosarcoma cells. Inhibitors of ADAM17, but not of other ADAM family members, prevented the effects of iRhom-ΔN expression. iRhom1 and iRhom2 were functionally redundant, suggesting a conserved role for the iRhom amino termini. Cells from patients with a dominantly inherited cancer susceptibility syndrome called tylosis with esophageal cancer (TOC) have amino-terminal mutations in iRhom2. Keratinocytes from TOC patients exhibited increased TNFR1 shedding compared with cells from healthy donors. Our results explain how loss of the amino terminus in iRhom1 and iRhom2 impairs TNF signaling, despite enhancing ADAM17 activity, and may explain how mutations in the amino-terminal region contribute to the cancer predisposition syndrome TOC.

  View details for DOI 10.1126/scisignal.aac5356

  View details for PubMedID 26535007

• Mass cytometry as a platform for the discovery of cellular biomarkers to guide effective rheumatic disease therapy. Arthritis research & therapy Nair, N., Mei, H. E., Chen, S., Hale, M., Nolan, G. P., Maecker, H. T., Genovese, M., Fathman, C. G., Whiting, C. C. 2015; 17: 127-?

  Abstract

  The development of biomarkers for autoimmune diseases has been hampered by a lack of understanding of disease etiopathogenesis and of the mechanisms underlying the induction and maintenance of inflammation, which involves complex activation dynamics of diverse cell types. The heterogeneous nature and suboptimal clinical response to treatment observed in many autoimmune syndromes highlight the need to develop improved strategies to predict patient outcome to therapy and personalize patient care. Mass cytometry, using CyTOF®, is an advanced technology that facilitates multiparametric, phenotypic analysis of immune cells at single-cell resolution. In this review, we outline the capabilities of mass cytometry and illustrate the potential of this technology to enhance the discovery of cellular biomarkers for rheumatoid arthritis, a prototypical autoimmune disease.

  View details for DOI 10.1186/s13075-015-0644-z

  View details for PubMedID 25981462

• Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining. Cytometry. Part A : the journal of the International Society for Analytical Cytology Behbehani, G. K., Thom, C., Zunder, E. R., Finck, R., Gaudilliere, B., Fragiadakis, G. K., Fantl, W. J., Nolan, G. P. 2014; 85 (12): 1011-1019

  Abstract

  Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression. © 2014 International Society for Advancement of Cytometry.

  View details for DOI 10.1002/cyto.a.22573

  View details for PubMedID 25274027

• Conditional density-based analysis of T cell signaling in single-cell data SCIENCE Krishnaswamy, S., Spitzer, M. H., Mingueneau, M., Bendall, S. C., Litvin, O., Stone, E., Pe'er, D., Nolan, G. P. 2014; 346 (6213): 1079-?

  View details for DOI 10.1126/science/1250689

  View details for Web of Science ID 000345763400033

• Systems biology. Conditional density-based analysis of T cell signaling in single-cell data. Science Krishnaswamy, S., Spitzer, M. H., Mingueneau, M., Bendall, S. C., Litvin, O., Stone, E., Pe'er, D., Nolan, G. P. 2014; 346 (6213)

  Abstract

  Cellular circuits sense the environment, process signals, and compute decisions using networks of interacting proteins. To model such a system, the abundance of each activated protein species can be described as a stochastic function of the abundance of other proteins. High-dimensional single-cell technologies, such as mass cytometry, offer an opportunity to characterize signaling circuit-wide. However, the challenge of developing and applying computational approaches to interpret such complex data remains. Here, we developed computational methods, based on established statistical concepts, to characterize signaling network relationships by quantifying the strengths of network edges and deriving signaling response functions. In comparing signaling between naïve and antigen-exposed CD4(+) T lymphocytes, we find that although these two cell subtypes had similarly wired networks, naïve cells transmitted more information along a key signaling cascade than did antigen-exposed cells. We validated our characterization on mice lacking the extracellular-regulated mitogen-activated protein kinase (MAPK) ERK2, which showed stronger influence of pERK on pS6 (phosphorylated-ribosomal protein S6), in naïve cells as compared with antigen-exposed cells, as predicted. We demonstrate that by using cell-to-cell variation inherent in single-cell data, we can derive response functions underlying molecular circuits and drive the understanding of how cells process signals.

  View details for DOI 10.1126/science.1250689

  View details for PubMedID 25342659

• NRAS9(G12V) oncogene facilitates self-renewal in a murine model of acute myelogenous leukemia BLOOD Sachs, Z., LaRue, R. S., Nguyen, H. T., Sachs, K., Noble, K. E., Hassan, N. A., Diaz-Flores, E., Rathe, S. K., Sarver, A. L., Bendall, S. C., Ha, N. A., Diers, M. D., Nolan, G. P., Shannon, K. M., Largaespada, D. A. 2014; 124 (22): 3274-3283

  Abstract

  Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. To elucidate the downstream functions of activated NRAS in AML, we used a murine model that harbors Mll-AF9 and a tetracycline-repressible, activated NRAS (NRAS(G12V)). Using computational approaches to explore our gene-expression data sets, we found that NRAS(G12V) enforced the leukemia self-renewal gene-expression signature and was required to maintain an MLL-AF9- and Myb-dependent leukemia self-renewal gene-expression program. NRAS(G12V) was required for leukemia self-renewal independent of its effects on growth and survival. Analysis of the gene-expression patterns of leukemic subpopulations revealed that the NRAS(G12V)-mediated leukemia self-renewal signature is preferentially expressed in the leukemia stem cell-enriched subpopulation. In a multiplexed analysis of RAS-dependent signaling, Mac-1(Low) cells, which harbor leukemia stem cells, were preferentially sensitive to NRAS(G12V) withdrawal. NRAS(G12V) maintained leukemia self-renewal through mTOR and MEK pathway activation, implicating these pathways as potential targets for cancer stem cell-specific therapies. Together, these experimental results define a RAS oncogene-driven function that is critical for leukemia maintenance and represents a novel mechanism of oncogene addiction.

  View details for DOI 10.1182/blood-2013-08-521708

  View details for Web of Science ID 000347463100016

  View details for PubMedCentralID PMC4239336

• NRASG12V oncogene facilitates self-renewal in a murine model of acute myelogenous leukemia. Blood Sachs, Z., LaRue, R. S., Nguyen, H. T., Sachs, K., Noble, K. E., Mohd Hassan, N. A., Diaz-Flores, E., Rathe, S. K., Sarver, A. L., Bendall, S. C., Ha, N. A., Diers, M. D., Nolan, G. P., Shannon, K. M., Largaespada, D. A. 2014; 124 (22): 3274-3283

  Abstract

  Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. To elucidate the downstream functions of activated NRAS in AML, we used a murine model that harbors Mll-AF9 and a tetracycline-repressible, activated NRAS (NRAS(G12V)). Using computational approaches to explore our gene-expression data sets, we found that NRAS(G12V) enforced the leukemia self-renewal gene-expression signature and was required to maintain an MLL-AF9- and Myb-dependent leukemia self-renewal gene-expression program. NRAS(G12V) was required for leukemia self-renewal independent of its effects on growth and survival. Analysis of the gene-expression patterns of leukemic subpopulations revealed that the NRAS(G12V)-mediated leukemia self-renewal signature is preferentially expressed in the leukemia stem cell-enriched subpopulation. In a multiplexed analysis of RAS-dependent signaling, Mac-1(Low) cells, which harbor leukemia stem cells, were preferentially sensitive to NRAS(G12V) withdrawal. NRAS(G12V) maintained leukemia self-renewal through mTOR and MEK pathway activation, implicating these pathways as potential targets for cancer stem cell-specific therapies. Together, these experimental results define a RAS oncogene-driven function that is critical for leukemia maintenance and represents a novel mechanism of oncogene addiction.

  View details for DOI 10.1182/blood-2013-08-521708

  View details for PubMedID 25316678

• Single-cell mass cytometry of TCR signaling: Amplification of small initial differences results in low ERK activation in NOD mice PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mingueneau, M., Krishnaswamy, S., Spitzer, M. H., Bendall, S. C., Stone, E. L., Hedrick, S. M., Pe'er, D., Mathis, D., Nolan, G. P., Benoist, C. 2014; 111 (46): 16466-16471

  View details for DOI 10.1073/pnas.1419337111

  View details for Web of Science ID 000345153300062

• Clinical recovery from surgery correlates with single-cell immune signatures SCIENCE TRANSLATIONAL MEDICINE Gaudilliere, B., Fragiadakis, G. K., Bruggner, R. V., Nicolau, M., Finck, R., Tingle, M., Silva, J., Ganio, E. A., Yeh, C. G., Maloney, W. J., Huddleston, J. I., Goodman, S. B., Davis, M. M., Bendall, S. C., Fantl, W. J., Angst, M. S., Nolan, G. P. 2014; 6 (255)

  Abstract

  Delayed recovery from surgery causes personal suffering and substantial societal and economic costs. Whether immune mechanisms determine recovery after surgical trauma remains ill-defined. Single-cell mass cytometry was applied to serial whole-blood samples from 32 patients undergoing hip replacement to comprehensively characterize the phenotypic and functional immune response to surgical trauma. The simultaneous analysis of 14,000 phosphorylation events in precisely phenotyped immune cell subsets revealed uniform signaling responses among patients, demarcating a surgical immune signature. When regressed against clinical parameters of surgical recovery, including functional impairment and pain, strong correlations were found with STAT3 (signal transducer and activator of transcription), CREB (adenosine 3',5'-monophosphate response element-binding protein), and NF-κB (nuclear factor κB) signaling responses in subsets of CD14(+) monocytes (R = 0.7 to 0.8, false discovery rate <0.01). These sentinel results demonstrate the capacity of mass cytometry to survey the human immune system in a relevant clinical context. The mechanistically derived immune correlates point to diagnostic signatures, and potential therapeutic targets, that could postoperatively improve patient recovery.

  View details for DOI 10.1126/scitranslmed.3009701

  View details for Web of Science ID 000343316800006

• Clinical recovery from surgery correlates with single-cell immune signatures. Science translational medicine Gaudillière, B., Fragiadakis, G. K., Bruggner, R. V., Nicolau, M., Finck, R., Tingle, M., Silva, J., Ganio, E. A., Yeh, C. G., Maloney, W. J., Huddleston, J. I., Goodman, S. B., Davis, M. M., Bendall, S. C., Fantl, W. J., Angst, M. S., Nolan, G. P. 2014; 6 (255): 255ra131-?

  Abstract

  Delayed recovery from surgery causes personal suffering and substantial societal and economic costs. Whether immune mechanisms determine recovery after surgical trauma remains ill-defined. Single-cell mass cytometry was applied to serial whole-blood samples from 32 patients undergoing hip replacement to comprehensively characterize the phenotypic and functional immune response to surgical trauma. The simultaneous analysis of 14,000 phosphorylation events in precisely phenotyped immune cell subsets revealed uniform signaling responses among patients, demarcating a surgical immune signature. When regressed against clinical parameters of surgical recovery, including functional impairment and pain, strong correlations were found with STAT3 (signal transducer and activator of transcription), CREB (adenosine 3',5'-monophosphate response element-binding protein), and NF-κB (nuclear factor κB) signaling responses in subsets of CD14(+) monocytes (R = 0.7 to 0.8, false discovery rate <0.01). These sentinel results demonstrate the capacity of mass cytometry to survey the human immune system in a relevant clinical context. The mechanistically derived immune correlates point to diagnostic signatures, and potential therapeutic targets, that could postoperatively improve patient recovery.

  View details for DOI 10.1126/scitranslmed.3009701

  View details for PubMedID 25253674

• Single-cell mass cytometry analysis of human tonsil T cell remodeling by varicella zoster virus. Cell reports Sen, N., Mukherjee, G., Sen, A., Bendall, S. C., Sung, P., Nolan, G. P., Arvin, A. M. 2014; 8 (2): 633-645

  Abstract

  Although pathogens must infect differentiated host cells that exhibit substantial diversity, documenting the consequences of infection against this heterogeneity is challenging. Single-cell mass cytometry permits deep profiling based on combinatorial expression of surface and intracellular proteins. We used this method to investigate varicella-zoster virus (VZV) infection of tonsil T cells, which mediate viral transport to skin. Our results indicate that VZV induces a continuum of changes regardless of basal phenotypic and functional T cell characteristics. Contrary to the premise that VZV selectively infects T cells with skin trafficking profiles, VZV infection altered T cell surface proteins to enhance or induce these properties. Zap70 and Akt signaling pathways that trigger such surface changes were activated in VZV-infected naive and memory cells by a T cell receptor (TCR)-independent process. Single-cell mass cytometry is likely to be broadly relevant for demonstrating how intracellular pathogens modulate differentiated cells to support pathogenesis in the natural host.

  View details for DOI 10.1016/j.celrep.2014.06.024

  View details for PubMedID 25043183

  View details for PubMedCentralID PMC4127309

• Single-Cell Mass Cytometry Analysis of Human Tonsil T Cell Remodeling by Varicella Zoster Virus CELL REPORTS Sen, N., Mukherjee, G., Sen, A., Bendall, S. C., Sung, P., Nolan, G. P., Arvin, A. M. 2014; 8 (2): 632-644

  Abstract

  Although pathogens must infect differentiated host cells that exhibit substantial diversity, documenting the consequences of infection against this heterogeneity is challenging. Single-cell mass cytometry permits deep profiling based on combinatorial expression of surface and intracellular proteins. We used this method to investigate varicella-zoster virus (VZV) infection of tonsil T cells, which mediate viral transport to skin. Our results indicate that VZV induces a continuum of changes regardless of basal phenotypic and functional T cell characteristics. Contrary to the premise that VZV selectively infects T cells with skin trafficking profiles, VZV infection altered T cell surface proteins to enhance or induce these properties. Zap70 and Akt signaling pathways that trigger such surface changes were activated in VZV-infected naive and memory cells by a T cell receptor (TCR)-independent process. Single-cell mass cytometry is likely to be broadly relevant for demonstrating how intracellular pathogens modulate differentiated cells to support pathogenesis in the natural host.

  View details for DOI 10.1016/j.celrep.2014.06.024

  View details for Web of Science ID 000341569800030

  View details for PubMedCentralID PMC4127309

• Automated identification of stratifying signatures in cellular subpopulations PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bruggner, R. V., Bodenmiller, B., Dill, D. L., Tibshirani, R. J., Nolan, G. P. 2014; 111 (26): E2770-E2777

  View details for DOI 10.1073/pnas.1408792111

  View details for Web of Science ID 000338118900020

• Joint Modeling and Registration of Cell Populations in Cohorts of High-Dimensional Flow Cytometric Data PLOS ONE Pyne, S., Lee, S. X., Wang, K., Irish, J., Tamayo, P., Nazaire, M., Duong, T., Shu-Kay Ng, S. K., Hafler, D., Levy, R., Nolan, G. P., Mesirov, J., McLachlan, G. J. 2014; 9 (7)

  Abstract

  In biomedical applications, an experimenter encounters different potential sources of variation in data such as individual samples, multiple experimental conditions, and multivariate responses of a panel of markers such as from a signaling network. In multiparametric cytometry, which is often used for analyzing patient samples, such issues are critical. While computational methods can identify cell populations in individual samples, without the ability to automatically match them across samples, it is difficult to compare and characterize the populations in typical experiments, such as those responding to various stimulations or distinctive of particular patients or time-points, especially when there are many samples. Joint Clustering and Matching (JCM) is a multi-level framework for simultaneous modeling and registration of populations across a cohort. JCM models every population with a robust multivariate probability distribution. Simultaneously, JCM fits a random-effects model to construct an overall batch template--used for registering populations across samples, and classifying new samples. By tackling systems-level variation, JCM supports practical biomedical applications involving large cohorts. Software for fitting the JCM models have been implemented in an R package EMMIX-JCM, available from http://www.maths.uq.edu.au/~gjm/mix_soft/EMMIX-JCM/.

  View details for DOI 10.1371/journal.pone.0100334

  View details for Web of Science ID 000339635000020

  View details for PubMedID 24983991

  View details for PubMedCentralID PMC4077578

• Automated identification of stratifying signatures in cellular subpopulations. Proceedings of the National Academy of Sciences of the United States of America Bruggner, R. V., Bodenmiller, B., Dill, D. L., Tibshirani, R. J., Nolan, G. P. 2014; 111 (26): E2770-7

  Abstract

  Elucidation and examination of cellular subpopulations that display condition-specific behavior can play a critical contributory role in understanding disease mechanism, as well as provide a focal point for development of diagnostic criteria linking such a mechanism to clinical prognosis. Despite recent advancements in single-cell measurement technologies, the identification of relevant cell subsets through manual efforts remains standard practice. As new technologies such as mass cytometry increase the parameterization of single-cell measurements, the scalability and subjectivity inherent in manual analyses slows both analysis and progress. We therefore developed Citrus (cluster identification, characterization, and regression), a data-driven approach for the identification of stratifying subpopulations in multidimensional cytometry datasets. The methodology of Citrus is demonstrated through the identification of known and unexpected pathway responses in a dataset of stimulated peripheral blood mononuclear cells measured by mass cytometry. Additionally, the performance of Citrus is compared with that of existing methods through the analysis of several publicly available datasets. As the complexity of flow cytometry datasets continues to increase, methods such as Citrus will be needed to aid investigators in the performance of unbiased--and potentially more thorough--correlation-based mining and inspection of cell subsets nested within high-dimensional datasets.

  View details for DOI 10.1073/pnas.1408792111

  View details for PubMedID 24979804

• Single-Cell Trajectory Detection Uncovers Progression and Regulatory Coordination in Human B Cell Development CELL Bendall, S. C., Davis, K. L., Amir, E. D., Tadmor, M. D., Simonds, E. F., Chen, T. J., Shenfeld, D. K., Nolan, G. P., Pe'er, D. 2014; 157 (3): 714-725

  Abstract

  Tissue regeneration is an orchestrated progression of cells from an immature state to a mature one, conventionally represented as distinctive cell subsets. A continuum of transitional cell states exists between these discrete stages. We combine the depth of single-cell mass cytometry and an algorithm developed to leverage this continuum by aligning single cells of a given lineage onto a unified trajectory that accurately predicts the developmental path de novo. Applied to human B cell lymphopoiesis, the algorithm (termed Wanderlust) constructed trajectories spanning from hematopoietic stem cells through to naive B cells. This trajectory revealed nascent fractions of B cell progenitors and aligned them with developmentally cued regulatory signaling including IL-7/STAT5 and cellular events such as immunoglobulin rearrangement, highlighting checkpoints across which regulatory signals are rewired paralleling changes in cellular state. This study provides a comprehensive analysis of human B lymphopoiesis, laying a foundation to apply this approach to other tissues and "corrupted" developmental processes including cancer.

  View details for DOI 10.1016/j.cell.2014.04.005

  View details for Web of Science ID 000335392100019

  View details for PubMedID 24766814

  View details for PubMedCentralID PMC4045247

• Multiplexed ion beam imaging of human breast tumors. Nature medicine Angelo, M., Bendall, S. C., Finck, R., Hale, M. B., Hitzman, C., Borowsky, A. D., Levenson, R. M., Lowe, J. B., Liu, S. D., Zhao, S., Natkunam, Y., Nolan, G. P. 2014; 20 (4): 436-442

  Abstract

  Immunohistochemistry (IHC) is a tool for visualizing protein expression that is employed as part of the diagnostic workup for the majority of solid tissue malignancies. Existing IHC methods use antibodies tagged with fluorophores or enzyme reporters that generate colored pigments. Because these reporters exhibit spectral and spatial overlap when used simultaneously, multiplexed IHC is not routinely used in clinical settings. We have developed a method that uses secondary ion mass spectrometry to image antibodies tagged with isotopically pure elemental metal reporters. Multiplexed ion beam imaging (MIBI) is capable of analyzing up to 100 targets simultaneously over a five-log dynamic range. Here, we used MIBI to analyze formalin-fixed, paraffin-embedded human breast tumor tissue sections stained with ten labels simultaneously. The resulting data suggest that MIBI can provide new insights into disease pathogenesis that will be valuable for basic research, drug discovery and clinical diagnostics.

  View details for DOI 10.1038/nm.3488

  View details for PubMedID 24584119

• Multiplexed ion beam imaging of human breast tumors. Nature medicine Angelo, M., Bendall, S. C., Finck, R., Hale, M. B., Hitzman, C., Borowsky, A. D., Levenson, R. M., Lowe, J. B., Liu, S. D., Zhao, S., Natkunam, Y., Nolan, G. P. 2014; 20 (4): 436-442

  Abstract

  Immunohistochemistry (IHC) is a tool for visualizing protein expression that is employed as part of the diagnostic workup for the majority of solid tissue malignancies. Existing IHC methods use antibodies tagged with fluorophores or enzyme reporters that generate colored pigments. Because these reporters exhibit spectral and spatial overlap when used simultaneously, multiplexed IHC is not routinely used in clinical settings. We have developed a method that uses secondary ion mass spectrometry to image antibodies tagged with isotopically pure elemental metal reporters. Multiplexed ion beam imaging (MIBI) is capable of analyzing up to 100 targets simultaneously over a five-log dynamic range. Here, we used MIBI to analyze formalin-fixed, paraffin-embedded human breast tumor tissue sections stained with ten labels simultaneously. The resulting data suggest that MIBI can provide new insights into disease pathogenesis that will be valuable for basic research, drug discovery and clinical diagnostics.

  View details for DOI 10.1038/nm.3488

  View details for PubMedID 24584119

• Antigen-Dependent Integration of Opposing Proximal TCR-Signaling Cascades Determines the Functional Fate of T Lymphocytes JOURNAL OF IMMUNOLOGY Wolchinsky, R., Hod-Marco, M., Oved, K., Shen-Orr, S. S., Bendall, S. C., Nolan, G. P., Reiter, Y. 2014; 192 (5): 2109-2119

  Abstract

  T cell anergy is a key tolerance mechanism to mitigate unwanted T cell activation against self by rendering lymphocytes functionally inactive following Ag encounter. Ag plays an important role in anergy induction where high supraoptimal doses lead to the unresponsive phenotype. How T cells "measure" Ag dose and how this determines functional output to a given antigenic dose remain unclear. Using multiparametric phospho-flow and mass cytometry, we measured the intracellular phosphorylation-dependent signaling events at a single-cell resolution and studied the phosphorylation levels of key proximal human TCR activation- and inhibition-signaling molecules. We show that the intracellular balance and signal integration between these opposing signaling cascades serve as the molecular switch gauging Ag dose. An Ag density of 100 peptide-MHC complexes/cell was found to be the transition point between dominant activation and inhibition cascades, whereas higher Ag doses induced an anergic functional state. Finally, the neutralization of key inhibitory molecules reversed T cell unresponsiveness and enabled maximal T cell functions, even in the presence of very high Ag doses. This mechanism permits T cells to make integrated "measurements" of Ag dose that determine subsequent functional outcomes.

  View details for DOI 10.4049/jimmunol.1301142

  View details for Web of Science ID 000332701400015

  View details for PubMedID 24489091

• Mass cytometry to decipher the mechanism of nongenetic drug resistance in cancer. Current topics in microbiology and immunology Fienberg, H. G., Nolan, G. P. 2014; 377: 85-94

  Abstract

  Nongenetic resistance has recently been described as a major impediment to effective cancer therapy. Nongenetic resistance is challenging to study since it occurs nonuniformly, even in cell lines, and can involve the interplay of multiple survival pathways. Until recently, no technology allowed measurement of large-scale alterations in survival pathways with single-cell resolution. Mass cytometry, a flow-based technique in which the activation of up to 50 proteins can be measured simultaneously in single-cell, now provides the ability to examine nongenetic resistance on the functional level on a cell-by-cell basis. The application of mass cytometry, in combination with new bioinformatic techniques, will allow fundamental questions on nongenetic resistance to be addressed: Is resistance caused by selection of cells with a pre-existing survival phenotype or induction of a survival program? Which survival pathways are necessary for nongenetic resistance and how do they interact? Currently, mass cytometry is being used to investigate the mechanism of nongenetic resistance to TRAIL-induced apoptosis. The approaches being developed to understand resistance to TRAIL will likely be applied to elucidate the mechanisms of nongenetic resistance broadly and in the clinic.

  View details for DOI 10.1007/82_2014_365

  View details for PubMedID 24578267

• Snapin, Positive Regulator of Stimulation- Induced Ca2+ Release through RyR, Is Necessary for HIV-1 Replication in T Cells PLOS ONE Kinoshita, S. M., Kogure, A., Taguchi, S., Nolan, G. P. 2013; 8 (10)

  Abstract

  To identify critical host factors necessary for human immunodeficiency virus 1 (HIV-1) replication, large libraries of short-peptide-aptamers were expressed retrovirally. The target of one inhibitor peptide, Pep80, identified in this screen was determined to be Snapin, a protein associated with the soluble N-ethyl maleimide sensitive factor adaptor protein receptor (SNARE) complex that is critical for calcium-dependent exocytosis during neurotransmission. Pep80 inhibited Ca²⁺ release from intracellular stores and blocked downstream signaling by direct interruption of the association between Snapin and an intracellular calcium release channel, the ryanodine receptor (RyR). NFAT signaling was preferentially abolished by Pep80. Expression of Snapin overcame Pep80-mediated inhibition of Ca²⁺/NFAT signaling and HIV-1 replication. Furthermore, Snapin induced HIV-1 replication in primary CD4⁺ T cells. Thus, through its interaction with RyR, Snapin is a critical regulator of Ca²⁺ signaling and T cell activation. Use of the genetically selected intracellular aptamer inhibitors allowed us to define unique mechanisms important to HIV-1 replication and T cell biology.

  View details for DOI 10.1371/journal.pone.0075297

  View details for Web of Science ID 000325814200009

  View details for PubMedID 24130701

• Single timepoint models of dynamic systems INTERFACE FOCUS Sachs, K., Itani, S., FitzGerald, J., Schoeberl, B., Nolan, G. P., Tomlin, C. J. 2013; 3 (4)

  View details for DOI 10.1098/rsfs.2013.0019

  View details for Web of Science ID 000320853900009

• Single-cell mass cytometry for analysis of immune system functional states. Current opinion in immunology Bjornson, Z. B., Nolan, G. P., Fantl, W. J. 2013; 25 (4): 484-494

  Abstract

  Mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on cell populations at single-cell resolution. Datasets are generated with panels of up to 45 antibodies. Each antibody is conjugated to a polymer chelated with a stable metal isotope, usually in the lanthanide series of the periodic table. Antibody panels recognize surface markers to delineate cell types simultaneously with intracellular signaling molecules to measure biological functions, such as metabolism, survival, DNA damage, cell cycle and apoptosis, to provide an overall determination of the network state of an individual cell. This review will cover the basics of mass cytometry as well as outline assays developed for the platform that enhance the immunologist's analytical arsenal.

  View details for DOI 10.1016/j.coi.2013.07.004

  View details for PubMedID 23999316

• The Systemic Immune State of Super-shedder Mice Is Characterized by a Unique Neutrophil-dependent Blunting of TH1 Responses. PLoS pathogens Gopinath, S., Hotson, A., Johns, J., Nolan, G., Monack, D. 2013; 9 (6)

  Abstract

  Host-to-host transmission of a pathogen ensures its successful propagation and maintenance within a host population. A striking feature of disease transmission is the heterogeneity in host infectiousness. It has been proposed that within a host population, 20% of the infected hosts, termed super-shedders, are responsible for 80% of disease transmission. However, very little is known about the immune state of these super-shedders. In this study, we used the model organism Salmonella enterica serovar Typhimurium, an important cause of disease in humans and animal hosts, to study the immune state of super-shedders. Compared to moderate shedders, super-shedder mice had an active inflammatory response in both the gastrointestinal tract and the spleen but a dampened TH1 response specific to the secondary lymphoid organs. Spleens from super-shedder mice had higher numbers of neutrophils, and a dampened T cell response, characterized by higher levels of regulatory T cells (Tregs), fewer T-bet(+) (TH1) T cells as well as blunted cytokine responsiveness. Administration of the cytokine granulocyte colony stimulating factor (G-CSF) and subsequent neutrophilia was sufficient to induce the super-shedder immune phenotype in moderate-shedder mice. Similar to super-shedders, these G-CSF-treated moderate-shedders had a dampened TH1 response with fewer T-bet(+) T cells and a loss of cytokine responsiveness. Additionally, G-CSF treatment inhibited IL-2-mediated TH1 expansion. Finally, depletion of neutrophils led to an increase in the number of T-bet(+) TH1 cells and restored their ability to respond to IL-2. Taken together, we demonstrate a novel role for neutrophils in blunting IL-2-mediated proliferation of the TH1 immune response in the spleens of mice that are colonized by high levels of S. Typhimurium in the gastrointestinal tract.

  View details for DOI 10.1371/journal.ppat.1003408

  View details for PubMedID 23754944

• The transcriptional landscape of aß T cell differentiation. Nature immunology Mingueneau, M., Kreslavsky, T., Gray, D., Heng, T., Cruse, R., Ericson, J., Bendall, S., Spitzer, M. H., Nolan, G. P., Kobayashi, K., Von Boehmer, H., Mathis, D., Benoist, C., Best, A. J., Knell, J., Goldrath, A., Jojic, V., Koller, D., Shay, T., Regev, A., Cohen, N., Brennan, P., Brenner, M., Kim, F., Rao, T. N., Wagers, A., Heng, T., Ericson, J., Rothamel, K., Ortiz-Lopez, A., Mathis, D., Benoist, C., Bezman, N. A., Sun, J. C., Min-Oo, G., Kim, C. C., Lanier, L. L., Miller, J., Brown, B., Merad, M., Gautier, E. L., Jakubzick, C., Randolph, G. J., Monach, P., Blair, D. A., Dustin, M. L., Shinton, S. A., Hardy, R. R., Laidlaw, D., Collins, J., Gazit, R., Rossi, D. J., Malhotra, N., Sylvia, K., Kang, J., Kreslavsky, T., Fletcher, A., Elpek, K., Bellemare-Pelletier, A., Malhotra, D., Turley, S. 2013; 14 (6): 619-632

  Abstract

  The differentiation of αβT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4(+)CD8(+) stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4(+) or CD8(+) lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation.

  View details for DOI 10.1038/ni.2590

  View details for PubMedID 23644507

• viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nature biotechnology Amir, E. D., Davis, K. L., Tadmor, M. D., Simonds, E. F., Levine, J. H., Bendall, S. C., Shenfeld, D. K., Krishnaswamy, S., Nolan, G. P., Pe'er, D. 2013; 31 (6): 545-552

  Abstract

  New high-dimensional, single-cell technologies offer unprecedented resolution in the analysis of heterogeneous tissues. However, because these technologies can measure dozens of parameters simultaneously in individual cells, data interpretation can be challenging. Here we present viSNE, a tool that allows one to map high-dimensional cytometry data onto two dimensions, yet conserve the high-dimensional structure of the data. viSNE plots individual cells in a visual similar to a scatter plot, while using all pairwise distances in high dimension to determine each cell's location in the plot. We integrated mass cytometry with viSNE to map healthy and cancerous bone marrow samples. Healthy bone marrow automatically maps into a consistent shape, whereas leukemia samples map into malformed shapes that are distinct from healthy bone marrow and from each other. We also use viSNE and mass cytometry to compare leukemia diagnosis and relapse samples, and to identify a rare leukemia population reminiscent of minimal residual disease. viSNE can be applied to any multi-dimensional single-cell technology.

  View details for DOI 10.1038/nbt.2594

  View details for PubMedID 23685480

• The transcriptional landscape of alpha beta T cell differentiation NATURE IMMUNOLOGY Mingueneau, M., Kreslavsky, T., Gray, D., Heng, T., Cruse, R., Ericson, J., Bendall, S., Spitzer, M., Nolan, G., Kobayashi, K., Von Boehmer, H., Mathis, D., Benoist, C., Best, A. J., Knell, J., Goldrath, A., Jojic, V., Koller, D., Shay, T., Regev, A., Cohen, N., Brennan, P., Brenner, M., Kim, F., Rao, T. N., Wagers, A., Heng, T., Ericson, J., Rothamel, K., Ortiz-Lopez, A., Mathis, D., Bezman, N. A., Sun, J. C., Min-Oo, G., Kim, C. C., Lanier, L. L., Miller, J., Brown, B., Merad, M., Gautier, E. L., Jakubzick, C., Randolph, G. J., Monach, P., Blair, D. A., Dustin, M. L., Shinton, S. A., Hardy, R. R., Laidlaw, D., Collins, J., Gazit, R., Rossi, D. J., Malhotra, N., Sylvia, K., Kang, J., Kreslavsky, T., Fletcher, A., Elpek, K., Bellemare-Pelletier, A., Malhotra, D., Turley, S. 2013; 14 (6): 619-?

  Abstract

  The differentiation of αβT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4(+)CD8(+) stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4(+) or CD8(+) lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation.

  View details for DOI 10.1038/ni.2590

  View details for Web of Science ID 000319107600015

  View details for PubMedCentralID PMC3660436

• Inner-outer beauty: DNA-binding surface tags as cellular barcodes. Nature methods Nolan, G. 2013; 10 (5): 399-401

  View details for DOI 10.1038/nmeth.2452

  View details for PubMedID 23629416

• Normalization of mass cytometry data with bead standards. Cytometry. Part A : the journal of the International Society for Analytical Cytology Finck, R., Simonds, E. F., Jager, A., Krishnaswamy, S., Sachs, K., Fantl, W., Pe'er, D., Nolan, G. P., Bendall, S. C. 2013; 83 (5): 483-494

  Abstract

  Mass cytometry uses atomic mass spectrometry combined with isotopically pure reporter elements to currently measure as many as 40 parameters per single cell. As with any quantitative technology, there is a fundamental need for quality assurance and normalization protocols. In the case of mass cytometry, the signal variation over time due to changes in instrument performance combined with intervals between scheduled maintenance must be accounted for and then normalized. Here, samples were mixed with polystyrene beads embedded with metal lanthanides, allowing monitoring of mass cytometry instrument performance over multiple days of data acquisition. The protocol described here includes simultaneous measurements of beads and cells on the mass cytometer, subsequent extraction of the bead-based signature, and the application of an algorithm enabling correction of both short- and long-term signal fluctuations. The variation in the intensity of the beads that remains after normalization may also be used to determine data quality. Application of the algorithm to a one-month longitudinal analysis of a human peripheral blood sample reduced the range of median signal fluctuation from 4.9-fold to 1.3-fold.

  View details for DOI 10.1002/cyto.a.22271

  View details for PubMedID 23512433

• Involvement of Toso in activation of monocytes, macrophages, and granulocytes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lang, K. S., Lang, P. A., Meryk, A., Pandyra, A. A., Boucher, L., Pozdeev, V. I., Tusche, M. W., Goethert, J. R., Haight, J., Wakeham, A., You-Ten, A. J., McIlwain, D. R., Merches, K., Khairnar, V., Recher, M., Nolan, G. P., Hitoshi, Y., Funkner, P., Navarini, A. A., Verschoor, A., Shaabani, N., Honke, N., Penn, L. Z., Ohashi, P. S., Haeussinger, D., Lee, K., Mak, T. W. 2013; 110 (7): 2593-2598

  Abstract

  Rapid activation of immune responses is necessary for antibacterial defense, but excessive immune activation can result in life-threatening septic shock. Understanding how these processes are balanced may provide novel therapeutic potential in treating inflammatory disease. Fc receptors are crucial for innate immune activation. However, the role of the putative Fc receptor for IgM, known as Toso/Faim3, has to this point been unclear. In this study, we generated Toso-deficient mice and used them to uncover a critical regulatory function of Toso in innate immune activation. Development of innate immune cells was intact in the absence of Toso, but Toso-deficient neutrophils exhibited more reactive oxygen species production and reduced phagocytosis of pathogens compared with controls. Cytokine production was also decreased in Toso(-/-) mice compared with WT animals, rendering them resistant to septic shock induced by lipopolysaccharide. However, Toso(-/-) mice also displayed limited cytokine production after infection with the bacterium Listeria monocytogenes that was correlated with elevated presence of Listeria throughout the body. Accordingly, Toso(-/-) mice succumbed to infections of L. monocytogenes, whereas WT mice successfully eliminated the infection. Taken together, our data reveal Toso to be a unique regulator of innate immune responses during bacterial infection and septic shock.

  View details for DOI 10.1073/pnas.1222264110

  View details for Web of Science ID 000315812800047

  View details for PubMedID 23359703

  View details for PubMedCentralID PMC3574925

• Single Cell Mass Cytometry of Dysregulated Signaling Networks in Myeloproliferative Neoplasms and Secondary Acute Myeloid Leukemia 54th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Fisher, D. A., Simonds, E. F., Behbehani, G. K., Nolan, G. P., Bendall, S. C., Oh, S. T. AMER SOC HEMATOLOGY. 2012

  View details for Web of Science ID 000313838900254

• Short Term Signalling Responses of the Most Primitive Subsets of Human Hematopoietic Cells Stimulated in Vitro Correlate with Their Subsequent Self-Renewal Behaviour 54th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Knapp, D. J., Bendall, S. C., Miller, P. H., Cheung, A. M., Imren, S., Humphries, R. K., Brinkman, R. R., Oostendorp, R. A., Nolan, G. P., Eaves, C. J. AMER SOC HEMATOLOGY. 2012

  View details for Web of Science ID 000314049601119

• MASS CYTOMETRY TO COMPREHENSIVELY STUDY SINGLE CELL SIGNALING IN BIOLOGY AND DISEASE 12th Euroconference on Clinical Cell Analysis / 8th European Clinical Cytometry Course Bodenmiller, B., Zunder, E., Finck, R., Chen, T., Savig, E., Bruggner, R., Simonds, E., Bendall, S., Sachs, K., Krutzik, P., Nolan, G. WILEY-BLACKWELL. 2012: 376–77

  View details for Web of Science ID 000310386300014

• CytoSPADE: high-performance analysis and visualization of high-dimensional cytometry data BIOINFORMATICS Linderman, M. D., Bjornson, Z., Simonds, E. F., Qiu, P., Bruggner, R. V., Sheode, K., Meng, T. H., Plevritis, S. K., Nolan, G. P. 2012; 28 (18): 2400-2401

  Abstract

  MOTIVATION: Recent advances in flow cytometry enable simultaneous single-cell measurement of 30+ surface and intracellular proteins. CytoSPADE is a high-performance implementation of an interface for the Spanning-tree Progression Analysis of Density-normalized Events algorithm for tree-based analysis and visualization of this high-dimensional cytometry data. AVAILABILITY: Source code and binaries are freely available at http://cytospade.org and via Bioconductor version 2.10 onwards for Linux, OSX and Windows. CytoSPADE is implemented in R, C++ and Java. CONTACT: michael.linderman@mssm.edu SUPPLEMENTARY INFORMATION: Additional documentation available at http://cytospade.org.

  View details for DOI 10.1093/bioinformatics/bts425

  View details for Web of Science ID 000308532300067

  View details for PubMedID 22782546

  View details for PubMedCentralID PMC3436846

• Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators NATURE BIOTECHNOLOGY Bodenmiller, B., Zunder, E. R., Finck, R., Chen, T. J., Savig, E. S., Bruggner, R. V., Simonds, E. F., Bendall, S. C., Sachs, K., Krutzik, P. O., Nolan, G. P. 2012; 30 (9): 858-U89

  Abstract

  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.

  View details for DOI 10.1038/nbt.2317

  View details for Web of Science ID 000308705700020

  View details for PubMedID 22902532

  View details for PubMedCentralID PMC3627543

• COP9 Signalosome Component JAB1/CSN5 Is Necessary for T Cell Signaling through LFA-1 and HIV-1 Replication PLOS ONE Kinoshita, S. M., Krutzik, P. O., Nolan, G. P. 2012; 7 (7)

  Abstract

  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.

  View details for DOI 10.1371/journal.pone.0041725

  View details for Web of Science ID 000306751300067

  View details for PubMedID 22911848

• Raman labeled nanoparticles: characterization of variability and improved method for unmixing JOURNAL OF RAMAN SPECTROSCOPY Kode, K., Shachaf, C., Elchuri, S., Nolan, G., Paik, D. S. 2012; 43 (7): 895-905

  View details for DOI 10.1002/jrs.3114

  View details for Web of Science ID 000306570400012

• Single-cell mass cytometry adapted to measurements of the cell cycle CYTOMETRY PART A Behbehani, G. K., Bendall, S. C., Clutter, M. R., Fantl, W. J., Nolan, G. P. 2012; 81A (7): 552-566

  View details for DOI 10.1002/cyto.a.22075

  View details for Web of Science ID 000305558700006

• From single cells to deep phenotypes in cancer NATURE BIOTECHNOLOGY Bendall, S. C., Nolan, G. P. 2012; 30 (7): 639-647

  Abstract

  In recent years, major advances in single-cell measurement systems have included the introduction of high-throughput versions of traditional flow cytometry that are now capable of measuring intracellular network activity, the emergence of isotope labels that can enable the tracking of a greater variety of cell markers and the development of super-resolution microscopy techniques that allow measurement of RNA expression in single living cells. These technologies will facilitate our capacity to catalog and bring order to the inherent diversity present in cancer cell populations. Alongside these developments, new computational approaches that mine deep data sets are facilitating the visualization of the shape of the data and enabling the extraction of meaningful outputs. These applications have the potential to reveal new insights into cancer biology at the intersections of stem cell function, tumor-initiating cells and multilineage tumor development. In the clinic, they may also prove important not only in the development of new diagnostic modalities but also in understanding how the emergence of tumor cell clones harboring different sets of mutations predispose patients to relapse or disease progression.

  View details for DOI 10.1038/nbt.2283

  View details for Web of Science ID 000306293400023

  View details for PubMedID 22781693

• Single-cell mass cytometry adapted to measurements of the cell cycle. Cytometry. Part A : the journal of the International Society for Analytical Cytology Behbehani, G. K., Bendall, S. C., Clutter, M. R., Fantl, W. J., Nolan, G. P. 2012; 81 (7): 552-566

  Abstract

  Mass cytometry is a recently introduced technology that utilizes transition element isotope-tagged antibodies for protein detection on a single-cell basis. By circumventing the limitations of emission spectral overlap associated with fluorochromes utilized in traditional flow cytometry, mass cytometry currently allows measurement of up to 40 parameters per cell. Recently, a comprehensive mass cytometry analysis was described for the hematopoietic differentiation program in human bone marrow from a healthy donor. The current study describes approaches to delineate cell cycle stages utilizing 5-iodo-2-deoxyuridine (IdU) to mark cells in S phase, simultaneously with antibodies against cyclin B1, cyclin A, and phosphorylated histone H3 (S28) that characterize the other cell cycle phases. Protocols were developed in which an antibody against phosphorylated retinoblastoma protein (Rb) at serines 807 and 811 was used to separate cells in G0 and G1 phases of the cell cycle. This mass cytometry method yielded cell cycle distributions of both normal and cancer cell populations that were equivalent to those obtained by traditional fluorescence cytometry techniques. We applied this to map the cell cycle phases of cells spanning the hematopoietic hierarchy in healthy human bone marrow as a prelude to later studies with cancers and other disorders of this lineage.

  View details for DOI 10.1002/cyto.a.22075

  View details for PubMedID 22693166

• A deep profiler's guide to cytometry TRENDS IN IMMUNOLOGY Bendall, S. C., Nolan, G. P., Roederer, M., Chattopadhyay, P. K. 2012; 33 (7): 323-332

  Abstract

  In recent years, advances in technology have provided us with tools to quantify the expression of multiple genes in individual cells. The ability to measure simultaneously multiple genes in the same cell is necessary to resolve the great diversity of cell subsets, as well as to define their function in the host. Fluorescence-based flow cytometry is the benchmark for this; with it, we can quantify 18 proteins per cell, at >10 000 cells/s. Mass cytometry is a new technology that promises to extend these capabilities significantly. Immunophenotyping by mass spectrometry provides the ability to measure >36 proteins at a rate of 1000 cells/s. We review these cytometric technologies, capable of high-content, high-throughput single-cell assays.

  View details for DOI 10.1016/j.it.2012.02.010

  View details for Web of Science ID 000306639500001

  View details for PubMedID 22476049

• A platinum-based covalent viability reagent for single-cell mass cytometry. Cytometry. Part A : the journal of the International Society for Analytical Cytology Fienberg, H. G., Simonds, E. F., Fantl, W. J., Nolan, G. P., Bodenmiller, B. 2012; 81 (6): 467-475

  Abstract

  In fluorescence-based flow cytometry, cellular viability is determined with membrane-impermeable fluorescent reagents that specifically enter and label plasma membrane-compromised nonviable cells. A recent technological advance in flow cytometry uses antibodies conjugated to elemental metal isotopes, rather than to fluorophores, to allow signal detection by atomic mass spectrometry. Unhampered by the limitations of overlapping emission fluorescence, mass cytometry increases the number of parameters that can be measured in single cells. However, mass cytometry is unable to take advantage of current fluorescent viability dyes. An alternative methodology was therefore developed here in which the platinum-containing chemotherapy drug cisplatin was used to resolve live and dead cells by mass cytometry. In a 1-min incubation step, cisplatin preferentially labeled nonviable cells from both adherent and suspension cultures, resulting in a platinum signal quantifiable by mass cytometry. This protocol was compatible with established sample processing steps for intracellular cytometry. Furthermore, the live/dead ratios were comparable between mass- and fluorescence-based cytometry. Importantly, although cisplatin is a known DNA-damaging agent, a 1-min "pulse" of cisplatin did not induce observable DNA damage or apoptotic responses even within 6-h post-exposure. Cisplatin can therefore be used as a viability reagent for a wide range of mass cytometry protocols.

  View details for DOI 10.1002/cyto.a.22067

  View details for PubMedID 22577098

• A platinum-based covalent viability reagent for single-cell mass cytometry CYTOMETRY PART A Fienberg, H. G., Simonds, E. F., Fantl, W. J., Nolan, G. P., Bodenmiller, B. 2012; 81A (6): 467-475

  View details for DOI 10.1002/cyto.a.22067

  View details for Web of Science ID 000304258400008

• Decoupling of Tumor-Initiating Activity from Stable Immunophenotype in HoxA9-Meis1-Driven AML CELL STEM CELL Gibbs, K. D., Jager, A., Crespo, O., Goltsev, Y., Trejo, A., Richard, C. E., Nolan, G. P. 2012; 10 (2): 210-217

  Abstract

  Increasing evidence suggests tumors are maintained by cancer stem cells; however, their nature remains controversial. In a HoxA9-Meis1 (H9M) model of acute myeloid leukemia (AML), we found that tumor-initiating activity existed in three, immunophenotypically distinct compartments, corresponding to disparate lineages on the normal hematopoietic hierarchy--stem/progenitor cells (Lin(-)kit(+)) and committed progenitors of the myeloid (Gr1(+)kit(+)) and lymphoid lineages (Lym(+)kit(+)). These distinct tumor-initiating cells (TICs) clonally recapitulated the immunophenotypic spectrum of the original tumor in vivo (including cells with a less-differentiated immunophenotype) and shared signaling networks, such that in vivo pharmacologic targeting of conserved TIC survival pathways (DNA methyltransferase and MEK phosphorylation) significantly increased survival. Collectively, H9M AML is organized as an atypical hierarchy that defies the strict lineage marker boundaries and unidirectional differentiation of normal hematopoiesis. Moreover, this suggests that in certain malignancies tumor-initiation activity (or "cancer stemness") can represent a cellular state that exists independently of distinct immunophenotypic definition.

  View details for DOI 10.1016/j.stem.2012.01.004

  View details for Web of Science ID 000300272100013

  View details for PubMedID 22305570

• Single-cell phospho-protein analysis by flow cytometry. Current protocols in immunology / edited by John E. Coligan ... [et al.] Schulz, K. R., Danna, E. A., Krutzik, P. O., Nolan, G. P. 2012; Chapter 8: Unit 8 17 1-20

  Abstract

  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.

  View details for DOI 10.1002/0471142735.im0817s96

  View details for PubMedID 22314834

• Cytometry by Time-of-Flight Shows Combinatorial Cytokine Expression and Virus-Specific Cell Niches within a Continuum of CD8(+) T Cell Phenotypes IMMUNITY Newell, E. W., Sigal, N., Bendall, S. C., Nolan, G. P., Davis, M. M. 2012; 36 (1): 142-152

  Abstract

  Cytotoxic CD8(+) T lymphocytes directly kill infected or aberrant cells and secrete proinflammatory cytokines. By using metal-labeled probes and mass spectrometric analysis (cytometry by time-of-flight, or CyTOF) of human CD8(+) T cells, we analyzed the expression of many more proteins than previously possible with fluorescent labels, including surface markers, cytokines, and antigen specificity with modified peptide-MHC tetramers. With 3-dimensional principal component analysis (3D-PCA) to display phenotypic diversity, we observed a relatively uniform pattern of variation in all subjects tested, highlighting the interrelatedness of previously described subsets and the continuous nature of CD8(+) T cell differentiation. These data also showed much greater complexity in the CD8(+) T cell compartment than previously appreciated, including a nearly combinatorial pattern of cytokine expression, with distinct niches occupied by virus-specific cells. This large degree of functional diversity even between cells with the same specificity gives CD8(+) T cells a remarkable degree of flexibility in responding to pathogens.

  View details for DOI 10.1016/j.immuni.2012.01.002

  View details for Web of Science ID 000299766000017

  View details for PubMedID 22265676

• A novel splice donor mutation in the thrombopoietin gene leads to exon 2 skipping in a Filipino family with hereditary thrombocythemia. Blood Zhang, B., Ng, D., Jones, C., Oh, S. T., Nolan, G. P., Salehi, S., Wong, W., Zehnder, J. L., Gotlib, J. 2011; 118 (26): 6988-6990

  View details for DOI 10.1182/blood-2011-10-386177

  View details for PubMedID 22194398

• A novel splice donor mutation in the thrombopoietin gene leads to exon 2 skipping in a Filipino family with hereditary thrombocythemia BLOOD Ng, D., Jones, C., Oh, S. T., Nolan, G. P., Salehi, S., Wong, W., Zehnder, J. L., Gotlib, J. 2011; 118 (26): 6988-?

  View details for DOI 10.1182/blood-2011-10-386177

  View details for Web of Science ID 000298401000038

• Flow cytometry in the post fluorescence era BEST PRACTICE & RESEARCH CLINICAL HAEMATOLOGY Nolan, G. R. 2011; 24 (4): 505-508

  Abstract

  While flow cytometry once enabled researchers to examine 10--15 cell surface parameters, new mass flow cytometry technology enables interrogation of up to 45 parameters on a single cell. This new technology has increased understanding of cell expression and how cells differentiate during hematopoiesis. Using this information, knowledge of leukemia cell biology has also increased. Other new technologies, such as SPADE analysis and single cell network profiling (SCNP), are enabling researchers to put different cancers into more biologically similar categories and have the potential to enable more personalized medicine.

  View details for DOI 10.1016/j.beha.2011.09.005

  View details for Web of Science ID 000298462000003

  View details for PubMedID 22127312

• Signaling and Immunophenotypic Diversity in Pediatric Acute Myeloid Leukemia As Defined by 31-Parameter Single-Cell Mass Cytometry 53rd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Simonds, E. F., Bendall, S. C., Davis, K. L., Fienberg, H. G., Finck, R. L., Amir, E. D., Radtke, I., Downing, J. R., Pe'er, D., Nolan, G. P. AMER SOC HEMATOLOGY. 2011: 1100–1101

  View details for Web of Science ID 000299597103431

• Novel Hematopoietic Progenitor Populations Revealed by Direct Assessment of GATA1 Protein Expression and cMPL Signaling Events STEM CELLS Heffner, G. C., Clutter, M. R., Nolan, G. P., Weissman, I. L. 2011; 29 (11): 1774-1782

  Abstract

  Hematopoietic stem cells (HSCs) must exhibit tight regulation of both self-renewal and differentiation to maintain homeostasis of the hematopoietic system as well as to avoid aberrations in growth that may result in leukemias or other disorders. In this study, we sought to understand the molecular basis of lineage determination, with particular focus on factors that influence megakaryocyte/erythrocyte-lineage commitment, in hematopoietic stem and progenitor cells. We used intracellular flow cytometry to identify two novel hematopoietic progenitor populations within the mouse bone-marrow cKit(+) Lineage (-) Sca1(+) (KLS) Flk2 (+) compartment that differ in their protein-level expression of GATA1, a critical megakaryocyte/erythrocyte-promoting transcription factor. GATA1-high repopulating cells exhibited the cell surface phenotype KLS Flk2(+ to int), CD150(int), CD105(+), cMPL(+), and were termed "FSE cells." GATA1-low progenitors were identified as KLS Flk2(+), CD150(-), and cMPL(-), and were termed "Flk(+) CD150(-) cells." FSE cells had increased megakaryocyte/platelet potential in culture and transplant settings and exhibited a higher clonal frequency of colony-forming unit-spleen activity compared with Flk(+) CD150(-) cells, suggesting functional consequences of GATA1 upregulation in promoting megakaryocyte and erythroid lineage priming. Activation of ERK and AKT signal-transduction cascades was observed by intracellular flow cytometry in long-term HSCs and FSE cells, but not in Flk(+) CD150(-) cells in response to stimulation with thrombopoietin, an important megakaryocyte-promoting cytokine. We provide a mechanistic rationale for megakaryocyte/erythroid bias within KLS Flk2(+) cells, and demonstrate how assessment of intracellular factors and signaling events can be used to refine our understanding of lineage commitment during early definitive hematopoiesis.

  View details for DOI 10.1002/stem.719

  View details for Web of Science ID 000296565500014

  View details for PubMedID 21898686

• Association of Reactive Oxygen Species-Mediated Signal Transduction with In Vitro Apoptosis Sensitivity in Chronic Lymphocytic Leukemia B Cells PLOS ONE Palazzo, A. L., Evensen, E., Huang, Y., Cesano, A., Nolan, G. P., Fantl, W. J. 2011; 6 (10)

  Abstract

  Chronic lymphocytic leukemia (CLL) is a B cell malignancy with a variable clinical course and unpredictable response to therapeutic agents. Single cell network profiling (SCNP) utilizing flow cytometry measures alterations in signaling biology in the context of molecular changes occurring in malignancies. In this study SCNP was used to identify proteomic profiles associated with in vitro apoptotic responsiveness of CLL B cells to fludarabine, as a basis for ultimately linking these with clinical outcome.SCNP was used to quantify modulated-signaling of B cell receptor (BCR) network proteins and in vitro F-ara-A mediated apoptosis in 23 CLL samples. Of the modulators studied the reactive oxygen species, hydrogen peroxide (H₂O₂), a known intracellular second messenger and a general tyrosine phosphatase inhibitor stratified CLL samples into two sub-groups based on the percentage of B cells in a CLL sample with increased phosphorylation of BCR network proteins. Separately, in the same patient samples, in vitro exposure to F-ara-A also identified two sub-groups with B cells showing competence or refractoriness to apoptotic induction. Statistical analysis showed that in vitro F-ara-A apoptotic proficiency was highly associated with the proficiency of CLL B cells to undergo H₂O₂-augmented signaling.This linkage in CLL B cells among the mechanisms governing chemotherapy-induced apoptosis increased signaling of BCR network proteins and a likely role of phosphatase activity suggests a means of stratifying patients for their response to F-ara-A based regimens. Future studies will examine the clinical applicability of these findings and also the utility of this approach in relating mechanism to function of therapeutic agents.

  View details for DOI 10.1371/journal.pone.0024592

  View details for Web of Science ID 000295971700001

  View details for PubMedID 22016760

• Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE NATURE BIOTECHNOLOGY Qiu, P., Simonds, E. F., Bendall, S. C., Gibbs, K. D., Bruggner, R. V., Linderman, M. D., Sachs, K., Nolan, G. P., Plevritis, S. K. 2011; 29 (10): 886-U181

  Abstract

  The ability to analyze multiple single-cell parameters is critical for understanding cellular heterogeneity. Despite recent advances in measurement technology, methods for analyzing high-dimensional single-cell data are often subjective, labor intensive and require prior knowledge of the biological system. To objectively uncover cellular heterogeneity from single-cell measurements, we present a versatile computational approach, spanning-tree progression analysis of density-normalized events (SPADE). We applied SPADE to flow cytometry data of mouse bone marrow and to mass cytometry data of human bone marrow. In both cases, SPADE organized cells in a hierarchy of related phenotypes that partially recapitulated well-described patterns of hematopoiesis. We demonstrate that SPADE is robust to measurement noise and to the choice of cellular markers. SPADE facilitates the analysis of cellular heterogeneity, the identification of cell types and comparison of functional markers in response to perturbations.

  View details for DOI 10.1038/nbt.1991

  View details for Web of Science ID 000296273000015

  View details for PubMedID 21964415

  View details for PubMedCentralID PMC3196363

• A regression model approach to enable cell morphology correction in high-throughput flow cytometry MOLECULAR SYSTEMS BIOLOGY Knijnenburg, T. A., Roda, O., Wan, Y., Nolan, G. P., Aitchison, J. D., Shmulevich, I. 2011; 7

  Abstract

  Cells exposed to stimuli exhibit a wide range of responses ensuring phenotypic variability across the population. Such single cell behavior is often examined by flow cytometry; however, gating procedures typically employed to select a small subpopulation of cells with similar morphological characteristics make it difficult, even impossible, to quantitatively compare cells across a large variety of experimental conditions because these conditions can lead to profound morphological variations. To overcome these limitations, we developed a regression approach to correct for variability in fluorescence intensity due to differences in cell size and granularity without discarding any of the cells, which gating ipso facto does. This approach enables quantitative studies of cellular heterogeneity and transcriptional noise in high-throughput experiments involving thousands of samples. We used this approach to analyze a library of yeast knockout strains and reveal genes required for the population to establish a bimodal response to oleic acid induction. We identify a group of epigenetic regulators and nucleoporins that, by maintaining an 'unresponsive population,' may provide the population with the advantage of diversified bet hedging.

  View details for DOI 10.1038/msb.2011.64

  View details for Web of Science ID 000295578500003

  View details for PubMedID 21952134

• Structural Linkage between Ligand Discrimination and Receptor Activation by Type I Interferons CELL Thomas, C., Moraga, I., Levin, D., Krutzik, P. O., Podoplelova, Y., Trejo, A., Lee, C., Yarden, G., Vleck, S. E., Glenn, J. S., Nolan, G. P., Piehler, J., Schreiber, G., Garcia, K. C. 2011; 146 (4): 621-632

  Abstract

  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.

  View details for DOI 10.1016/j.cell.2011.06.048

  View details for Web of Science ID 000294043600016

  View details for PubMedID 21854986

  View details for PubMedCentralID PMC3166218

• Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum SCIENCE Bendall, S. C., Simonds, E. F., Qiu, P., Amir, E. D., Krutzik, P. O., Finck, R., Bruggner, R. V., Melamed, R., Trejo, A., Ornatsky, O. I., Balderas, R. S., Plevritis, S. K., Sachs, K., Pe'er, D., Tanner, S. D., Nolan, G. P. 2011; 332 (6030): 687-696

  Abstract

  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.

  View details for DOI 10.1126/science.1198704

  View details for Web of Science ID 000290265800035

  View details for PubMedID 21551058

  View details for PubMedCentralID PMC3273988

• Single-cell phospho-specific flow cytometric analysis demonstrates biochemical and functional heterogeneity in human hematopoietic stem and progenitor compartments BLOOD Gibbs, K. D., Gilbert, P. M., Sachs, K., Zhao, F., Blau, H. M., Weissman, I. L., Nolan, G. P., Majeti, R. 2011; 117 (16): 4226-4233

  Abstract

  The low frequency of hematopoietic stem and progenitor cells (HSPCs) in human BM has precluded analysis of the direct biochemical effects elicited by cytokines in these populations, and their functional consequences. Here, single-cell phospho-specific flow cytometry was used to define the signaling networks active in 5 previously defined human HSPC subsets. This analysis revealed that the currently defined HSC compartment is composed of biochemically distinct subsets with the ability to respond rapidly and directly in vitro to a broader array of cytokines than previously appreciated, including G-CSF. The G-CSF response was physiologically relevant-driving cell-cycle entry and increased proliferation in a subset of single cells within the HSC compartment. The heterogeneity in the single-cell signaling and proliferation responses prompted subfractionation of the adult BM HSC compartment by expression of CD114 (G-CSF receptor). Xenotransplantation assays revealed that HSC activity is significantly enriched in the CD114(neg/lo) compartment, and almost completely absent in the CD114(pos) subfraction. The single-cell analyses used here can be adapted for further refinement of HSPC surface immunophenotypes, and for examining the direct regulatory effects of other factors on the homeostasis of stem and progenitor populations in normal or diseased states.

  View details for DOI 10.1182/blood-2010-07-298232

  View details for Web of Science ID 000289807600012

  View details for PubMedID 21357764

• Cloud and heterogeneous computing solutions exist today for the emerging big data problems in biology NATURE REVIEWS GENETICS Schadt, E. E., Linderman, M. D., Sorenson, J., Lee, L., Nolan, G. P. 2011; 12 (3)

  View details for DOI 10.1038/nrg2857-c2

  View details for Web of Science ID 000287471500015

  View details for PubMedID 21301474

• Fluorescent cell barcoding for multiplex flow cytometry. Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] Krutzik, P. O., Clutter, M. R., Trejo, A., Nolan, G. P. 2011; Chapter 6: Unit 6 31-?

  Abstract

  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.

  View details for DOI 10.1002/0471142956.cy0631s55

  View details for PubMedID 21207359

• Phospho flow cytometry methods for the analysis of kinase signaling in cell lines and primary human blood samples. Methods in molecular biology (Clifton, N.J.) Krutzik, P. O., Trejo, A., Schulz, K. R., Nolan, G. P. 2011; 699: 179-202

  Abstract

  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.

  View details for DOI 10.1007/978-1-61737-950-5_9

  View details for PubMedID 21116984

• Phosphoproteomic Analysis Reveals Interconnected System-Wide Responses to Perturbations of Kinases and Phosphatases in Yeast SCIENCE SIGNALING Bodenmiller, B., Wanka, S., Kraft, C., Urban, J., Campbell, D., Pedrioli, P. G., Gerrits, B., Picotti, P., Lam, H., Vitek, O., Brusniak, M., Roschitzki, B., Zhang, C., Shokat, K. M., Schlapbach, R., Colman-Lerner, A., Nolan, G. P., Nesvizhskii, A. I., Peter, M., Loewith, R., von Mering, C., Aebersold, R. 2010; 3 (153)

  Abstract

  The phosphorylation and dephosphorylation of proteins by kinases and phosphatases constitute an essential regulatory network in eukaryotic cells. This network supports the flow of information from sensors through signaling systems to effector molecules and ultimately drives the phenotype and function of cells, tissues, and organisms. Dysregulation of this process has severe consequences and is one of the main factors in the emergence and progression of diseases, including cancer. Thus, major efforts have been invested in developing specific inhibitors that modulate the activity of individual kinases or phosphatases; however, it has been difficult to assess how such pharmacological interventions would affect the cellular signaling network as a whole. Here, we used label-free, quantitative phosphoproteomics in a systematically perturbed model organism (Saccharomyces cerevisiae) to determine the relationships between 97 kinases, 27 phosphatases, and more than 1000 phosphoproteins. We identified 8814 regulated phosphorylation events, describing the first system-wide protein phosphorylation network in vivo. Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery-and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis.

  View details for DOI 10.1126/scisignal.2001182

  View details for Web of Science ID 000285511300005

  View details for PubMedID 21177495

• Identification of a Novel Splice Donor Mutation In the Thrombopoietin Gene In a Philippine Family with Hereditary Thrombocythemia 52nd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Gotlib, J., Zhang, B., Jones, C. D., Riess, J., Wong, W. B., Simonds, E. F., Hale, M. B., Abidi, P., McClung, J., Nolan, G. P., Oh, S. T., Zehnder, J. L. AMER SOC HEMATOLOGY. 2010: 1272–72

  View details for Web of Science ID 000289662203418

• Oncogene Withdrawal Selectively Alters Phosphoprotein States and Shifts Differentiation Status In Myeloid Leukemia Subpopulations 52nd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Sachs, Z., Diaz-Flores, E., Diers, M. D., Sachs, K., Bendall, S. C., Fienberg, H. G., Rathe, S. K., Nolan, G. P., Shannon, K., Largaespada, D. AMER SOC HEMATOLOGY. 2010: 1300–1300

  View details for Web of Science ID 000289662203491

• Identification of Novel LNK Mutations In Patients with Chronic Myeloproliferative Neoplasms and Related Disorders 52nd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Oh, S. T., Zahn, J. M., Jones, C. D., Zhang, B., Loh, M. L., Kantarjian, H., Simonds, E. F., Bruggner, R. V., Abidi, P., Natsoulis, G., Bell, J., Buenrostro, J., Nolan, G. P., Zehnder, J. L., Ji, H. P., Gotlib, J. AMER SOC HEMATOLOGY. 2010: 143–44

  View details for Web of Science ID 000289662200316

• Clinical Translation of a Prognostic Follicular Lymphoma Signaling Profile 52nd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Irish, J. M., Czerwinski, D. K., Nolan, G. P., Levy, R. AMER SOC HEMATOLOGY. 2010: 279–79

  View details for Web of Science ID 000289662200637

• Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry. Cytometry. Part A : the journal of the International Society for Analytical Cytology Clutter, M. R., Heffner, G. C., Krutzik, P. O., Sachen, K. L., Nolan, G. P. 2010; 77 (11): 1020-1031

  Abstract

  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.

  View details for DOI 10.1002/cyto.a.20970

  View details for PubMedID 20824632

• Tyramide Signal Amplification for Analysis of Kinase Activity by Intracellular Flow Cytometry CYTOMETRY PART A Clutter, M. R., Heffner, G. C., Krutzik, P. O., Sachen, K. L., Nolan, G. P. 2010; 77A (11): 1020-1031

  View details for DOI 10.1002/cyto.a.20970

  View details for Web of Science ID 000284383400004

• Mammalian Target of Rapamycin Controls Dendritic Cell Development Downstream of Flt3 Ligand Signaling IMMUNITY Sathaliyawala, T., O'Gorman, W. E., Greter, M., Bogunovic, M., Konjufca, V., Hou, Z. E., Nolan, G. P., Miller, M. J., Merad, M., Reizis, B. 2010; 33 (4): 597-606

  Abstract

  Dendritic cells (DCs) comprise distinct functional subsets including CD8⁻ and CD8(+) classical DCs (cDCs) and interferon-secreting plasmacytoid DCs (pDCs). The cytokine Flt3 ligand (Flt3L) controls the development of DCs and is particularly important for the pDC and CD8(+) cDC and their CD103(+) tissue counterparts. We report that mammalian target of rapamycin (mTOR) inhibitor rapamycin impaired Flt3L-driven DC development in vitro, with the pDCs and CD8(+)-like cDCs most profoundly affected. Conversely, deletion of the phosphoinositide 3-kinase (PI3K)-mTOR negative regulator Pten facilitated Flt3L-driven DC development in culture. DC-specific Pten targeting in vivo caused the expansion of CD8(+) and CD103(+) cDC numbers, which was reversible by rapamycin. The increased CD8(+) cDC numbers caused by Pten deletion correlated with increased susceptibility to the intracellular pathogen Listeria. Thus, PI3K-mTOR signaling downstream of Flt3L controls DC development, and its restriction by Pten ensures optimal DC pool size and subset composition.

  View details for DOI 10.1016/j.immuni.2010.09.012

  View details for Web of Science ID 000284300200016

  View details for PubMedID 20933441

• Duration of antigen receptor signaling determines T-cell tolerance or activation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Katzman, S. D., O'Gorman, W. E., Villarino, A. V., Gallo, E., Friedman, R. S., Krummel, M. F., Nolan, G. P., Abbas, A. K. 2010; 107 (42): 18085-18090

  Abstract

  The early events that determine the decision between lymphocyte tolerance and activation are not well-understood. Using a model of systemic self-antigen recognition by CD4(+) T cells, we show, using single-cell biochemical analyses, that tolerance is characterized by transient signaling events downstream of T-cell receptor engagement in the mammalian target of rapamycin (mTOR) and NF-κB pathways. Parallel studies done by live cell imaging show that the key difference between tolerance and activation is the duration of the T cell-antigen presenting cell (APC) interaction, as revealed by stable T-cell immobilization on antigen encounter. Brief T cell-APC interactions result in tolerance, and prolonged interactions are associated with activation and the development of effector cells. These studies show that the duration of T cell-APC interactions and magnitude of associated TCR-mediated signaling are key determinants of lymphocyte tolerance vs. activation.

  View details for DOI 10.1073/pnas.1010560107

  View details for Web of Science ID 000283184800045

  View details for PubMedID 20921406

• Computational solutions to large-scale data management and analysis NATURE REVIEWS GENETICS Schadt, E. E., Linderman, M. D., Sorenson, J., Lee, L., Nolan, G. P. 2010; 11 (9): 647-657

  Abstract

  Today we can generate hundreds of gigabases of DNA and RNA sequencing data in a week for less than US$5,000. The astonishing rate of data generation by these low-cost, high-throughput technologies in genomics is being matched by that of other technologies, such as real-time imaging and mass spectrometry-based flow cytometry. Success in the life sciences will depend on our ability to properly interpret the large-scale, high-dimensional data sets that are generated by these technologies, which in turn requires us to adopt advances in informatics. Here we discuss how we can master the different types of computational environments that exist - such as cloud and heterogeneous computing - to successfully tackle our big data problems.

  View details for DOI 10.1038/nrg2857

  View details for Web of Science ID 000281115600013

  View details for PubMedID 20717155

  View details for PubMedCentralID PMC3124937

• Distinct Patterns of DNA Damage Response and Apoptosis Correlate with Jak/Stat and PI3Kinase Response Profiles in Human Acute Myelogenous Leukemia PLOS ONE Rosen, D. B., Putta, S., Covey, T., Huang, Y., Nolan, G. P., Cesano, A., Minden, M. D., Fantl, W. J. 2010; 5 (8)

  Abstract

  Single cell network profiling (SCNP) utilizing flow cytometry measures alterations in intracellular signaling responses. Here SCNP was used to characterize Acute Myeloid Leukemia (AML) disease subtypes based on survival, DNA damage response and apoptosis pathways.Thirty four diagnostic non-M3 AML samples from patients with known clinical outcome were treated with a panel of myeloid growth factors and cytokines, as well as with apoptosis-inducing agents. Analysis of induced Jak/Stat and PI3K pathway responses in blasts from individual patient samples identified subgroups with distinct signaling profiles that were not seen in the absence of a modulator. In vitro exposure of patient samples to etoposide, a DNA damaging agent, revealed three distinct "DNA damage response (DDR)/apoptosis" profiles: 1) AML blasts with a defective DDR and failure to undergo apoptosis; 2) AML blasts with proficient DDR and failure to undergo apoptosis; 3) AML blasts with proficiency in both DDR and apoptosis pathways. Notably, AML samples from clinical responders fell within the "DDR/apoptosis" proficient profile and, as well, had low PI3K and Jak/Stat signaling responses. In contrast, samples from clinical non responders had variable signaling profiles often with in vitro apoptotic failure and elevated PI3K pathway activity. Individual patient samples often harbored multiple, distinct, leukemia-associated cell populations identifiable by their surface marker expression, functional performance of signaling pathway in the face of cytokine or growth factor stimulation, as well as their response to apoptosis-inducing agents.Characterizing and tracking changes in intracellular pathway profiles in cell subpopulations both at baseline and under therapeutic pressure will likely have important clinical applications, potentially informing the selection of beneficial targeted agents, used either alone or in combination with chemotherapy.

  View details for DOI 10.1371/journal.pone.0012405

  View details for Web of Science ID 000281234700027

  View details for PubMedID 20811632

• Alternate Mechanisms of Initial Pattern Recognition Drive Differential Immune Responses to Related Poxviruses CELL HOST & MICROBE O'Gorman, W. E., Sampath, P., Simonds, E. F., Sikorski, R., O'Malley, M., Krutzik, P. O., Chen, H., Panchanathan, V., Chaudhri, G., Karupiah, G., Lewis, D. B., Thorne, S. H., Nolan, G. P. 2010; 8 (2): 174-185

  Abstract

  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.

  View details for DOI 10.1016/j.chom.2010.07.008

  View details for Web of Science ID 000281169600007

  View details for PubMedID 20709294

  View details for PubMedCentralID PMC2940993

• Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms BLOOD Oh, S. T., Simonds, E. F., Jones, C., Hale, M. B., Goltsev, Y., Gibbs, K. D., Merker, J. D., Zehnder, J. L., Nolan, G. P., Gotlib, J. 2010; 116 (6): 988-992

  Abstract

  Dysregulated Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling due to activation of tyrosine kinases is a common feature of myeloid malignancies. Here we report the first human disease-related mutations in the adaptor protein LNK, a negative regulator of JAK-STAT signaling, in 2 patients with JAK2 V617F-negative myeloproliferative neoplasms (MPNs). One patient exhibited a 5 base-pair deletion and missense mutation leading to a premature stop codon and loss of the pleckstrin homology (PH) and Src homology 2 (SH2) domains. A second patient had a missense mutation (E208Q) in the PH domain. BaF3-MPL cells transduced with these LNK mutants displayed augmented and sustained thrombopoietin-dependent growth and signaling. Primary samples from MPN patients bearing LNK mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34(+) early progenitors were abnormally abundant in both patients. These findings indicate that JAK-STAT activation due to loss of LNK negative feedback regulation is a novel mechanism of MPN pathogenesis.

  View details for DOI 10.1182/blood-2010-02-270108

  View details for Web of Science ID 000280881700021

  View details for PubMedID 20404132

  View details for PubMedCentralID PMC2924231

• New technologies for autoimmune disease monitoring CURRENT OPINION IN ENDOCRINOLOGY DIABETES AND OBESITY Maecker, H. T., Nolan, G. P., Fathman, C. G. 2010; 17 (4): 322-328

  Abstract

  This article will review new technologies used to characterize the immune phenotype of cells and serum for potential use in studies of autoimmunity.One area of recent development in studies of immune phenotyping is the contrast between cells of the immune system at rest and following activation. This simply involves comparing these cells at rest and following ligand-induced activation and measuring signaling system activation (phosphoepitope identification) or intracellular cytokine production or activation-induced gene expression. Preliminary data using these techniques have begun to identify signatures of disease (biomarkers) that are only seen when using these activation-induced assays. One of the most exciting new technologies, cytometry by time-of-flight mass spectrometry, couples a flow cytometer to a mass spectrometer, allowing many more parameters to be analyzed per cell, and without spillover between assay reagents, compared to conventional optical flow cytometry (heavy ions, mass, replaces fluorophore readout). Another new technology to analyze soluble proteins, bead-based immunoassays, can simultaneously measure up to 75 soluble analytes in a multiplexed array. Other technologies provide similar innovations in terms of analytical content, throughput, and miniaturization.We believe that new cellular genomic and protein-based technologies can provide key insights into autoimmune disease pathogenesis, progression, and therapy, and that these assays need to be applied in a systematic way to samples from patients with autoimmune diseases.

  View details for DOI 10.1097/MED.0b013e32833ada91

  View details for Web of Science ID 000285063800003

  View details for PubMedID 20531181

• B-cell signaling networks reveal a negative prognostic human lymphoma cell subset that emerges during tumor progression PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Irish, J. M., Myklebust, J. H., Alizadeh, A. A., Houot, R., Sharman, J. P., Czerwinski, D. K., Nolan, G. P., Levy, R. 2010; 107 (29): 12747-12754

  Abstract

  Human tumors contain populations of both cancerous and host immune cells whose malignant signaling interactions may define each patient's disease trajectory. We used multiplexed phospho-flow cytometry to profile single cells within human follicular lymphoma tumors and discovered a subpopulation of lymphoma cells with impaired B cell antigen receptor (BCR) signaling. The abundance of BCR-insensitive cells in each tumor negatively correlated with overall patient survival. These lymphoma negative prognostic (LNP) cells increased as tumors relapsed following chemotherapy. Loss of antigen receptor expression did not explain the absence of BCR signaling in LNP tumor cells, and other signaling responses were intact in these cells. Furthermore, BCR signaling responses could be reactivated in LNP cells, indicating that BCR signaling is not missing but rather specifically suppressed. LNP cells were also associated with changes to signaling interactions in the tumor microenvironment. Lower IL-7 signaling in tumor infiltrating T cells was observed in tumors with high LNP cell counts. The strength of signaling through T cell mediator of B cell function CD40 also stratified patient survival, particularly for those whose tumors contained few LNP cells. Thus, analysis of cell-cell interactions in heterogeneous primary tumors using signaling network profiles can identify and mechanistically define new populations of rare and clinically significant cells. Both the existence of these LNP cells and their aberrant signaling profiles provide targets for new therapies for follicular lymphoma.

  View details for DOI 10.1073/pnas.1002057107

  View details for Web of Science ID 000280144500010

  View details for PubMedID 20543139

  View details for PubMedCentralID PMC2919949

• Towards Program Optimization through Automated Analysis of Numerical Precision 8th International Symposium on Code Generation and Optimization Linderman, M. D., Ho, M., Dill, D. L., Meng, T. H., Nolan, G. P. IEEE COMPUTER SOC. 2010: 230–237

  View details for Web of Science ID 000286903200026

• Altered T Cell Antigen-specific Receptor Signaling in Tumor Infiltrating T Cells in Follicular Lymphomas 10th Annual Meeting of the Federation-of-Clinical-Immunology-Societies Hsu, F., Irish, J., Sachs, K., Nolan, G. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2010: S66–S66

  View details for DOI 10.1016/j.clim.2010.03.202

  View details for Web of Science ID 000277953700186

• Single Cell Phospho-Flow Analysis of Cytokine Stimulation in Human Hematopoietic Progenitors Reveals That G-CSF Acts Directly On Human Hematopoietic Stem Cells. 51st Annual Meeting and Exposition of the American-Society-of-Hematology Gibbs, K., Gilbert, P., Weissman, I. L., Blau, H. M., Nolan, G. P., Majeti, R. AMER SOC HEMATOLOGY. 2009: 1398–98

  View details for Web of Science ID 000272725804281

• Stage Dependent Aberrant Regulation of Cytokine-STAT Signaling in Murine Systemic Lupus Erythematosus PLOS ONE Hale, M. B., Krutzik, P. O., Samra, S. S., Crane, J. M., Nolan, G. P. 2009; 4 (8)

  Abstract

  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.

  View details for DOI 10.1371/journal.pone.0006756

  View details for Web of Science ID 000269278400021

  View details for PubMedID 19707593

• The Initial Phase of an Immune Response Functions to Activate Regulatory T Cells JOURNAL OF IMMUNOLOGY O'Gorman, W. E., Dooms, H., Thorne, S. H., Kuswanto, W. F., Simonds, E. F., Krutzik, P. O., Nolan, G. P., Abbas, A. K. 2009; 183 (1): 332-339

  Abstract

  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.

  View details for DOI 10.4049/jimmunol.0900691

  View details for Web of Science ID 000275119400037

  View details for PubMedID 19542444

• The T Cell STAT Signaling Network Is Reprogrammed within Hours of Bacteremia via Secondary Signals JOURNAL OF IMMUNOLOGY Hotson, A. N., Hardy, J. W., Hale, M. B., Contag, C. H., Nolan, G. P. 2009; 182 (12): 7558-7568

  Abstract

  The delicate balance between protective immunity and inflammatory disease is challenged during sepsis, a pathologic state characterized by aspects of both a hyperactive immune response and immunosuppression. The events driven by systemic infection by bacterial pathogens on the T cell signaling network that likely control these responses have not been illustrated in great detail. We characterized how intracellular signaling within the immune compartment is reprogrammed at the single cell level when the host is challenged with a high level of pathogen. To accomplish this, we applied flow cytometry to measure the phosphorylation potential of key signal transduction proteins during acute bacterial challenge. We modeled the onset of sepsis by i.v. administration of avirulent strains of Listeria monocytogenes and Escherichia coli to mice. Within 6 h of bacterial challenge, T cells were globally restricted in their ability to respond to specific cytokine stimulations as determined by assessing the extent of STAT protein phosphorylation. Mechanisms by which this negative feedback response occurred included SOCS1 and SOCS3 gene up-regulation and IL-6-induced endocystosis of the IL-6 receptor. Additionally, macrophages were partially tolerized in their ability to respond to TLR agonists. Thus, in contrast to the view that there is a wholesale immune activation during sepsis, one immediate host response to blood-borne bacteria was induction of a refractory period during which leukocyte activation by specific stimulations was attenuated.

  View details for DOI 10.4049/jimmunol.0803666

  View details for Web of Science ID 000266833900026

  View details for PubMedID 19494279

• A Novel Method for Detection of Phosphorylation in Single Cells by Surface Enhanced Raman Scattering (SERS) using Composite Organic-Inorganic Nanoparticles (COINs) PLOS ONE Shachaf, C. M., Elchuri, S. V., Koh, A. L., Zhu, J., Nguyen, L. N., Mitchell, D. J., Zhang, J., Swartz, K. B., Sun, L., Chan, S., Sinclair, R., Nolan, G. P. 2009; 4 (4)

  Abstract

  Detection of single cell epitopes has been a mainstay of immunophenotyping for over three decades, primarily using fluorescence techniques for quantitation. Fluorescence has broad overlapping spectra, limiting multiplexing abilities.To expand upon current detection systems, we developed a novel method for multi-color immuno-detection in single cells using "Composite Organic-Inorganic Nanoparticles" (COINs) Raman nanoparticles. COINs are Surface-Enhanced Raman Scattering (SERS) nanoparticles, with unique Raman spectra. To measure Raman spectra in single cells, we constructed an automated, compact, low noise and sensitive Raman microscopy device (Integrated Raman BioAnalyzer). Using this technology, we detected proteins expressed on the surface in single cells that distinguish T-cells among human blood cells. Finally, we measured intracellular phosphorylation of Stat1 (Y701) and Stat6 (Y641), with results comparable to flow cytometry.Thus, we have demonstrated the practicality of applying COIN nanoparticles for measuring intracellular phosphorylation, offering new possibilities to expand on the current fluorescent technology used for immunoassays in single cells.

  View details for DOI 10.1371/journal.pone.0005206

  View details for Web of Science ID 000265510400006

  View details for PubMedID 19367337

  View details for PubMedCentralID PMC2666268

• WebFlow: A Software Package for High-Throughput Analysis of Flow Cytometry Data ASSAY AND DRUG DEVELOPMENT TECHNOLOGIES Hammer, M. M., Kotecha, N., Irish, J. M., Nolan, G. P., Krutzik, P. O. 2009; 7 (1): 44-55

  Abstract

  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.

  View details for DOI 10.1089/adt.2008.174

  View details for Web of Science ID 000265635500003

  View details for PubMedID 19187010

• Learning Signaling Network Structures with Sparsely Distributed Data JOURNAL OF COMPUTATIONAL BIOLOGY Sachs, K., Itani, S., Carlisle, J., Nolan, G. P., Pe'er, D., Lauffenburger, D. A. 2009; 16 (2): 201-212

  Abstract

  Flow cytometric measurement of signaling protein abundances has proved particularly useful for elucidation of signaling pathway structure. The single cell nature of the data ensures a very large dataset size, providing a statistically robust dataset for structure learning. Moreover, the approach is easily scaled to many conditions in high throughput. However, the technology suffers from a dimensionality constraint: at the cutting edge, only about 12 protein species can be measured per cell, far from sufficient for most signaling pathways. Because the structure learning algorithm (in practice) requires that all variables be measured together simultaneously, this restricts structure learning to the number of variables that constitute the flow cytometer's upper dimensionality limit. To address this problem, we present here an algorithm that enables structure learning for sparsely distributed data, allowing structure learning beyond the measurement technology's upper dimensionality limit for simultaneously measurable variables. The algorithm assesses pairwise (or n-wise) dependencies, constructs "Markov neighborhoods" for each variable based on these dependencies, measures each variable in the context of its neighborhood, and performs structure learning using a constrained search.

  View details for DOI 10.1089/cmb.2008.07TT

  View details for Web of Science ID 000263057400007

  View details for PubMedID 19193145

• Characterization of Patient Specific Signaling via Augmentation of Bayesian Networks with Disease and Patient State Nodes Annual International Conference of the IEEE-Engineering-in-Medicine-and-Biology-Society Sachs, K., Gentles, A. J., Youland, R., Itani, S., Irish, J., Nolan, G. P., Plevritis, S. K. IEEE. 2009: 6624–6627

  Abstract

  Characterization of patient-specific disease features at a molecular level is an important emerging field. Patients may be characterized by differences in the level and activity of relevant biomolecules in diseased cells. When high throughput, high dimensional data is available, it becomes possible to characterize differences not only in the level of the biomolecules, but also in the molecular interactions among them. We propose here a novel approach to characterize patient specific signaling, which augments high throughput single cell data with state nodes corresponding to patient and disease states, and learns a Bayesian network based on this data. Features distinguishing individual patients emerge as downstream nodes in the network. We illustrate this approach with a six phospho-protein, 30,000 cell-per-patient dataset characterizing three comparably diagnosed follicular lymphoma, and show that our approach elucidates signaling differences among them.

  View details for Web of Science ID 000280543605113

  View details for PubMedID 19963681

  View details for PubMedCentralID PMC3124088

• LEARNING CYCLIC SIGNALING PATHWAY STRUCTURES WHILE MINIMIZING DATA REQUIREMENTS Pacific Symposium on Biocomputing Sachs, K., Itani, S., Fitzgerald, J., Wille, L., Schoeberl, B., Dahleh, M. A., Nolan, G. P. WORLD SCIENTIFIC PUBL CO PTE LTD. 2009: 63–74

  Abstract

  Bayesian network structure learning is a useful tool for elucidation of regulatory structures of biomolecular pathways. The approach however is limited by its acyclicity constraint, a problematic one in the cycle-containing biological domain. Here, we introduce a novel method for modeling cyclic pathways in biology, by employing our newly introduced Generalized Bayesian Networks (GBNs). Our novel algorithm enables cyclic structure learning while employing biologically relevant data, as it extends our cycle-learning algorithm to permit learning with singly perturbed samples. We present theoretical arguments as well as structure learning results from realistic, simulated data of a biological system. We also present results from a real world dataset, involving signaling pathways in T-cells.

  View details for Web of Science ID 000263639700007

  View details for PubMedID 19209696

• Conditional protein stabilization via the small molecules Shld-1 and rapamycin increases the signal-to-noise ratio with tet-inducible gene expression BIOTECHNIQUES Almogy, G., Nolan, G. P. 2009; 46 (1): 44-50

  Abstract

  Cellular mechanisms control one or more of the three basic levels of regulation (transcription, translation, and protein activity/locality), allowing for finely tuned spatial and temporal regulation of protein expression patterns. Gene regulation constructs in wide use today often employ a constitutively expressed transcription factor whose activity is determined by the presence or absence of a small molecule. A case in point is the tet transcription system, wherein transcription is regulated by doxycycline (Dox), allowing the researcher to turn protein expression on or off depending on the presence/absence of Dox. However in many applications of that system, there is basal transcription from the promoter element that is independent of Dox. Moreover, in vivo, heterogeneous distribution of Dox leads to concurrent differences in gene expression. We addressed these limitations by introducing conditional destabilizing elements to the system. First, we created a transactivator protein fusion regulated at the additional level of protein stability. This modification enabled a system that demonstrated an off state that is less sensitive to variations in Dox concentrations. We also regulated the stability of the protein expressed from the tet operator cassette, observing greatly improved signal-to-noise ratios. The results underscore how investigator-defined regulation at multiple levels of protein expression can attain afiner degree of control over the final expression of introduced genes.

  View details for DOI 10.2144/000113030

  View details for Web of Science ID 000263637300004

  View details for PubMedID 19301621

• Electron microscopy localization and characterization of functionalized composite organic-inorganic SERS nanoparticles on leukemia cells ULTRAMICROSCOPY Koh, A. L., Shachaf, C. M., Elchuri, S., Nolan, G. P., Sinclair, R. 2008; 109 (1): 111-121

  Abstract

  We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic nanoparticle (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet scanning electron microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron (BSE) detector was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution transmission electron microscopy (TEM) images and scanning Auger electron spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens.

  View details for DOI 10.1016/j.ultramic.2008.09.004

  View details for Web of Science ID 000261750500016

  View details for PubMedID 18995965

• Signaling Diversity in Human Lymphoma B Cells and in Tumor Infiltrating T Cells Correlates with Follicular Lymphoma Patient Clinical Outcomes 50th Annual Meeting of the American-Society-of-Hematology/ASH/ASCO Joint Symposium Irish, J. M., Houot, R., Myklebust, J. H., Czerwinski, D. K., Nolan, G. P., Levy, R. AMER SOC HEMATOLOGY. 2008: 146–46

  View details for Web of Science ID 000262104700378

• Single-cell profiling identifies aberrant STAT5 activation in myeloid malignancies with specific clinical and biologic correlates CANCER CELL Kotecha, N., Floress, N. J., Irish, J. M., Simonds, E. F., Sakai, D. S., Archambeault, S., Diaz-Flores, E., Coram, M., Shannon, K. M., Nolan, G. P., Loh, M. L. 2008; 14 (4): 335-343

  Abstract

  Progress in understanding the molecular pathogenesis of human myeloproliferative disorders (MPDs) has led to guidelines incorporating genetic assays with histopathology during diagnosis. Advances in flow cytometry have made it possible to simultaneously measure cell type and signaling abnormalities arising as a consequence of genetic pathologies. Using flow cytometry, we observed a specific evoked STAT5 signaling signature in a subset of samples from patients suspected of having juvenile myelomonocytic leukemia (JMML), an aggressive MPD with a challenging clinical presentation during active disease. This signature was a specific feature involving JAK-STAT signaling, suggesting a critical role of this pathway in the biological mechanism of this disorder and indicating potential targets for future therapies.

  View details for DOI 10.1016/j.ccr.2008.08.014

  View details for Web of Science ID 000259896500008

  View details for PubMedID 18835035

  View details for PubMedCentralID PMC2647559

• MIFlowCyt: The Minimum Information about a Flow Cytometry Experiment CYTOMETRY PART A Lee, J. A., Spidlen, J., Boyce, K., Cai, J., Crosbie, N., Dalphin, M., Furlong, J., Gasparetto, M., Goldberg, M., Goralczyk, E. M., Hyun, B., Jansen, K., Kollmann, T., Kong, M., Leif, R., McWeeney, S., Moloshok, T. D., Moore, W., Nolan, G., Nolan, J., Nikolich-Zugich, J., Parrish, D., Purcell, B., Qian, Y., Selvaraj, B., Smith, C., Tchuvatkina, O., Wertheimer, A., Wilkinson, P., Wilson, C., Wood, J., Zigon, R., Scheuermann, R. H., Brinkman, R. R. 2008; 73A (10): 926-930

  Abstract

  A fundamental tenet of scientific research is that published results are open to independent validation and refutation. Minimum data standards aid data providers, users, and publishers by providing a specification of what is required to unambiguously interpret experimental findings. Here, we present the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, stating the minimum information required to report flow cytometry (FCM) experiments. We brought together a cross-disciplinary international collaborative group of bioinformaticians, computational statisticians, software developers, instrument manufacturers, and clinical and basic research scientists to develop the standard. The standard was subsequently vetted by the International Society for Advancement of Cytometry (ISAC) Data Standards Task Force, Standards Committee, membership, and Council. The MIFlowCyt standard includes recommendations about descriptions of the specimens and reagents included in the FCM experiment, the configuration of the instrument used to perform the assays, and the data processing approaches used to interpret the primary output data. MIFlowCyt has been adopted as a standard by ISAC, representing the FCM scientific community including scientists as well as software and hardware manufacturers. Adoptionof MIFlowCyt by the scientific and publishing communities will facilitate third-party understanding and reuse of FCM data.

  View details for DOI 10.1002/cyto.a.20623

  View details for Web of Science ID 000259750300008

  View details for PubMedCentralID PMC2773297

• MIFlowCyt: the minimum information about a Flow Cytometry Experiment. Cytometry. Part A : the journal of the International Society for Analytical Cytology Lee, J. A., Spidlen, J., Boyce, K., Cai, J., Crosbie, N., Dalphin, M., Furlong, J., Gasparetto, M., Goldberg, M., Goralczyk, E. M., Hyun, B., Jansen, K., Kollmann, T., Kong, M., Leif, R., McWeeney, S., Moloshok, T. D., Moore, W., Nolan, G., Nolan, J., Nikolich-Zugich, J., Parrish, D., Purcell, B., Qian, Y., Selvaraj, B., Smith, C., Tchuvatkina, O., Wertheimer, A., Wilkinson, P., Wilson, C., Wood, J., Zigon, R., Scheuermann, R. H., Brinkman, R. R. 2008; 73 (10): 926-930

  Abstract

  A fundamental tenet of scientific research is that published results are open to independent validation and refutation. Minimum data standards aid data providers, users, and publishers by providing a specification of what is required to unambiguously interpret experimental findings. Here, we present the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, stating the minimum information required to report flow cytometry (FCM) experiments. We brought together a cross-disciplinary international collaborative group of bioinformaticians, computational statisticians, software developers, instrument manufacturers, and clinical and basic research scientists to develop the standard. The standard was subsequently vetted by the International Society for Advancement of Cytometry (ISAC) Data Standards Task Force, Standards Committee, membership, and Council. The MIFlowCyt standard includes recommendations about descriptions of the specimens and reagents included in the FCM experiment, the configuration of the instrument used to perform the assays, and the data processing approaches used to interpret the primary output data. MIFlowCyt has been adopted as a standard by ISAC, representing the FCM scientific community including scientists as well as software and hardware manufacturers. Adoptionof MIFlowCyt by the scientific and publishing communities will facilitate third-party understanding and reuse of FCM data.

  View details for DOI 10.1002/cyto.a.20623

  View details for PubMedID 18752282

• Genomic and proteomic analysis reveals a threshold level of MYC required for tumor maintenance CANCER RESEARCH Shachaf, C. M., Gentles, A. J., Elchuri, S., Sahoo, D., Soen, Y., Sharpe, O., Perez, O. D., Chang, M., Mitchel, D., Robinson, W. H., Dill, D., Nolan, G. P., Plevritis, S. K., Felsher, D. W. 2008; 68 (13): 5132-5142

  Abstract

  MYC overexpression has been implicated in the pathogenesis of most types of human cancers. MYC is likely to contribute to tumorigenesis by its effects on global gene expression. Previously, we have shown that the loss of MYC overexpression is sufficient to reverse tumorigenesis. Here, we show that there is a precise threshold level of MYC expression required for maintaining the tumor phenotype, whereupon there is a switch from a gene expression program of proliferation to a state of proliferative arrest and apoptosis. Oligonucleotide microarray analysis and quantitative PCR were used to identify changes in expression in 3,921 genes, of which 2,348 were down-regulated and 1,573 were up-regulated. Critical changes in gene expression occurred at or near the MYC threshold, including genes implicated in the regulation of the G(1)-S and G(2)-M cell cycle checkpoints and death receptor/apoptosis signaling. Using two-dimensional protein analysis followed by mass spectrometry, phospho-flow fluorescence-activated cell sorting, and antibody arrays, we also identified changes at the protein level that contributed to MYC-dependent tumor regression. Proteins involved in mRNA translation decreased below threshold levels of MYC. Thus, at the MYC threshold, there is a loss of its ability to maintain tumorigenesis, with associated shifts in gene and protein expression that reestablish cell cycle checkpoints, halt protein translation, and promote apoptosis.

  View details for DOI 10.1158/0008-5472.CAN-07-6192

  View details for Web of Science ID 000257415300024

  View details for PubMedID 18593912

• Single-cell, phosphoepitope-specific analysis demonstrates cell type- and pathway-specific dysregulation of Jak/STAT and MAPK signaling associated with in vivo human immunodeficiency virus type 1 infection JOURNAL OF VIROLOGY Lee, A. W., Sharp, E. R., O'Mahony, A., Rosenberg, M. G., Israelski, D. M., Nolan, G. P., Nixon, D. F. 2008; 82 (7): 3702-3712

  Abstract

  Despite extensive evidence of cell signaling alterations induced by human immunodeficiency virus type 1 (HIV-1) in vitro, the relevance of these changes to the clinical and/or immunologic status of HIV-1-infected individuals is often unclear. As such, mapping the details of cell type-specific degradation of immune function as a consequence of changes to signaling network responses has not been readily accessible. We used a flow cytometric-based assay of signaling to determine Janus kinase/signal transducers and activators of transcription (Jak/STAT) signaling changes at the single-cell level within distinct cell subsets from the primary immune cells of HIV-1-infected donors. We identified a specific defect in granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven Stat5 phosphorylation in the monocytes of HIV-1+ donors. This inhibition was statistically significant in a cohort of treated and untreated individuals. Ex vivo Stat5 phosphorylation levels varied among HIV-1+ donors but did not correlate with CD4(+) T-cell counts or HIV-1 plasma viral load. Low Stat5 activation occurred in HIV-1-infected donors despite normal GM-CSF receptor levels. Investigation of mitogen-activated protein kinase (MAPK) pathways, also stimulated by GM-CSF, led to the observation that lipopolysaccharide-stimulated extracellular signal-regulated kinase phosphorylation is enhanced in monocytes. Thus, we have identified a specific, imbalanced monocyte signaling profile, with inhibition of STAT and enhancement of MAPK signaling, associated with HIV-1 infection. This understanding of altered monocyte signaling responses that contribute to defective antigen presentation during HIV-1 infection could lead to immunotherapeutic approaches that compensate for the deficiency.

  View details for DOI 10.1128/JVI.01582-07

  View details for Web of Science ID 000254139800049

  View details for PubMedID 18216116

  View details for PubMedCentralID PMC2268489

• High-content single-cell drug screening with phosphospecific flow cytometry NATURE CHEMICAL BIOLOGY Krutzik, P. O., Crane, J. M., Clutter, M. R., Nolan, G. P. 2008; 4 (2): 132-142

  Abstract

  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.

  View details for DOI 10.1038/nchembio.2007.59

  View details for Web of Science ID 000252585400013

  View details for PubMedID 18157122

• Structure learning for biomolecular pathways containing cycles 8th IEEE International Conference on Bioinformatics and Bioengineering Itani, S., Sachs, K., Nolan, G. P., Dahleh, M. A. IEEE. 2008: 486–491

  View details for Web of Science ID 000263828500081

• A profile of 648 signaling network events identifies cell subsets with diverse, abnormal responses to lymphocyte stimuli within follicular lymphoma tumors 49th Annual Meeting of the American-Society-of-Hematology Irish, J. M., Hsu, F. Y., Sharman, J. P., Houot, R., Brody, J. D., Czerwinski, D. K., Nolan, G. P., Levy, R. AMER SOC HEMATOLOGY. 2007: 111A–112A

  View details for Web of Science ID 000251100800357

• A novel assay for juvenile myelomonocytic leukemia based on aberrant signaling networks measured via phospho-specific flow cytometry reduces diagnosis time from weeks to days. 49th Annual Meeting of the American-Society-of-Hematology Kotecha, N., Flores, N. J., Irish, J. M., Sakaguchi, D., Archambeault, S., Diaz-Flores, E., Coram, M., Shannon, K. M., Nolan, G. P., Loh, M. L. AMER SOC HEMATOLOGY. 2007: 168A–168A

  View details for Web of Science ID 000251100800547

• Inhibition of HMGcoA reductase by atorvastatin prevents and reverses MYC-induced lymphomagenesis BLOOD Shachaf, C. M., Perez, O. D., Youssef, S., Fan, A. C., Elchuri, S., Goldstein, M. J., Shirer, A. E., Sharpe, O., Chen, J., Mitchell, D. J., Chang, M., Nolan, G. P., Steinman, L., Felsher, D. W. 2007; 110 (7): 2674-2684

  Abstract

  Statins are a class of drugs that inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMGcoA) reductase, a critical enzyme in the mevalonate pathway. Several reports document that statins may prevent different human cancers. However, whether or not statins can prevent cancer is controversial due to discordant results. One possible explanation for these conflicting conclusions is that only some tumors or specific statins may be effective. Here, we demonstrate in an in vivo transgenic model in which atorvastatin reverses and prevents the onset of MYC-induced lymphomagenesis, but fails to reverse or prevent tumorigenesis in the presence of constitutively activated K-Ras (G12D). Using phosphoprotein fluorescence-activated cell sorter (FACS) analysis, atorvastatin treatment was found to result in the inactivation of the Ras and ERK1/2 signaling pathways associated with the dephosphorylation and inactivation of MYC. Correspondingly, tumors with a constitutively activated K-Ras (G12D) did not exhibit dephosphorylation of ERK1/2 and MYC. Atorvastatin's effects on MYC were specific to the inhibition of HMGcoA reductase, as treatment with mevalonate, the product of HMG-CoA reductase activity, abrogated these effects and inhibited the ability of atorvastatin to reverse or suppress tumorigenesis. Also, RNAi directed at HMGcoA reductase was sufficient to abrogate the neoplastic properties of MYC-induced tumors. Thus, atorvastatin, by inhibiting HMGcoA reductase, induces changes in phosphoprotein signaling that in turn prevent MYC-induced lymphomagenesis.

  View details for DOI 10.1182/blood-2006-09-048033

  View details for Web of Science ID 000249800900069

  View details for PubMedID 17622571

• Toward understanding the molecular mechanisms of lineage determination in hematopoietic stem cells 36th Annual Meeting of the International-Society-for-Experimental-Hematology Heffner, G. C., Clutter, M. R., Nolan, G. P., Weissman, I. L. ELSEVIER SCIENCE INC. 2007: 19–19

  View details for Web of Science ID 000249535200043

• K-Ras(G12D) expression induces hyperproliferation and aberrant signaling in primary hematopoietic stem/progenitor cells BLOOD Van Meter, M. E., Diaz-Flores, E., Archard, J. A., Passegue, E., Irish, J. M., Kotecha, N., Nolan, G. P., Shannon, K., Braun, B. S. 2007; 109 (9): 3945-3952

  Abstract

  Defining how cancer-associated mutations perturb signaling networks in stem/progenitor populations that are integral to tumor formation and maintenance is a fundamental problem with biologic and clinical implications. Point mutations in RAS genes contribute to many cancers, including myeloid malignancies. We investigated the effects of an oncogenic Kras(G12D) allele on phosphorylated signaling molecules in primary c-kit(+) lin(-/low) hematopoietic stem/progenitor cells. Comparison of wild-type and Kras(G12D) c-kit(+) lin(-/low) cells shows that K-Ras(G12D) expression causes hyperproliferation in vivo and results in abnormal levels of phosphorylated STAT5, ERK, and S6 under basal and stimulated conditions. Whereas Kras(G12D) cells demonstrate hyperactive signaling after exposure to granulocyte-macrophage colony-stimulating factor, we unexpectedly observe a paradoxical attenuation of ERK and S6 phosphorylation in response to stem cell factor. These studies provide direct biochemical evidence that cancer stem/progenitor cells remodel signaling networks in response to oncogenic stress and demonstrate that multi-parameter flow cytometry can be used to monitor the effects of targeted therapeutics in vivo. This strategy has broad implications for defining the architecture of signaling networks in primary cancer cells and for implementing stem cell-targeted interventions.

  View details for DOI 10.1182/blood-2006-09-047530

  View details for Web of Science ID 000246091400052

  View details for PubMedID 17192389

• What's wrong with drug screening today NATURE CHEMICAL BIOLOGY Nolan, G. P. 2007; 3 (4): 187-191

  Abstract

  Drug screening in the immediate term will be best accomplished by early use of primary cells in which the target of the screen is a network of proteins measured in populations of single cells.

  View details for Web of Science ID 000245103000002

  View details for PubMedID 17372598

• Flt3 Y591 duplication and Bc1-2 overexpression are detected in acute myeloid leukemia cells with high levels of phosphorylated wild-type p53 BLOOD Irish, J. M., Anensen, N., Hovland, R., Skavland, J., Borresen-Dale, A., Bruserud, O., Nolan, G. P., Gjertsen, B. T. 2007; 109 (6): 2589-2596

  Abstract

  Loss or mutation of the TP53 tumor suppressor gene is not commonly observed in acute myeloid leukemia (AML), suggesting that there is an alternate route for cell transformation. We investigated the hypothesis that previously observed Bcl-2 family member overexpression suppresses wild-type p53 activity in AML. We demonstrate that wild-type p53 protein is expressed in primary leukemic blasts from patients with de novo AML using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and phospho-specific flow cytometry. We found that p53 was heterogeneously expressed and phosphorylated in AML patient samples and could accumulate following DNA damage. Overexpression of antiapoptosis protein Bcl-2 in AML cells was directly correlated with p53 expression and phosphorylation on serine residues 15, 46, and 392. Within those patients with the highest levels of Bcl-2 expression, we identified a mutation in FLT3 that duplicated phosphorylation site Y591. The presence of this mutation correlated with greater than normal Bcl-2 expression and with previously observed profiles of potentiated STAT and MAPK signaling. These results support the hypothesis that Flt3-mediated signaling in AML enables accumulation of Bcl-2 and maintains a downstream block to p53 pathway apoptosis. Bcl-2 inhibition might therefore improve the efficacy of existing AML therapies by inactivating this suppression of wild-type p53 activity.

  View details for DOI 10.1182/blood-2006-02-004234

  View details for Web of Science ID 000245004700050

  View details for PubMedID 17105820

• RACK1 regulates Ki-Ras-mediated signaling and morphological transformation of NIH 3T3 cells INTERNATIONAL JOURNAL OF CANCER Bjorndal, B., Myklebust, L. M., Rosendal, K. R., Myromslien, F. D., Lorens, J. B., Nolan, G., Bruland, O., Lillehaug, J. R. 2007; 120 (5): 961-969

  Abstract

  Activating Ras mutations are involved in a significant fraction of human tumors. A suppressor screen using a retroviral mouse fibroblast cDNA library was performed to identify novel factors in Ras-mediated transformation. We identified a novel potent inhibitor of Ras-mediated morphological transformation encoded by a truncated version of the receptor for activated C-kinase (RACK1). The truncated protein, designated RACK1DeltaWD1, lacked the N-terminal 49 amino acids encoding the first of the 7 WD40 repeats in RACK1. RACK1DeltaWD1 expression restored contact inhibition, stress fiber formation and reduced ERK phosphorylation in Ki-Ras transformed NIH 3T3 cells. We demonstrate that truncated RACK1 is involved in complexes consisting of wild-type RACK1 and protein kinase C isoforms alpha, betaI and delta, compromising the transduction of an activated Ras signal to the Raf-MEK-ERK pathway. The cellular localization of RACK1DeltaWD1 differed from wtRACK1, indicating that signaling complexes containing the truncated version of RACK1 are incorrectly localized. Notably, 12-O-tetradecanoyl-13-phorbol acetate (TPA) mediated intracellular translocation of RACK1-interacting PKC alpha and delta was abrogated in RACK1DeltaWD1-expressing cells. Our data support a model where RACK1 acts as a key factor in Ki-Ras-mediated morphological transformation.

  View details for DOI 10.1002/ijc.22373

  View details for Web of Science ID 000243892700002

  View details for PubMedID 17149700

• The ERK mitogen-activated protein kinase pathway contributes to Ebola virus glycoprotein-induced cytotoxicity JOURNAL OF VIROLOGY Zampieri, C. A., Fortin, J., Nolan, G. P., Nabel, G. J. 2007; 81 (3): 1230-1240

  Abstract

  Ebola virus is a highly lethal pathogen that causes hemorrhagic fever in humans and nonhuman primates. Among the seven known viral gene products, the envelope glycoprotein (GP) alone induces cell rounding and detachment that ultimately leads to cell death. Cellular cytoxicity is not seen with comparable levels of expression of a mutant form of GP lacking a mucin-like domain (GPDeltamuc). GP-induced cell death is nonapoptotic and is preceded by downmodulation of cell surface molecules involved in signaling pathways, including certain integrins and epidermal growth factor receptor. To investigate the mechanism of GP-induced cellular toxicity, we analyzed the activation of several signal transduction pathways involved in cell growth and survival. The active form of extracellular signal-regulated kinases types 1 and 2 (ERK1/2), phospho-ERK1/2, was reduced in cells expressing GP compared to those expressing GPDeltamuc as determined by flow cytometry, in contrast to the case for several other signaling proteins. Subsequent analysis of the activation states and kinase activities of related kinases revealed a more pronounced effect on the ERK2 kinase isoform. Disruption of ERK2 activity by a dominant negative ERK or by small interfering RNA-mediated ERK2 knockdown potentiated the decrease in alphaV integrin expression associated with toxicity. Conversely, activation of the pathway through the expression of a constitutively active form of ERK2 significantly protected against this effect. These results indicate that the ERK signaling cascade mediates GP-mediated cytotoxicity and plays a role in pathogenicity induced by this gene product.

  View details for DOI 10.1128/JVI.01586-06

  View details for Web of Science ID 000243766800017

  View details for PubMedID 17108034

• Differential role of ICAM ligands in determination of human memory T cell differentiation BMC IMMUNOLOGY Perez, O. D., Mitchell, D., Nolan, G. P. 2007; 8

  Abstract

  Leukocyte Function Antigen-1 (LFA-1) is a primary adhesion molecule that plays important roles in T cell activation, leukocyte recirculation, and trans-endothelial migration. By applying a multivariate intracellular phospho-proteomic analysis, we demonstrate that LFA-1 differentially activates signaling molecules.Signal intensity was dependent on both ICAM ligand and LFA-1 concentration. In the presence of CD3 and CD28 stimulation, ICAM-2 and ICAM-3 decreased TGFbeta1 production more than ICAM-1. In long-term differentiation experiments, stimulation with ICAM-3, CD3, and CD28 generated IFNgamma producing CD4+CD45RO+CD62L-CD11aBrightCD27- cells that had increased expression of intracellular BCL2, displayed distinct chemokine receptor profiles, and exhibited distinct migratory characteristics. Only CD3/CD28 with ICAM-3 generated CD4+CD45RO+CD62L-CD11aBrightCD27- cells that were functionally responsive to chemotaxis and exhibited higher frequencies of cells that signaled to JNK and ERK1/2 upon stimulation with MIP3alpha. Furthermore, these reports identify that the LFA-1 receptor, when presented with multiple ligands, can result in distinct T cell differentiation states and suggest that the combinatorial integration of ICAM ligand interactions with LFA-1 have functional consequences for T cell biology.Thus, the ICAM ligands, differentially modulate LFA-1 signaling in T cells and potentiate the development of memory human T cells in vitro. These findings are of importance in a mechanistic understanding of memory cell differentiation and ex vivo generation of memory cell subsets for therapeutic applications.

  View details for DOI 10.1186/1471-2172-8-2

  View details for Web of Science ID 000244057800001

  View details for PubMedID 17233909

• Visualizing inside the cell BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Nolan, G. 2007; 13 (1): 120-123

  View details for DOI 10.1016/j.bbmt.2006.11.010

  View details for Web of Science ID 000244043800025

• Altered B-cell receptor signaling kinetics distinguish human follicular lymphoma. B cells from tumor-infiltrating nonmalignant B cells BLOOD Irish, J. M., Czerwinski, D. K., Nolan, G. P., Levy, R. 2006; 108 (9): 3135-3142

  Abstract

  The B-cell receptor (BCR) transmits life and death signals throughout B-cell development, and altered BCR signaling may be required for survival of B-lymphoma cells. We used single-cell signaling profiles to compare follicular lymphoma (FL) B cells and nonmalignant host B cells within individual patient biopsies and identified BCR-mediated signaling events specific to lymphoma B cells. Expression of CD20, Bcl-2, and BCR light chain isotype (kappa or lambda) distinguished FL tumor B-cell and nontumor host B-cell subsets within FL patient biopsies. BCR-mediated signaling via phosphorylation of Btk, Syk, Erk1/2, and p38 occurred more rapidly in tumor B cells from FL samples than in infiltrating nontumor B cells, achieved greater levels of per-cell signaling, and sustained this level of signaling for hours longer than nontumor B cells. The timing and magnitude of BCR-mediated signaling in nontumor B cells within an FL sample instead resembled that observed in mature B cells from the peripheral blood of healthy subjects. BCR signaling pathways that are potentiated specifically in lymphoma cells should provide new targets for therapeutic attention.

  View details for DOI 10.1182/blood-2006-02-003921

  View details for Web of Science ID 000241586100043

  View details for PubMedID 16835385

• Kinetics of B cell receptor signaling in human B cell subsets mapped by phosphospecific flow cytometry JOURNAL OF IMMUNOLOGY Irish, J. M., Czerwinski, D. K., Nolan, G. P., Levy, R. 2006; 177 (3): 1581-1589

  Abstract

  Differences in BCR signaling may govern outcomes as diverse as proliferation and cell death. We profiled BCR signaling kinetics in subsets of primary human B cells using flow cytometry. In the predominant population expressing IgM, BCR cross-linking led to a quick burst of Syk, ERK1/2, and p38 signaling. In contrast, IgG B cells sustained higher per-cell ERK1/2 phosphorylation over time. This dichotomy suggested a mechanism for dampening signals transmitted by IgM. Regulatory phosphatase activity in IgM B cells was BCR-mediated and initiated more slowly than kinase activity. This BCR-mediated phosphatase activity was sensitive to inhibition by H(2)O(2) and required to attenuate IgM BCR signaling. These results provide the first kinetic maps of BCR signaling in primary human B cell subsets and enable new studies of signaling in B cell disorders, such as autoimmunity and cancer.

  View details for Web of Science ID 000239140300032

  View details for PubMedID 16849466

• Global transcriptional response to interferon is a determinant of HCV treatment outcome and is modified by race HEPATOLOGY He, X., Ji, X., Hale, M. B., Cheung, R., Ahmed, A., Guo, Y., Nolan, G. P., Pfeffer, L. M., Wright, T. L., Risch, N., Tibshirani, R., Greenberg, H. B. 2006; 44 (2): 352-359

  Abstract

  Interferon (IFN)-alpha-based therapy for chronic hepatitis C is effective in fewer than 50% of all treated patients, with a substantially lower response rate in black patients. The goal of this study was to investigate the underlying host transcriptional response associated with interferon treatment outcomes. We collected peripheral blood mononuclear cells from chronic hepatitis C patients before initiation of IFN-alpha therapy and incubated the cells with or without IFN-alpha for 6 hours, followed by microarray assay to identify IFN-induced gene transcription. The microarray datasets were analyzed statistically according to the patients' race and virological responses to subsequent IFN-alpha treatment. The global induction of IFN-stimulated genes (ISGs) was significantly greater in sustained virological responders compared with nonresponders and in white patients compared with black patients. In addition, a significantly greater global induction of ISGs was observed in sustained virological responders compared with nonresponders within the group of white patients. The level of IFN-induced signal transducer and activator of transcription (STAT) 1 activation, a key component of the Janus kinase (JAK)-STAT signaling pathway, correlated with the global induction of ISGs and was significantly higher in white patients than in black patients. In conclusion, both treatment outcome and race are associated with different transcriptional responses to IFN-alpha. Because this difference is evident in the global induction of ISGs rather than a selective effect on a subset of such genes, key factors affecting the outcome of IFN-alpha therapy are likely to act at the JAK-STAT pathway that controls transcription of downstream ISGs.

  View details for DOI 10.1002/hep.21267

  View details for Web of Science ID 000239523200009

  View details for PubMedID 16871572

• Chemical labeling strategies for cell biology NATURE METHODS Marks, K. M., Nolan, G. P. 2006; 3 (8): 591-596

  Abstract

  Methods to visualize, track, measure and perturb proteins in living cells are central to biomedicine's efforts to characterize and understand the spatial and temporal underpinnings of life inside cells. Although fluorescent proteins have revolutionized such studies, they have shortcomings, which have spurred the creation of alternative approaches to chemically label proteins in live cells. In this review we highlight research questions that can be addressed using site-specific chemical labeling and present a comparison of the various labeling techniques that have been developed. We also provide a 'roadmap' for selection of appropriate labeling techniques(s) and outline generalized strategies to validate and troubleshoot chemical labeling experiments.

  View details for DOI 10.1038/NMETH906

  View details for Web of Science ID 000239598600007

  View details for PubMedID 16862131

• Phospho-specific flow cytometry: Intersection of immunology and biochemistry at the single-cell level CURRENT OPINION IN MOLECULAR THERAPEUTICS Hale, M. B., Nolan, G. P. 2006; 8 (3): 215-224

  Abstract

  Striving to achieve greater clinical relevance, researchers in basic science and in drug discovery are transitioning from biochemical investigations using cell lines to technologies that garner mechanistic information from primary patient material. Such studies can be broad in scope, despite limited sample material and cell-type heterogeneity. The development of flow cytometry for following intracellular signaling has met some of these demands and opened new avenues for mechanistic exploration. This review covers some of the most recent research to leverage this new technology and follows two new developments: increasing interest in JAK/STAT signaling, and experimental strategies that reveal disease-induced modulation of signaling networks.

  View details for Web of Science ID 000237946300006

  View details for PubMedID 16774041

• Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling NATURE METHODS Krutzik, P. O., Nolan, G. P. 2006; 3 (5): 361-368

  Abstract

  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.

  View details for DOI 10.1038/NMETH872

  View details for Web of Science ID 000237210700015

  View details for PubMedID 16628206

• Luminescent imaging of beta-galactosidase activity in living subjects using sequential reporter-enzyme luminescence NATURE METHODS Wehrman, T. S., von Degenfeld, G., Krutzik, P., Nolan, G. P., Blau, H. M. 2006; 3 (4): 295-301

  Abstract

  We generated a sequential reporter-enzyme luminescence (SRL) technology for in vivo detection of beta-galactosidase (beta-gal) activity. The substrate, a caged D-luciferin-galactoside conjugate, must first be cleaved by beta-gal before it can be catalyzed by firefly luciferase (FLuc) to generate light. As a result, luminescence is dependent on beta-gal activity. Using this technology, constitutive beta-gal activity in engineered cells and inducible tissue-specific beta-gal expression in transgenic mice can now be visualized noninvasively over time. A substantial advantage of beta-gal as a bioluminescent probe is that the enzyme retains full activity outside of cells, unlike FLuc, which requires intracellular cofactors. As a result, antibodies conjugated to the recombinant beta-gal enzyme can be used to detect endogenous cells and extracellular antigens in vivo. Thus, coupling the properties of FLuc to the advantages of beta-gal permits bioluminescent imaging applications that previously were not possible.

  View details for DOI 10.1038/NMETH868

  View details for Web of Science ID 000236501400017

  View details for PubMedID 16554835

• Phospho-proteomic immune analysis by flow cytometry: from mechanism to translational medicine at the single-cell level IMMUNOLOGICAL REVIEWS Perez, O. D., Nolan, G. P. 2006; 210: 208-228

  Abstract

  Understanding a molecular basis for cellular function is a common goal of biomedicine. The complex and dynamic cellular processes underlying physiological processes become subtly or grossly perturbed in human disease. A primary objective is to demystify this complexity by creating and establishing relevant model systems to study important aspects of human disease. Although significant technological advancements over the last decade in both genomic and proteomic arenas have enabled progress, accessing the complexity of cellular interactions that occur in vivo has been a difficult arena in which to make progress. Moreover, there are extensive challenges in translating research tools to clinical applications. Flow cytometry, over the course of the last 40 years, has revolutionized the field of immunology, in both the basic science and clinical settings, as well as having been instrumental to new and exciting areas of discovery such as stem cell biology. Multiparameter machinery and systems exist now to access the heterogeneity of cellular subsets and enable phenotypic characterization and functional assays to be performed on material from both animal models and humans. This review focuses primarily on the development and application of using activation-state readouts of intracellular activity for phospho-epitopes. We present recent work on how a flow cytometric platform is used to obtain mechanistic insight into cellular processes as well as highlight the clinical applications that our laboratory has explored. Furthermore, this review discusses the challenges faced with processing high-content multidimensional and multivariate data sets. Flow cytometry, as a platform that is well situated in both the research and clinical settings, can contribute to drug discovery as well as having utility for both biomarker and patient-stratification.

  View details for Web of Science ID 000236327600014

  View details for PubMedID 16623773

• Transcending the biomarker mindset: deciphering disease mechanisms at the single cell level CURRENT OPINION IN CHEMICAL BIOLOGY Danna, E. A., Nolan, G. P. 2006; 10 (1): 20-27

  Abstract

  The application of proteomics to disease research promises to enhance the understanding and treatment of many human maladies through the identification of molecular profiles associated with each disease. However, although much is made of the utility of molecular signatures as markers of disease state, insufficient emphasis is often placed on the simultaneous need for biological mechanism inquiry. Focused and detailed analyses of disease-associated signaling networks have the potential to be more mechanistically informative than large-scale proteomic profiling approaches, providing insight into the cellular processes involved in pathogenesis, disease progression and therapeutic resistance; while still providing diagnostic or clinical management direction. Phospho-specific flow cytometry provides a method for the analysis of pathological signaling networks, enabling the investigation of disease mechanisms at the single-cell level.

  View details for DOI 10.1016/j.cbpa.2005.12.021

  View details for Web of Science ID 000235858200004

  View details for PubMedID 16406766

• Innovation - Mapping normal and cancer cell signalling networks: towards single-cell proteomics NATURE REVIEWS CANCER Irish, J. M., Kotecha, N., Nolan, G. P. 2006; 6 (2): 146-155

  View details for DOI 10.1038/nrc1804

  View details for Web of Science ID 000234844000013

• Mapping normal and cancer cell signalling networks: towards single-cell proteomics. Nature reviews. Cancer Irish, J. M., Kotecha, N., Nolan, G. P. 2006; 6 (2): 146-155

  Abstract

  Oncogenesis and tumour progression are supported by alterations in cell signalling. Using flow cytometry, it is now possible to track and analyse signalling events in individual cancer cells. Data from this type of analysis can be used to create a network map of signalling in each cell and to link specific signalling profiles with clinical outcomes. This form of 'single-cell proteomics' can identify pathways that are activated in therapy-resistant cells and can provide biomarkers for cancer diagnosis and for determining patient prognosis.

  View details for PubMedID 16491074

• Deeper insights into hematological oncology disorders via single-cell phospho-signaling analysis. Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program Nolan, G. P. 2006: 123-?

  Abstract

  An understanding of hematological cancer cell signaling processes poses one of the most complex and intractable problems in modern biomedical inquiry. While we understand some of the fundamental players that contribute to oncogenic processes, significant effort is focused upon determining how these individual players relay information to each other to create the composite functions of a cancer cell. Efforts designed to understand these processes at the single cell level will undoubtedly allow for understanding of the heterogeneity of hematological tumors as well as, simultaneously, the function of the 'responding' immune system. I will relate some of the insights our laboratory has developed over the last several years applying single-cell phospho-flow cytometry to the study of signaling in primary patient material and murine models. While it is clear that this analysis now allows us to accomplish phospho-signaling biochemistry at the single cell level with primary cell material, we are only beginning to develop some of the bioinformatics tools to appropriately display the vast amount of information collected by such approaches. These approaches, however, have already allowed us to develop approaches that prognosticate patient outcomes based on signaling status, prior to any treatment, as well as subgroup patient subtypes according to signaling states. The modest efforts to date presage a time where it should be possible to provide far more tailored therapies specific to the varied diseases represented by the hematological malignancies.

  View details for PubMedID 17124050

• Rapid, sustained B cell receptor signaling in lymphoma B cells differs from normal signaling in tumor infiltrating nonmalignant B cells. 47th Annual Meeting of the American-Society-of-Hematology Irish, J. M., Czerwinski, D. K., Nolan, G. P., Levy, R. AMER SOC HEMATOLOGY. 2005: 86A–87A

  View details for Web of Science ID 000233426000284

• Multiparameter flow cytometric analysis reveals aberrant phosphorylation of a network of effector molecules in defined populations of Kras mutant bone marrow. 47th Annual Meeting of the American-Society-of-Hematology Van Meter, M. E., Diaz-Flores, E., Archard, J. A., Kotecha, N., Sabnis, A. J., Irish, J. M., Nolan, G. P., Shannon, K. M., Braun, B. S. AMER SOC HEMATOLOGY. 2005: 73A–73A

  View details for Web of Science ID 000233426000240

• T-Cell tropism and the role of ORF66 protein in pathogenesis of varicella-zoster virus infection JOURNAL OF VIROLOGY Schaap, A., Fortin, J. F., Sommer, M., Zerboni, L., Stamatis, S., Ku, C. C., Nolan, G. P., Arvin, A. M. 2005; 79 (20): 12921-12933

  Abstract

  The pathogenesis of varicella-zoster virus (VZV) involves a cell-associated viremia during which infectious virus is carried from sites of respiratory mucosal inoculation to the skin. We now demonstrate that VZV infection of T cells is associated with robust virion production and modulation of the apoptosis and interferon pathways within these cells. The VZV serine/threonine protein kinase encoded by ORF66 is essential for the efficient replication of VZV in T cells. Preventing ORF66 protein expression by stop codon insertion (pOka66S) impaired the growth of the parent Oka (pOka) strain in T cells in SCID-hu T-cell xenografts in vivo and reduced formation of VZV virions. The lack of ORF66 protein also increased the susceptibility of infected T cells to apoptosis and reduced the capacity of the virus to interfere with induction of the interferon (IFN) signaling pathway following exposure to IFN-gamma. However, preventing ORF66 protein expression only slightly reduced growth in melanoma cells in culture and did not diminish virion formation in these cells. The pOka66S virus showed only a slight defect in growth in SCID-hu skin implants compared with intact pOka. These observations suggest that the ORF66 kinase plays a unique role during infection of T cells and supports VZV T-cell tropism by contributing to immune evasion and enhancing survival of infected T cells.

  View details for DOI 10.1128/JVI.79.20.12921-12933.2005

  View details for Web of Science ID 000232243200032

  View details for PubMedID 16188994

  View details for PubMedCentralID PMC1235817

• Phospho-specific flow cytometry in drug discovery. Drug discovery today. Technologies Clutter, M. R., Krutzik, P. O., Nolan, G. P. 2005; 2 (3): 295-302

  Abstract

  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.:

  View details for DOI 10.1016/j.ddtec.2005.08.010

  View details for PubMedID 24981951

• Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry JOURNAL OF IMMUNOLOGY Krutzik, P. O., Clutter, M. R., Nolan, G. P. 2005; 175 (4): 2357-2365

  Abstract

  Recently, phosphospecific flow cytometry has emerged as a powerful tool to analyze intracellular signaling events in complex populations of cells because of its ability to simultaneously discriminate cell types based on surface marker expression and measure levels of intracellular phosphoproteins. This has provided novel insights into the cell- and pathway-specific nature of immune signaling. However, we and others have found that the fixation and permeabilization steps necessary for phosphoprotein analysis often negatively affect the resolution of cell types based on surface marker analysis and light scatter characteristics. Therefore, we performed a comprehensive profile of >35 different murine surface marker Abs to understand the effects of fixation and permeabilization on surface Ag staining. Fortuitously, approximately 80% of the Abs tested resolved cell populations of interest, although with decreased separation between positive and negative populations and at very different titers than those used on live cells. The other 20% showed either complete loss of separation between populations or loss of intermediately staining populations. We were able to rescue staining of several of these Ags by performing staining after fixation, but before permeabilization, although with limited fluorophore choices. Scatter characteristics of lymphocytes were well retained, but changed dramatically for monocyte and neutrophil populations. These results compile a comprehensive resource for researchers interested in applying phosphospecific flow cytometry to complex populations of cells while outlining steps necessary to successfully apply new surface marker Abs to this platform.

  View details for Web of Science ID 000232010400043

  View details for PubMedID 16081806

• Characterization of the murine immunological signaling network with phosphospecific flow cytometry JOURNAL OF IMMUNOLOGY Krutzik, P. O., Hale, M. B., Nolan, G. P. 2005; 175 (4): 2366-2373

  Abstract

  The immune system is a multitiered network that at the first level uses changes to intracellular signaling proteins to commit cells to determined fates. At the second tier, cells interact with one another via specifically expressed surface receptors and their cognate signaling molecules. At the third level, the local environments of immune cells change the outcomes of intracellular signaling pathways and thereby the role of cells during immune challenge. The interplay among these three tiers allows the distinct cell types of the immune system to respond cohesively to eliminate foreign Ags. In this study, using phosphospecific flow cytometry, we analyze elements of these network tiers by generating profiles of single-cell phosphoprotein responses in B cells, T cells, and myeloid cells to a number of mechanistically and clinically relevant cytokines (IFN-gamma, GM-CSF, IL-2, and IL-10) as well as LPS at key regulatory interfaces (Jak-Stat and MAPK pathways). The stimuli typically induced phosphorylation of specific signaling pathways and exerted their effects on distinct subsets of immune cells. However, upon comparison of stimulation in vitro and in vivo, we noted that signaling pathway specificity and cell type specificity were influenced strongly by the external environment. When taken from the in vivo environment, certain cell subsets became hypo- or hyper-responsive, showed profound differences in sensitivity to cytokine levels, or displayed altered phosphorylation kinetics. Thus, simultaneous analysis of the three tiers of the immune system network illustrates the principles by which immune regulation is context dependent and how in vitro culture systems compare with the in vivo environment.

  View details for Web of Science ID 000232010400044

  View details for PubMedID 16081807

• Multiparameter analysis of intracellular phosphoepitopes in immunophenotyped cell populations by flow cytometry. Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] Perez, O. D., Mitchell, D., Campos, R., Gao, G., Li, L., Nolan, G. P. 2005; Chapter 6: Unit 6 20-?

  Abstract

  This unit presents protocols for measuring intracellular phosphoepitopes by flow cytometry for biochemical investigations in both human and murine primary cells as well as in cell lines. Conventional methods that require cellular lysis cannot discriminate between proteins from different cellular subsets. Intracellular detection of phosphorylated and nonphosphorylated signaling molecules, on the other hand, has recently exposed the heterogeneity that can be observed upon signal transduction. Although staining methodologies for cytokines and cell cycle antigens have been appreciated for years, detection of phosphorylated molecules presents several new challenges, including generation of reagents and details of the staining technique. As these techniques are adapted to new applications, the protocols continue to be refined. This unit describes signal amplification of intracellular signals upon detergent-based permeabilizations, staining protocol for adherent cells, methodology for whole-blood staining, and multiparameter staining procedures for surface and intracellular antigens.

  View details for DOI 10.1002/0471142956.cy0620s32

  View details for PubMedID 18770823

• Y-box-binding protein 1 confers EGF independence to human mammary epithelial cells ONCOGENE Berquin, I. M., Pang, B., Dziubinski, M. L., Scott, L. M., Chen, Y. Q., Nolan, G. P., Ethier, S. P. 2005; 24 (19): 3177-3186

  Abstract

  The epidermal growth factor receptor (EGFR) is linked to poor outcome in breast cancer, and resistance to hormonal therapy is often accompanied by activation of growth factor receptors. To investigate the mechanism(s) by which EGFR becomes activated in breast cancer, we screened a cDNA expression library for genes that mediate EGF-independent proliferation of human mammary epithelial cells (HMECs). We isolated the NSEP1 cDNA encoding Y-box-binding protein 1 (YB-1), a multifunctional transcriptional and translational regulator. This cDNA conferred growth factor independence to HMECs. YB-1-transduced cells overexpressed EGFR, but ErbB-2 (Her-2/neu) levels were unchanged. Moreover, EGFR was constitutively phosphorylated in the absence of exogenous ligand. In these cells, an EGFR-blocking antibody failed to inhibit proliferation, conditioned medium activity could not be detected, and the synthesis of EGFR ligands was reduced compared to parental cells. This suggests that EGFR is activated in a ligand-independent fashion. However, cell growth could be blocked with an ErbB kinase inhibitor, indicating that EGFR signaling plays a major role in YB-1-induced growth factor independence. Taken together, our results demonstrate that YB-1 overexpression can induce EGF independence in HMECs via activation of the EGFR pathway. This could represent one of the mechanisms by which YB-1 contributes to breast tumor aggressiveness.

  View details for DOI 10.1038/sj.onc.1208504

  View details for Web of Science ID 000228728100011

  View details for PubMedID 15735691

• Causal protein-signaling networks derived from multiparameter single-cell data SCIENCE Sachs, K., Perez, O., Pe'er, D., Lauffenburger, D. A., Nolan, G. P. 2005; 308 (5721): 523-529

  Abstract

  Machine learning was applied for the automated derivation of causal influences in cellular signaling networks. This derivation relied on the simultaneous measurement of multiple phosphorylated protein and phospholipid components in thousands of individual primary human immune system cells. Perturbing these cells with molecular interventions drove the ordering of connections between pathway components, wherein Bayesian network computational methods automatically elucidated most of the traditionally reported signaling relationships and predicted novel interpathway network causalities, which we verified experimentally. Reconstruction of network models from physiologically relevant primary single cells might be applied to understanding native-state tissue signaling biology, complex drug actions, and dysfunctional signaling in diseased cells.

  View details for DOI 10.1126/science.1105809

  View details for Web of Science ID 000228810500045

  View details for PubMedID 15845847

• A random peptide library fused to CCR5 for selection of mimetopes expressed on the mammalian cell surface via retroviral vectors JOURNAL OF BIOLOGICAL CHEMISTRY Wolkowicz, R., Jager, G. C., Nolan, G. P. 2005; 280 (15): 15195-15201

  Abstract

  A random peptide library was expressed on the surface of a mammalian cell by applying retroviral vectors. The random sequence was fused to the CCR5 chemokine receptor, which served as a scaffold to present the library at the cell surface. We used this library to isolate an epitope mimetope in a proof of principle system. This approach can become a tool for rapid creation of peptidic expression domains in a eukaryotic environment. Applications include the creation of decoys for receptors in cell-cell interactions, screening for molecules that drive ligand expression on target cells in two-cell interaction screens, among other utilities.

  View details for DOI 10.1074/jbc.M500254200

  View details for Web of Science ID 000228236800104

  View details for PubMedID 15657029

• Gene therapy progress and prospects: Novel gene therapy approaches for AIDS GENE THERAPY Wolkowicz, R., Nolan, G. P. 2005; 12 (6): 467-476

  Abstract

  Acquired immunodeficiency syndrome (AIDS), caused by human immunodeficiency virus (HIV), kills millions worldwide every year. Vaccines against HIV still seem a distant promise. Pharmaceutical treatments exist, but these are not always effective, and there is increasing prevalence of viral strains with multidrug resistance. Highly active antiretroviral therapy (HAART) consists of inhibitors of viral enzymes (reverse transcriptase (RT) and protease). Gene therapy, first introduced as intracellular immunization, may offer hopes for new treatments to be used alone, or in conjunction with, conventional small molecule drugs. Gene therapy approaches against HIV-1, including suicide genes, RNA-based technology, dominant negative viral proteins, intracellular antibodies, intrakines, and peptides, are the subject of this review.

  View details for DOI 10.1038/sj.gt.3302488

  View details for Web of Science ID 000227495400001

  View details for PubMedID 15703764

• Tadpoles by the tail NATURE METHODS Nolan, G. P. 2005; 2 (1): 11-12

  View details for Web of Science ID 000226754000009

  View details for PubMedID 15782163

• Screening of retroviral cDNA libraries for factors involved in protein phosphorylation in signaling cascades NUCLEIC ACIDS RESEARCH Stitz, J., Krutzik, P. O., Nolan, G. P. 2005; 33 (4)

  Abstract

  We report a novel approach that allows for the rapid identification of proteins mediating phosphorylation in signaling cascades after specific stimulation. As a proof of concept, we used the interferon- gamma (IFN-gamma)-induced phosphorylation of signal transducer and activator of transcription-1 (Stat1) in a human promonocytic cell line, which was previously shown to be deficient in this signaling pathway. By using retroviral cDNA expression libraries, transduced selector cells expressing single cDNAs were stimulated with IFN-gamma, then fixed, permeabilized and stained intracellularly for phospho-Stat1 levels. Cells responding to the stimulation, which showed increased levels of phosphorylated Stat1, were enriched using fluorescence activated cell sorting (FACS). Genomic DNA was isolated from the enriched cell population and served as a template for cDNA amplification using PCR. After only one round of selection, a cDNA encoding the beta-chain of the IFN-gamma receptor (IFNGR2) was obtained and demonstrated to restore the selected phenotype. The approach now allows one to use phospho-events as reporters, alone or in tandem, for screening of signaling network states, overcoming a prior need to rely on the reporter genes that are often only indirect measures of phenotypes desired in a screen.

  View details for DOI 10.1093/nar/gni039

  View details for Web of Science ID 000227565400006

  View details for PubMedID 15731332

• LFA-1 signaling through p44/42 is coupled to perforin degranulation in CD56(+)CD8(+) natural killer cells BLOOD Perez, O. D., Mitchell, D., Jager, G. C., Nolan, G. P. 2004; 104 (4): 1083-1093

  Abstract

  Leukocyte function antigen 1 (LFA-1) is essential for the formation of immune cell synapses and plays a role in the pathophysiology of various autoimmune diseases. We investigated the molecular details of LFA-1 activation during adhesion between cytotoxic cells and a target model leukemia cell. The cytolytic activity of a CD3-CD8+CD56+ natural killer (NK) subset was enhanced when LFA-1 was activated. In a comparison of LFA-1 ligands, intercellular adhesion molecule 2 (ICAM-2) and ICAM-3 promoted LFA-1-directed perforin release, whereas ICAM-1 had little effect. Ligand-induced LFA-1 clustering facilitated perforin release, demonstrating LFA-1 could regulate degranulation mechanisms. LFA-1 induced the activation of src family kinases, Vav1 and p44/42 mitogen-activated protein kinase (MAPK), in human CD56+ NK cells as evidenced by intracellular phospho-epitope measurements that correlated with effector-target cell binding and perforin-granzyme A-mediated cytolytic activity. These results identify novel, specific functional consequence of LFA-1-mediated cytolytic activity in perforin-containing human NK subsets.

  View details for DOI 10.1182/blood-2003-08-2652

  View details for Web of Science ID 000223145800032

  View details for PubMedID 15113754

• Prolonged liver-specific transgene expression by a non-primate lentiviral vector BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Condiotti, R., Curran, M. A., Nolan, G. P., Giladi, H., Ketzinel-Gilad, M., Gross, E., Galun, E. 2004; 320 (3): 998-1006

  Abstract

  Liver-directed gene therapy has the potential for treatment of numerous inherited diseases affecting metabolic functions. The aim of this study was to evaluate gene expression in hepatocytes using feline immunodeficiency virus-based lentiviral vectors, which may be potentially safer than those based on human immunodeficiency virus. In vitro studies revealed that gene expression was stable for up to 24 days post-transduction and integration into the host cell genome was suggested by Alu PCR and Southern blot analyses. Systemic in vivo administration of viral particles by the hydrodynamics method resulted in high levels of gene expression exclusively in the liver for over 7 months whereas injection of plasmid DNA by the same method led to transient expression levels. Our studies suggest that feline immunodeficiency-based lentiviral vectors specifically transduce liver cells and may be used as a novel vehicle of gene delivery for treatment of metabolic disease.

  View details for DOI 10.1016/j.bbrc.2004.06.044

  View details for Web of Science ID 000222723200054

  View details for PubMedID 15240147

• Single cell profiling of potentiated phospho-protein networks in cancer cells CELL Irish, J. M., Hovland, R., Krutzik, P. O., Perez, O. D., Bruserud, O., Gjertsen, B. T., Nolan, G. P. 2004; 118 (2): 217-228

  Abstract

  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.

  View details for Web of Science ID 000222966000010

  View details for PubMedID 15260991

• A general approach for chemical labeling and rapid, spatially controlled protein inactivation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Marks, K. M., Braun, P. D., Nolan, G. P. 2004; 101 (27): 9982-9987

  Abstract

  Chemical labeling of proteins inside of living cells can enable studies of the location, movement, and function of proteins in vivo. Here we demonstrate an approach for chemical labeling of proteins that uses the high-affinity interaction between an FKBP12 mutant (F36V) and a synthetic, engineered ligand (SLF'). A fluorescein conjugate to the engineered ligand (FL-SLF') retained binding to FKBP12(F36V) and possessed similar fluorescence properties as parental fluorescein. FL-SLF' labeled FKBP12(F36V) fusion proteins in live mammalian cells, and was used to monitor the subcellular localization of a membrane targeted FKBP12(F36V) construct. Chemical labeling of FKBP12(F36V) fusion proteins with FL-SLF' was readily detectable at low expression levels of the FKBP12(F36V) fusion, and the level of fluorescent staining with FL-SLF' was proportional to the FKBP12(F36V) expression level. This FL-SLF'-FKBP12(F36V) labeling technique was tested in fluorophore assisted laser inactivation (FALI), a light-mediated technique to rapidly inactivate fluorophore-labeled target proteins. FL-SLF' mediated FALI of a beta-galactosidase-FKBP12(F36V) fusion protein, causing rapid inactivation of >90% of enzyme activity upon irradiation in vitro. FL-SLF' also mediated FALI of a beta-galactosidase fusion expressed in living NIH 3T3 cells, where beta-galactosidase activity was reduced in 15 s. Thus, FL-SLF' can be used to monitor proteins in vivo and to target rapid, spatially and temporally defined inactivation of target proteins in living cells in a process that we call FK-FALI.

  View details for DOI 10.1073/pnas.0401609101

  View details for Web of Science ID 000222534200009

  View details for PubMedID 15218100

• Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications CLINICAL IMMUNOLOGY Krutzik, P. O., Irish, J. M., Nolan, G. P., Perez, O. D. 2004; 110 (3): 206-221

  Abstract

  Analysis of protein phosphorylation with flow cytometric techniques has emerged as a powerful tool in the field of immunological signaling, allowing cellular subsets in complex populations to be analyzed accurately and rapidly. In this review, we examine the development of phospho-epitope, or phospho-specific, flow cytometry and the premises upon which the technique is based. Phospho-specific flow cytometry is compared to traditional biochemical methods, and its advantages, such as single cell analysis, multiparameter data acquisition, rapid protocols, and the ability to analyze rare cell subsets, are detailed. We also discuss the many technical considerations that must be addressed when developing new antibodies or analyzing new epitopes including antigen accessibility, stability of the phospho-epitope, fluorophore selection, surface phenotype integrity, and antibody suitability for staining epitopes inside fixed and permeabilized cells. The methods that have been used to date are described in light of these technical considerations. The importance of developing bioinformatic platforms in parallel with these techniques is emphasized due to the large, multiparameter datasets that are rapidly accumulated and which require more efficient data viewing and complex clustering methods than currently available for flow cytometric data. Finally, we discuss the potential clinical applications of phospho-specific flow cytometry in analyzing immune cell development and antigen-specific immune responses, as well as pharmacodynamic profiling of disease states or drug efficacy and specificity against particular signaling proteins.

  View details for DOI 10.1016/j.clim.2003.11.009

  View details for Web of Science ID 000220831800003

  View details for PubMedID 15047199

• In vivo targeting of organic calcium sensors via genetically selected peptides CHEMISTRY & BIOLOGY Marks, K. M., Rosinov, M., Nolan, G. P. 2004; 11 (3): 347-356

  Abstract

  A library of constrained peptides that form stable folded structures was screened for aptamers that bind with high affinity to the fluorescent dye Texas red. Two selected clones had binding constants to Texas red of 25 and 80 pM as phage and binding had minimal effects on the fluorescence of Texas red. The peptides interact with distinct but overlapping regions of Texas red. One peptide bound to X-rhod calcium sensors, which share the same core fluorophore as Texas red. These dyes retained calcium sensitivity when bound to the peptide. This peptide was used to label a fusion protein with X-rhod-5F in vivo, and X-rhod sensed changes in calcium locally. Thus, minimal, constrained peptides can functionally bind to environmentally sensitive dyes or other organic agents in biological contexts, suggesting tools for in vivo imaging and analysis.

  View details for DOI 10.1016/j.chembiol.2004.03.004

  View details for Web of Science ID 000220502900011

  View details for PubMedID 15123264

• Oligonucleotide-directed site-specific integration of high complexity libraries into ssDNA templates NUCLEIC ACIDS RESEARCH Hale, M. B., Nolan, G. P., Wolkowicz, R. 2004; 32 (2)

  Abstract

  We present an approach that generates an oligomer-based library with minimal need for restriction site modification of sequences in the target vector. The technique has the advantage that it can be applied for generating peptide aptamer libraries at sites within proteins without the need for introducing flanking enzyme sites. As an example we present a phagemid retroviral shuttle vector that can be used to achieve stable expression of the library in mammalian cells for the purpose of screening for peptides with desired biological activity.

  View details for DOI 10.1093/nar/gnh021

  View details for Web of Science ID 000188989000008

  View details for PubMedID 14752044

• Analysis of signaling pathways in human T-Cells using Bayesian network modeling of single cell data IEEE Computational Systems Bioinformatics Conference (CSB 2004) Sachs, K., Perez, O. D., Pe'er, D., Nolan, G. P., Gifford, D. K., Jaakkola, T. S., Lauffenburger, D. A. IEEE COMPUTER SOC. 2004: 644–644

  View details for Web of Science ID 000224127800117

• Flow cytometric analysis of kinase signaling cascades. Methods in molecular biology (Clifton, N.J.) Perez, O. D., Krutzik, P. O., Nolan, G. P. 2004; 263: 67-94

  Abstract

  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.

  View details for PubMedID 14976361

• Lentiviral vectors for the delivery of DNA into mammalian cells. Methods in molecular biology (Clifton, N.J.) Wolkowicz, R., Nolan, G. P., Curran, M. A. 2004; 246: 391-411

  Abstract

  Vectors derived from oncoretroviruses, represented by the prototype Moloney murine leukemia virus (MMLV), are powerful tools for gene transfer into mammalian cells. Vectors derived from such viruses are able to carry an insert of up to 6.5 kb. Because Retroviridae and derived vectors insert their genome into the host chromosome, the transgene delivered by these viruses are stably expressed in the infected cells. From a safety standpoint, the vectors are designed to eliminate any need to carry viral genes or associated toxicities into the host cell. This also substantially reduces their potential immunogenicity. Finally, the titers achieved with these vectors can be very high, yielding efficient infection in a broad range of cell types.

  View details for PubMedID 14970606

• Leukocyte functional antigen 1 lowers T cell activation thresholds and signaling through cytohesin-1 and Jun-activating binding protein 1 NATURE IMMUNOLOGY Perez, O. D., Mitchell, D., Jager, G. C., South, S., Murriel, C., McBride, J., Herzenberg, L. A., Kinoshita, S., Nolan, G. P. 2003; 4 (11): 1083-1092

  Abstract

  Leukocyte functional antigen 1 (LFA-1), with intercellular adhesion molecule ligands, mediates T cell adhesion, but the signaling pathways and functional effects imparted by LFA-1 are unclear. Here, intracellular phosphoprotein staining with 13-dimensional flow cytometry showed that LFA-1 activation induced phosphorylation of the beta(2) integrin chain and release of Jun-activating binding protein 1 (JAB-1), and mediated signaling of kinase Erk1/2 through cytohesin-1. Dominant negatives of both JAB-1 and cytohesin-1 inhibited interleukin 2 production and impaired T helper type 1 differentiation. LFA-1 stimulation lowered the threshold of T cell activation. Thus, LFA-1 signaling contributes to T cell activation and effects T cell differentiation.

  View details for DOI 10.1038/ni984

  View details for Web of Science ID 000186241100012

  View details for PubMedID 14528303

• Intracellular phospho-protein staining techniques for flow cytometry: Monitoring single cell signaling events CYTOMETRY PART A Krutzik, P. O., Nolan, G. P. 2003; 55A (2): 61-70

  View details for DOI 10.1002/cyto.a.10072

  View details for Web of Science ID 000185594400001

• Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry. Part A : the journal of the International Society for Analytical Cytology Krutzik, P. O., Nolan, G. P. 2003; 55 (2): 61-70

  Abstract

  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.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.Fixing cells with 1.5% formaldehyde followed by permeabilization in methanol gave optimal results for pERK, pp38, pJNK, pStat1, pStat5, and pStat6 staining. Alteration of formaldehyde fixation and methanol permeabilization times affected measurements of phosphorylation induction. Phospho-specific flow cytometric analyses correlated well with Western blotting, providing cross platform validation of the technique.Measuring phosphorylation events by flow cytometry provides a rapid and efficient way to measure kinase cascades in individual cells. Stability of phospho-epitopes in methanol allows long-term storage of samples prior to analysis. Multiple signaling cascades can be monitored simultaneously through the use of different fluorophore labels to determine specificity of ligands or inhibitors. Application of optimized techniques to heterogeneous cell types such as peripheral blood or murine splenocytes may allow signaling to be analyzed simultaneously in immune cell subsets.

  View details for PubMedID 14505311

• Localized expression of an anti-TNF single-chain antibody prevents development of collagen-induced arthritis GENE THERAPY Smith, R., Tarner, I. H., Hollenhorst, M., Lin, C., Levicnik, A. U., Fathman, C. G., Nolan, G. P. 2003; 10 (15): 1248-1257

  Abstract

  Although systemic administration of neutralizing anti-TNF antibodies has been used successfully in treating rheumatoid arthritis, there is a potential for side effects. We transduced a collagen reactive T-cell hybridoma with tissue-specific homing properties to assess therapeutic effects of local delivery to inflamed joints of anti-TNF single-chain antibodies (scFv) by adoptive cellular gene therapy. Cell culture medium conditioned with 1 x 10(6) scFv producer cells/ml had TNF neutralizing capacity in vitro equivalent to 50 ng/ml anti-TNF monoclonal antibody. Adding a kappa chain constant domain to the basic scFv (construct TN3-Ckappa) gave increased in vitro stability and in vivo therapeutic effect. TN3-Ckappa blocked development of collagen-induced arthritis in DBA/1LacJ mice for >60 days. Transgene expression was detected in the paws but not the spleen of treated animals for up to 55 days postinjection. No significant variations in cell proliferation or cytokine secretion were found in splenocytes or peripheral lymphocytes. IL-6 expression was blocked in the diseased paws of mice in the scFv treatment groups compared to controls. In conclusion, we have shown that local expression of an anti-inflammatory agent blocks disease development without causing demonstrable systemic immune function changes. This is encouraging for the potential development of safe adoptive cellular therapies to treat autoimmunity.

  View details for DOI 10.1038/sj.gt.3301980

  View details for Web of Science ID 000184207000007

  View details for PubMedID 12858190

• Interaction of HLA-DR with an acidic face of HLA-DM disrupts sequence-dependent interactions with peptides IMMUNITY Pashine, A., Busch, R., Belmares, M. P., Munning, J. N., Doebele, R. C., Buckingham, M., Nolan, G. P., Mellins, E. D. 2003; 19 (2): 183-192

  Abstract

  HLA-DM (DM) edits major histocompatibility complex class II (MHCII)-bound peptides in endocytic compartments and stabilizes empty MHCII molecules. Crystal structures of DM have revealed similarity to MHCII but not how DM and MHCII interact. We used mutagenesis to map a MHCII-interacting surface on DM. Mutations on this surface impair DM action on HLA-DR and -DP in cells and DM-dependent peptide loading in vitro. The orientation of DM and MHCII predicted by these studies guided design of soluble DM and DR molecules fused to leucine zippers via their beta chains, resulting in stable DM/DR complexes. Peptide release from the complexes was fast and only weakly sequence dependent, arguing that DM diminishes the selectivity of the MHCII groove. Analysis of soluble DM action on soluble DR/peptide complexes corroborates this conclusion.

  View details for Web of Science ID 000184929000006

  View details for PubMedID 12932352

• Polyclonal antibodies to xenogeneic endothelial cells induce apoptosis and block support of tumor growth in mice VACCINE Scappaticci, F. A., CONTRERAS, A., Boswell, C. A., Lewis, J. S., Nolan, G. 2003; 21 (19-20): 2667-2677

  Abstract

  In this study, we demonstrate that vaccination of rabbits with murine endothelial cells yields polyclonal immunoglobulin (IgG) with potent antiangiogenic activity. The mechanism of this response appears to be through apoptosis of endothelial cells in vitro. Induction of polyclonal IgG in a xenogeneic host may be useful in passive immunotherapy of a variety of cancers. In fact, the antibody showed antitumor activity in three mouse tumor models (murine B16F10 melanoma, murine SVR angiosarcoma, and human DLD-1 colorectal adenocarcinoma). The polyclonal antibody generated here demonstrated utility in radioimaging of tumors in vivo, using positron emission tomography (PET) imaging, and suggested an antitumor effect in vivo. The results suggest that the antitumor effect in vivo may be related to antiangiogenic effects. Furthermore, anti-endothelial cell antibodies such as these could be useful reagents in isolating specific targets that comprise and induce the antiangiogenic effect.

  View details for DOI 10.1016/S0264-410X(02)00693-X

  View details for Web of Science ID 000183100600059

  View details for PubMedID 12744904

• Retroviral technology - Applications for expressed peptide libraries FRONTIERS IN BIOSCIENCE-LANDMARK Wolkowicz, R., Nolan, G. P. 2003; 8: D603-D619

  Abstract

  The cell is an extremely complex network of interactions between large numbers of molecules. Understanding this entire network and the information arising from it is an overwhelming and challenging task. Reverse genetics has given us the possibility to discover unknown interactions and their related pathways. With the help of peptide libraries, interactions between biomolecules can be disrupted or distorted and the signaling pathways where these proteins are involved, altered. Consequently, novel biological pathways can be discerned. The peptide libraries become a pool of shapes, some of them might behave as dominant effectors. With the use of retroviral transfer vectors those libraries can be expressed in a stable manner in the mammalian cell. A strong selection and screening process can finally lead to specific peptides. Novel high-throughput approaches might allow for the rapid creation of small-molecule switches in protein-protein interactions. Reverse genetics and as such the expression of small molecules that will have a specific biological outcome, can become an answer to our queries.

  View details for Web of Science ID 000182352300022

  View details for PubMedID 12700115

• Induction of anti-tumor immunity in mice using a syngeneic endothelial cell vaccine ANTICANCER RESEARCH Scappaticci, F. A., Nolan, G. P. 2003; 23 (2B): 1165-1172

  Abstract

  Tumor endothelium could represent a novel target for active and passive immunotherapies of cancer. Here, we show that endothelial cells can be used as a vaccine in mice. In this study, three endothelial cell vaccine preparations from syngeneic (SVR), allogeneic (ISOS-1) and xenogeneic (ISO-HAS) sources were used to vaccinate mice. All mice developed humoral immune responses to endothelial cells and showed lower basal serum VEGF levels (37-45% lower) compared with unvaccinated control mice. Mice receiving the syngeneic SVR vaccine showed substantial inhibition of tumor growth after B16F10 melanoma challenge (50% of the mice in this group were tumor-free). The tumors that developed in the few mice in the syngeneic group had lower microvessel density counts (4-5 fold) compared with the other groups. The data suggests an in vivo antiangiogenic effect as the potential mechanism for the anti-cancer effect. In summary, further studies using other tumor models to demonstrate broad protection of this novel type of antiangiogenic vaccine are warranted.

  View details for Web of Science ID 000183471600001

  View details for PubMedID 12820367

• Treatment of autoimmune disease by adoptive cellular gene therapy 10th International Conference on Myasthenia Gravis and Related Disorders Tarner, I. H., Slavin, A. J., McBride, J., Levicnik, A., Smith, R., Nolan, G. P., Contag, C. H., Fathman, C. G. NEW YORK ACAD SCIENCES. 2003: 512–519

  Abstract

  Autoimmune disorders represent inappropriate immune responses directed at self-tissue. Antigen-specific CD4+ T cells and antigen-presenting dendritic cells (DCs) are important mediators in the pathogenesis of auto-immune disease and thus are ideal candidates for adoptive cellular gene therapy, an ex vivo approach to therapeutic gene transfer. Using retrovirally transduced cells and luciferase bioluminescence, we have demonstrated that primary T cells, T cell hybridomas, and DCs rapidly and preferentially home to the sites of inflammation in animal models of multiple sclerosis, arthritis, and diabetes. These cells, transduced with retroviral vectors to drive expression of various "regulatory proteins" such as IL-4, IL-10, IL-12p40, and anti-TNF scFv, deliver these immunoregulatory proteins to the inflamed lesions, providing therapy for experimental autoimmune encephalitis (EAE), collagen-induced arthritis (CIA), and nonobese diabetic mice (NOD).

  View details for Web of Science ID 000186107400066

  View details for PubMedID 14592922

• Expression from second-generation feline immunodeficiency virus vectors is impaired in human hematopoietic cells MOLECULAR THERAPY Price, M. A., Case, S. S., Carbonaro, D. A., Yu, X. J., Petersen, D., Sabo, K. M., Curran, M. A., Engel, B. C., Margarian, H., Abkowitz, J. L., Nolan, G. P., Kohn, D. B., Crooks, G. M. 2002; 6 (5): 645-652

  Abstract

  Vectors based on the feline immunodeficiency virus (FIV) have been developed as an alternative to those based on another lentivirus, human immunodeficiency virus-1 (HIV-1), because of theoretical safety advantages. We compared the efficiency of gene transfer and expression in human and feline hematopoietic progenitors using second-generation HIV-1 and FIV-based vectors. Vector pairs were tested using either human cytomegalovirus or murine phospho-glycerate kinase (PGK) internal promoters and were pseudotyped with the vesicular stomatitis virus G protein (VSV-G). Vector proviral copy numbers were similar in human and feline hematopoietic primary cells and cell lines transduced by HIV-1 or FIV vectors, demonstrating that both vectors are able to transfer genes efficiently to these cell types. HIV-1 vectors were well expressed in human primary hematopoietic cells and cell lines. However, transgene expression from FIV vectors was almost undetectable in human hematopoietic cells. In contrast, the FIV vector was expressed well in primary hematopoietic feline cells and human non-hematopoietic cells, demonstrating that low transgene expression from the FIV vector is a phenomenon specific to human hematopoietic cells. Northern blot analysis demonstrated decreased vector transcript levels in human CEM cells transduced with FIV relative to cells transduced with HIV-1, despite high vector copy numbers. No evidence of vector transcript instability was seen in studies of transduced CEM cells treated with actinomycin D. We conclude that FIV vectors can transfer genes into human hematopoietic cells as effectively as HIV-1 vectors, but that unknown elements in the current FIV backbone inhibit expression from FIV vectors in human hematopoietic cells.

  View details for DOI 10.1006/mthe.2002.0725

  View details for Web of Science ID 000179260600017

  View details for PubMedID 12409263

• Efficient transduction of pancreatic islets by feline immunodeficiency virus vectors TRANSPLANTATION Curran, M. A., Ochoa, M. S., Molano, R. D., Pileggi, A., Inverardi, L., Kenyon, N. S., Nolan, G. P., Ricordi, C., Fenjves, E. S. 2002; 74 (3): 299-306

  Abstract

  Pancreatic islets transplanted into immunocompetent diabetic subjects are rapidly lost to apoptotic or lytic death or both. Genetic engineering of islets before transplantation with protective genes may enhance their posttransplantation survival. Accomplishing this goal requires the development of a safe, efficient vector for islet gene delivery.The ability of feline immunodeficiency virus (FIV) vectors to transfer a green fluorescent protein (GFP) gene to NIT-1 cells and primary islets was measured and compared with murine leukemia virus (MLV) and human immunodeficiency virus (HIV) vectors. Islets were examined using confocal microscopy to determine the extent and pattern of infection. Toxicity of the procedure was assessed via measurement of glucose stimulation indices and by reversion of diabetic mice using either FIV-infected or control islet transplants.FIV effectively transduces islets with no untoward effect on the insulin secretion capacity of the beta cells. When FIV, HIV, and MLV GFP vectors were standardized to the same 293 cell titer and used to infect NIT-1 cells or whole islets, the FIV transduced equal or greater numbers of cells relative to the HIV vector and significantly more than the MLV vector. Islets transduced with FIV GFP were transplanted in a murine model for diabetes and were shown to revert diabetes and express GFP 4 weeks after transduction and 3 weeks after transplantation.FIV transduction is a nontoxic and efficient method to genetically modify pancreatic islets and may prove promising for delivering genes to augment islet survival after transplantation.

  View details for Web of Science ID 000177496000003

  View details for PubMedID 12177606

• Functional cloning of SPIN-2, a nuclear anti-apoptotic protein with roles in cell cycle progression LEUKEMIA Fletcher, B. S., Dragstedt, C., Notterpek, L., Nolan, G. P. 2002; 16 (8): 1507-1518

  Abstract

  The balance between hematopoietic cell viability and apoptosis is regulated by exogenous growth factors, however, the molecular mechanisms by which these trophic factors exert their effects remain obscure. A functional retroviral cDNA library-based screen was employed to identify genes that prevent growth factor withdrawal-mediated apoptosis in the myeloid progenitor cell 32Dcl3. This approach identified three classes of genes: those with known roles in apoptosis (bcl-X(L) and ornithine decarboxylase); genes previously identified but not linked directly to apoptotic signaling (O-linked N-acetylglucosamine transferase); and a previously uncharacterized gene we termed SPIN-2. In 32Dcl3 cells, expression of exogenous SPIN-2 provides 25% protection from apoptosis following growth factor withdrawal compared to controls which show approximately 1-2% survival. SPIN-2 overexpression slows cell growth rates and increases the percentage of cells in G(2)/M (32% vs control cells at 12%). Immunolocalization studies indicate that myc-epitope tagged SPIN-2 proteins, which retain their anti-apoptotic function, reside in the nucleus, whereas a C-terminal deletion mutant that loses its anti-apoptotic activity is located in the cytoplasm. These studies suggest that SPIN-2 is a novel nuclear protein that functions to regulate cell cycle progression and this activity is related to the inhibition of apoptosis following the removal of essential growth factors.

  View details for DOI 10.1038/sj.leu.2402557

  View details for Web of Science ID 000177436500018

  View details for PubMedID 12145692

• Recognizing a something when your library sees it CHEMISTRY & BIOLOGY Fortin, J. F., Nolan, G. P. 2002; 9 (6): 670-672

  Abstract

  Advances are needed in random-display technologies to more tightly link drug actions and functions to the genes that control physiological processes. The reports discussed here explore two sides of these issues-generating new library formats and identifying the targets of drug ligands.

  View details for Web of Science ID 000176418100003

  View details for PubMedID 12079777

• Motexafin gadolinium (Gd-Tex) selectively induces apoptosis in HIV-1 infected CD4+T helper cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Perez, O. D., Nolan, G. P., Magda, D., MILLER, R. A., Herzenberg, L. A., Herzenberg, L. A. 2002; 99 (4): 2270-2274

  Abstract

  Here, we show that motexafin gadolinium (Gd-Tex), a compound that promotes intracellular oxidative stress, selectively induces apoptosis in HIV-1-infected CD4(+) T cells in IL-2-stimulated cultures of peripheral blood mononuclear cells infected in vitro with HIV-1. This selective induction of apoptosis, which we detect by FACS analysis of intracellular HIV/p24 and concomitant surface and apoptosis marker expression, is abrogated by the glutathione precursor, N-acetyl-l-cysteine. Importantly, it occurs at Gd-Tex concentrations that are not cytotoxic to uninfected cells in the culture. These findings suggest that Gd-Tex may have therapeutic utility as an anti-HIV agent capable of selectively targeting and removing HIV-infected cells in an infected host.

  View details for DOI 10.1073/pnas.261711499

  View details for Web of Science ID 000174031100093

  View details for PubMedID 11854523

• Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry NATURE BIOTECHNOLOGY Perez, O. D., Nolan, G. P. 2002; 20 (2): 155-162

  Abstract

  Intracellular assays of signaling systems have been limited by an inability to correlate functional subsets of cells in complex populations on the basis of active kinase states. Such correlations could be important in distinguishing changes in signaling status that arise in rare cell subsets during functional activation or in disease manifestation. Here we demonstrate the ability to simultaneously detect activated kinase members of the mitogen-activated protein kinases family (p38 MAPK, p44/42 MAPK, JNK/SAPK), members of cell survival pathways (AKT/PKB), and members of T-cell activation pathways (TYK2), among others, in subpopulations of complex cell populations by multiparameter flow-cytometric analysis. We demonstrate the utility of these probes in identifying distinct signaling cascades for (1) both artificial and physiological stimulatory conditions of peripheral blood mononuclear cells (PBMCs), (2) cytokine stimulation in human memory and naïve lymphocyte subsets as identified by five differentiation markers, and (3) ordering of kinase activation in potential signaling hierarchies. Polychromatic flow-cytometric active kinase measurements demonstrate that multidimensional analysis of signaling pathways can provide functional signaling pathway assessment on a single-cell level and allow for potential correlation with biological and clinical parameters.

  View details for Web of Science ID 000173601000024

  View details for PubMedID 11821861

• Activation of the PKB/AKT pathway by ICAM-2 IMMUNITY Perez, O. D., Kinoshita, S., Hitoshi, Y., Payan, D. G., Kitamura, T., Nolan, G. P., Lorens, J. B. 2002; 16 (1): 51-65

  Abstract

  We identified intracellular adhesion molecule-2 (ICAM-2) in a genetic screen as an activator of the PI3K/AKT pathway leading to inhibition of apoptosis. ICAM-2 induced tyrosine phosphorylation of ezrin and PI3K kinase membrane translocation, resulting in phosphatidylinositol 3,4,5 production, PDK-1 and AKT activation, and subsequent phosphorylation of AKT targets BAD, GSK3, and FKHR. ICAM-2 clustering protected primary human CD19+ cells from TNFalpha- and Fas-mediated apoptosis as determined by single-cell analysis. ICAM-2 engagement by CD19+ cells of its natural receptor, LFA-1, on CD4+ naive cells specifically induced AKT activity in the absence of an MHC-peptide interaction. These results attribute a novel signaling function to ICAM-2 that might suggest mechanisms by which ICAM-2 signals intracellular communication at various immunological synapses.

  View details for Web of Science ID 000173575700006

  View details for PubMedID 11825565

• Recombinant feline immunodeficiency virus vectors. Preparation and use. Methods in molecular medicine Curran, M. A., Nolan, G. P. 2002; 69: 335-350

  View details for PubMedID 11987787

• Nonprimate lentiviral vectors LENTIVIRAL VECTORS Curran, M. A., Nolan, G. P. 2002; 261: 75-105

  View details for Web of Science ID 000175080800004

  View details for PubMedID 11892254

• Resistance is futile: Assimilation of cellular machinery by HIV-1 IMMUNITY Perez, O. D., Nolan, G. P. 2001; 15 (5): 687-690

  Abstract

  HIV-1 budding appears to require Vps4 and Tsg101-two proteins that have links to endosomal sorting machinery. A picture emerges wherein divergent viruses recruit endosomal proteins like Tsg101 to gain access to ubiquitin processes that play a crucial role during viral budding.

  View details for Web of Science ID 000172528200002

  View details for PubMedID 11728331

• Genetic selection and the lure of SIN - A rapid method for exploring gene function uses alphavirus expression cloning. NATURE BIOTECHNOLOGY Nolan, G. P. 2001; 19 (9): 824-825

  View details for Web of Science ID 000170774000018

  View details for PubMedID 11533637

• A functional screen for genes inducing epidermal growth factor autonomy of human mammary epithelial cells confirms the role of amphiregulin ONCOGENE Berquin, I. M., Dziubinski, M. L., Nolan, G. P., Ethier, S. P. 2001; 20 (30): 4019-4028

  Abstract

  To gain better understanding of the molecular alterations responsible for the aggressive growth potential of epidermal growth factor receptor (EGFR)-positive breast cancers, we utilized an expression cloning strategy to seek gene products that mediate the EGF-independent growth of human breast cancer cells. A retroviral cDNA expression library was constructed from the EGFR-positive SUM-149PT cell line, and transduced into growth factor-dependent human mammary epithelial (HME) cells. Recipient cells were functionally selected for their ability to proliferate in serum-free, EGF-free medium. Library cDNAs were recovered from EGF-independent colonies by PCR amplification or by biological rescue. Clone H55a#1 contained a library insert encoding amphiregulin. This EGFR ligand was able to confer EGF independence when transduced into HME cells. SUM-149PT and H55a#1 cells overexpressed amphiregulin transcripts, and secreted moderate EGF-like activity in conditioned media, indicating a possible autocrine loop. EGFR membrane levels and constitutive phosphorylation were consistent with this hypothesis, as well as the sensitivity of the cells to an ErbB-specific kinase inhibitor. Expression of the WT1 Wilms' tumor suppressor gene, a transcriptional activator of amphiregulin, did not parallel amphiregulin transcript levels, suggesting that another factor regulates amphiregulin in SUM-149PT. Our data confirm the importance of amphiregulin in the etiology of breast cancer.

  View details for Web of Science ID 000169681500010

  View details for PubMedID 11494130

• Rapid production of retroviruses for efficient gene delivery to mammalian cells using 293T cell-based systems. Current protocols in immunology / edited by John E. Coligan ... [et al.] Swift, S., Lorens, J., Achacoso, P., Nolan, G. P. 2001; Chapter 10: Unit 10 17C-?

  Abstract

  This unit details the applications of one of the more common retroviral packaging systems, based on the highly transfectable 293T cell. The packaging system employs the use of the Phoenix cell lines. Calcium phosphate-mediated transfection is described for efficient introduction of retroviral vector plasmid DNA into the cells to generate high yields of virion-containing supernatant. An alternate protocol describes a method for transfecting retroviruses that contain a vesicular stomatitis virus G (VSV G) protein. Such virions are said to be "pseudotyped" with VSV G glycoprotein. Support protocols provide a simple method for concentrating VSV-G-pseudotyped retroviruses, as well as methods for culturing, cryopreserving, thawing, and drug selecting the Phoenix packaging cell lines. Finally, several methods for transfecting adherent or suspension cells with retroviruses are described.

  View details for DOI 10.1002/0471142735.im1017cs31

  View details for PubMedID 18432682

• Combination angiostatin and endostatin gene transfer induces synergistic antiangiogenic activity in vitro and antitumor efficacy in leukemia and solid tumors in mice MOLECULAR THERAPY Scappaticci, F. A., Smith, R., Pathak, A., Schloss, D., Lum, B., Cao, Y. H., Johnson, F., Engleman, E. G., Nolan, G. P. 2001; 3 (2): 186-196

  Abstract

  Angiostatin and endostatin are potent endothelial cell growth inhibitors that have been shown to inhibit angiogenesis in vivo and tumor growth in mice. However, tumor shrinkage requires chronic delivery of large doses of these proteins. Here we report synergistic antitumor activity and survival of animals when these factors are delivered in combination to tumors by retroviral gene transfer. We have demonstrated this efficacy in both murine leukemia and melanoma models. Complete loss of tumorigenicity was seen in 40% of the animals receiving tumors transduced by the combination of angiostatin and endostatin in the leukemia model. The synergy was also demonstrated in vitro on human umbilical vein endothelial cell differentiation and this antiangiogenic activity may suggest a mechanism for the antitumor activity in vivo. These findings imply separate pathways by which angiostatin and endostatin mediate their antiangiogenic effects. Together, these data suggest that a combination of antiangiogenic factors delivered by retroviral gene transfer may produce synergistic antitumor effects in both leukemia and solid tumors, thus avoiding long-term administration of recombinant proteins. The data also suggest that novel combinations of antiangiogenic factors delivered into tumors require further investigation as therapeutic modalities.

  View details for Web of Science ID 000167343800008

  View details for PubMedID 11237675

• Retroviral transduction of a T cell receptor specific for an Epstein-Barr virus-encoded peptide CLINICAL IMMUNOLOGY Orentas, R. J., Roskopf, S. J., Nolan, G. P., Nishimura, M. I. 2001; 98 (2): 220-228

  Abstract

  The Type II EBV malignancies nasopharyngeal carcinoma and EBV(+) Hodgkin's disease express three subdominant antigens, latency membrane protein (LMP) 1, LMP2, and EBNA-1. While adoptive immunotherapy with T cell lines for Type III EBV malignancy (such as posttransplant lymphoma, PTLD, which expresses the immunodominant EBNA-3 antigens) has been used to prevent and treat PTLD, the generation of class I MHC-restricted CTL suitable for the immunotherapy of Type II EBV malignancy is difficult. This is primarily due to the lack of anti-LMP or EBNA-1 CTL activity in many healthy volunteers. We have engineered, by retroviral transduction of the TCR, CTL that have the potential to recognize subdominant EBV latency antigens. Using the SAMEN retroviral vector we demonstrate the ability to transfer CTL activity from a LMP2 peptide-specific CTL clone to a stimulated PBMC population. TCR-transduced PBMC also secrete IFN-gamma upon coculture with LMP2 targets and maintain expression of the transduced TCR during subsequent mitogenic expansion.

  View details for DOI 10.1006/clim.2000.4977

  View details for Web of Science ID 000166952800009

  View details for PubMedID 11161978

• Statin-AE: a novel angiostatin-endostatin fusion protein with enhanced antiangiogenic and antitumor activity. Angiogenesis Scappaticci, F. A., CONTRERAS, A., Smith, R., Bonhoure, L., Lum, B., Cao, Y., Engleman, E. G., Nolan, G. P. 2001; 4 (4): 263-268

  Abstract

  The combination of angiostatin and endostatin has been shown to have synergistic antiangiogenic and antitumor effects when the genes for these proteins are delivered to tumor cells by retroviral gene transfer. Here we report the construction of a murine angiostatin-endostatin fusion gene (Statin-AE) which shows enhanced antiangiogenic activity on human umbilical vein endothelial cell (HUVEC) tube formation in vitro compared with angiostatin or endostatin alone. Similarly, the fusion gene demonstrates antiangiogenic effects in vivo and antitumor activity in a B16F10 melanoma model when co-delivered by retroviral packaging cell inoculation in mice. The fusion gene demonstrates significantly greater inhibition of tumor growth compared with angiostatin, endostatin or the combination of genes.

  View details for PubMedID 12197471

• Dominant effector genetics in mammalian cells NATURE GENETICS Xu, X., Leo, C., Jang, Y. J., Chan, E., Padilla, D., Huang, B. C., Lin, T., Gururaja, T., Hitoshi, Y., Lorens, J. B., Anderson, D. C., Sikic, B., Luo, Y., Payan, D. G., Nolan, G. P. 2001; 27 (1): 23-29

  Abstract

  We have expressed libraries of peptides in mammalian cells to select for trans-dominant effects on intracellular signaling systems. As an example-and to reveal pharmacologically relevant points in pathways that lead to Taxol resistance-we selected for peptide motifs that confer resistance to Taxol-induced cell death. Of several peptides selected, one, termed RGP8.5, was linked to upregulation of expression of the gene ABCB1 (also known as MDR1, for multiple drug resistance) in HeLa cells. Our data indicate that trans-dominant effector peptides can point to potential mechanisms by which signaling systems operate. Such tools may be useful in functional genomic analysis of signaling pathways in mammalian disease processes.

  View details for Web of Science ID 000166187900011

  View details for PubMedID 11137994

• Wnt signaling is required for thymocyte development and activates Tcf-1 mediated transcription EUROPEAN JOURNAL OF IMMUNOLOGY Staal, F. J., Meeldijk, J., Moerer, P., Jay, P., van de Weerdt, B. C., VAINIO, S., Nolan, G. P., Clevers, H. 2001; 31 (1): 285-293

  Abstract

  T cell factor / lymphocyte enhancer factor (Tcf/Lef) transcription factors complex with the transcriptional co-activator beta-catenin to transduce Wnt signals in a variety of developmental systems. The prototypic family member Tcf-1 is highly expressed in T lineage cells. Tcf1-/- mice are defective in cell cycling of early thymocyte stages. Here, we show that the interaction of beta-catenin with Tcf-1 is required for full thymocyte development. This interaction may be established by signals mediated by Wnt1 and Wnt4, leading to increased Tcf-dependent transcriptional activity in thymocytes, as demonstrated in Tcf-LacZ reporter mice. Transduction of fetal thymocytes with Wnt1 and Wnt4 results in increased survival in an in vitro cell culture system. Retroviral expression of soluble Wnt receptor mutants that block Wnt signaling inhibits thymocyte development. These results imply an important role for the Wnt cascade in thymocyte development.

  View details for Web of Science ID 000166487700032

  View details for PubMedID 11265645

• Neurotrophin dependence domain - A domain required for the mediation of apoptosis by the p75 neurotrophin receptor JOURNAL OF MOLECULAR NEUROSCIENCE Rabizadeh, S., Ye, X., Sperandio, S., Wang, J. J., Ellerby, H. M., Ellerby, L. M., Giza, C., Andrusiak, R. L., Frankowski, H., Yaron, Y., Moayeri, N. N., Rovelli, G., Evans, C. J., Butcher, L. L., Nolan, G. P., Assa-Munt, N., Bredesen, D. E. 2000; 15 (3): 215-229

  Abstract

  The mechanisms underlying neurotrophin dependence, and cellular dependent states in general, are unknown. We show that a 29 amino acid region in the intracellular domain of the common neurotrophin receptor, p75NTR, is required for the mediation of apoptosis by p75NTR. Furthermore, contrary to results obtained with Fas, monomeric p75NTR is required for apoptosis induction, whereas multimerization inhibits the pro-apoptotic effect. Within the 29-residue domain required for apoptosis induction by p75NTR, a 14-residue region is sufficient as a peptide inducer of apoptosis. This 14-residue peptide requires the positively charged carboxyterminal residues for its effect on cell death, and these same residues are required by the full-length p75NTR. These studies define a novel type of domain that mediates neurotrophin dependence, and suggest that other cellular dependent states may be mediated by proteins displaying similar domains.

  View details for Web of Science ID 000167749300008

  View details for PubMedID 11303785

• Expression of a peptide binding to receptor for activated C-kinase (RACK1) inhibits phorbol myristoyl acetate-stimulated phospholipase D activity in C3H/10T1/2 cells: dissociation of phospholipase D-mediated phosphatidylcholine breakdown from its synthesis BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS Thorsen, V. A., Bjorndal, B., Nolan, G., Fukami, M. H., Bruland, O., Lillehaug, J. R., Holmsen, H. 2000; 1487 (2-3): 163-176

  Abstract

  The C3H/10T1/2 Cl8 HAbetaC2-1 cells used in this study express a peptide with a sequence shown to bind receptor for activated C-kinase (RACK1) and inhibit cPKC-mediated cell functions. Phorbol myristoyl acetate (PMA) strongly stimulated phosphatidylcholine (PtdCho)-specific phospholipase D (PLD) activity in the C3H/10T1/2 Cl8 parental cell line, but not in Cl8 HAbetaC2-1 cells, indicating that full PLD activity in PMA-treated Cl8 cells is dependent on a functional interaction of alpha/betaPKC with RACK1. In contrast, the PMA-stimulated uptake of choline and its subsequent incorporation into PtdCho, were not inhibited in Cl8 HAbetaC2-1 cells as compared to Cl8 cells, indicating a RACK1-independent but PKC-mediated process. Increased incorporation of labelled choline into PtdCho upon PMA treatment was not associated with changes of either CDP-choline: 1,2-diacylglycerol cholinephosphotransferase activity or the CTP:phosphocholine cytidylyltransferase distribution between cytosol and membrane fractions in Cl8 and Cl8 HAbetaC2-1 cells. The major effect of PMA on the PtdCho synthesis in C3H/10T1/2 fibroblasts was to increase the cellular uptake of choline. As a supporting experiment, we inhibited PMA-stimulated PtdH formation by PLD, and also putatively PtdH-derived DAG, in Cl8 cells with 1-butanol. Butanol did not influence the incorporation of [(14)C]choline into PtdCho. The present study shows: (1) PMA-stimulated PLD activity is dependent on a functional interaction between alpha/betaPKC and RACK1 in C3H/10T1/2 Cl8 fibroblasts; and (2) inhibition of PLD activity and PtdH formation did not reduce the cellular uptake and incorporation of labelled choline into PtdCho, indicating that these processes are not directly regulated by PtdCho-PLD activity in PMA-treated C3H/10T1/2 Cl8 fibroblasts.

  View details for Web of Science ID 000089680200006

  View details for PubMedID 11018469

• Retroviral delivery of peptide modulators of cellular functions MOLECULAR THERAPY Lorens, J. B., Bennet, M. K., Pearsall, D. M., Throndset, W. R., Rossi, A. B., Armstrong, R. J., Fox, B. P., Chan, E. H., Luo, Y., Masuda, E., Ferrick, D. A., Anderson, D. C., Payan, D. G., Nolan, G. P. 2000; 1 (5): 438-447

  Abstract

  Stable transduction of genetic material, in combination with sensitive methodologies for in vivo study of cell physiology, provides an opportunity to efficiently evaluate the functions of regulatory proteins. To dissect the minimal therapeutic function of such proteins, we have stably expressed protein microdomains as fusions, composed of short peptides, and detected specific subfunctions distinct from holoprotein function, using flow cytometry and other techniques. We demonstrate that retroviral delivery of the 24-amino-acid proliferating cell nuclear antigen-binding motif (p21C), derived from the C-terminus of the cell cycle inhibitor protein, p21, is sufficient to induce cell cycle arrest. Cells expressing this peptide motif reversibly execute both G1- and G2-checkpoint controls that are normally activated subsequent to interference with DNA synthesis. The p21C effect is distinct from results obtained with an intact p21 protein that also binds cyclin-CDK complexes and arrested cells exclusively at the G1/S transition. Thus, microdomains can exert unique biological effects compared to the parental molecules from which they were derived. To further evaluate the peptide delivery strategy, we analyzed the role of various kinases in IgE-mediated stimulation of mast cell exocytosis. Primary bone marrow-derived mast cells were transduced with retroviral constructs encoding short-kinase inhibitor motifs and analyzed by flow cytometry for effects on exocytosis. We found that a specific protein kinase A (PKA) inhibitor peptide suppressed IgE-mediated stimulation of mast cell exocytosis. This anti-exocytotic effect was mimicked by a small molecule inhibitor of PKA (KT5720). Thus, the ability to express protein microdomains can be a powerful means to subtly perturb cellular physiology in manners that reveal new paths for therapeutic intervention. We believe that such approaches might allow for new forms of gene therapy to become available.

  View details for DOI 10.1006/mthe.2000.0063

  View details for Web of Science ID 000090048100009

  View details for PubMedID 10933965

• Targeting rare populations of murine antigen-specific T lymphocytes by retroviral transduction for potential application in gene therapy for autoimmune disease JOURNAL OF IMMUNOLOGY Costa, G. L., Benson, J. M., Seroogy, C. M., Achacoso, P., Fathman, C. G., Nolan, G. P. 2000; 164 (7): 3581-3590

  Abstract

  CD4+ T cells are important mediators in the pathogenesis of autoimmunity and would therefore provide ideal candidates for lymphocyte-based gene therapy. However, the number of Ag-specific T cells in any single lesion of autoimmunity may be quite low. Successful gene transfer into autoantigen-specific CD4+ T cells would serve as an ideal vehicle for site-targeted gene therapy if it were possible to transduce preferentially the small number of autoantigen-specific T cells. In this study we have demonstrated that retroviral infection of CD4+ lymphocytes from either autoantigen-stimulated TCR transgenic mice, or Ag-activated immunized nontransgenic mice, with a retroviral vector (pGCIRES), resulted in the transduction of only the limited number of Ag-reactive CD4+ T cells. In contrast, polyclonal activation of the same cultures resulted in transduction of non-antigen-specific lymphocytes. Transduction of Ag-reactive CD4+ T cells with pGCIRES retrovirus encoding the regulatory genes IL-4 (IL4) and soluble TNF receptor (STNFR) resulted in stable integration and long-term expression of recombinant gene products. Moreover, expression of the pGCIRES marker protein, GFP, directly correlated with the expression of the upstream regulatory gene. Retroviral transduction of CD4+ T cells targeted specifically Ag-reactive cells and was cell cycle-dependent and evident only during the mitosis phase. These studies suggest that retroviral transduction of autoantigen-specific murine CD4+ T cells, using the pGCIRES retroviral vector, may provide a potential method to target and isolate the low frequency of autoantigen-specific murine CD4+ T cells, and provides a rational approach to gene therapy in animal models of autoimmunity.

  View details for Web of Science ID 000086020700019

  View details for PubMedID 10725713

• Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors MOLECULAR THERAPY Curran, M. A., Kaiser, S. M., Achacoso, P. L., Nolan, G. P. 2000; 1 (1): 31-38

  Abstract

  Second- and third-generation three-plasmid vector systems, termed FELIX, were constructed from feline immunodeficiency virus (FIV). To enhance vector production, the weak FIV long terminal repeat promoter was replaced with the human cytomegalovirus enhancer/promoter. To construct a minimal system in which Gag-Pol was the only viral protein present, the cytoplasmic transport element was used in place of the FIV Rev-RRE system to facilitate nuclear export of Gag-Pol and the transfer vector. Unconcentrated vector titers routinely exceeded 1 x 10(6) IU/mL for most constructs tested. Second- and optimized third-generation vectors were capable of efficiently infecting G1/S- and G2/M-arrested cells. FIV-based FELIX vectors transduced human dendritic cells, hepatocytes, and aortic smooth muscle with efficiencies similar to that of a control 3T3 cell line. All three of these primary cell types were transducible by both the second- and third-generation FELIX vectors, demonstrating that FIV Gag-Pol alone contains the determinants necessary for transduction of primary cells. In cross-packaging tests, we observed that HIV Gag-Pol does not substantially package FIV vectors; consequently, use of such vectors in human immunodeficiency virus-infected cells should not lead to efficient mobilization of the inserted gene. Thus, this FIV-based vector system offers high efficiency and stable delivery of genes to numerous nondividing and primary cell types, opening new avenues for biological inquiry into normal human cells.

  View details for Web of Science ID 000090018300008

  View details for PubMedID 10933909

• Retroviral and lentiviral gene therapy for autoimmune disease. Current directions in autoimmunity Smith, R., Nolan, G. P. 2000; 2: 167-188

  View details for PubMedID 11791455

• Antiangiogenic gene therapy of murine B16F10 melanoma and L1210 leukemia tumors with angiostatin and endostatin containing MMuLV retroviral vectors. Scappaticci, F. A., Smith, R., Schloss, D., Cao, Y., Engleman, E. G., Nolan, G. P., Leung, L. AMER SOC HEMATOLOGY. 1999: 420B–420B

  View details for Web of Science ID 000083790701952

• NF-kappa B to the rescue - RELs, apoptosis and cellular transformation TRENDS IN GENETICS Foo, S. Y., Nolan, G. P. 1999; 15 (6): 229-235

  Abstract

  The REL/NF-kappaB/IkappaB superfamily of signal transducers and transcription factors are paradigmatic of molecular mechanisms by which rapid responses in the immune system can be achieved. NF-kappaB proteins have been implicated in diverse processes such as the ontogeny of the immune system, immune responses to pathogens and, importantly, in contributions to the multistage processes of oncogenesis, as described in this review. NF-kappaB and its regulators, the IkappaBs, are linked to pro- and anti-apoptotic events as well as signaling systems contributing to cellular transformation. How are these disparate events controlled to effect normal and abnormal processes in cells? Here we explore a few of the many events in which NF-kappaB appears to participate and processes that integrate signals to control important stages of oncogenesis.

  View details for Web of Science ID 000081380100010

  View details for PubMedID 10354583

• Identification of RIP3, a RIP-like kinase that activates apoptosis and NF kappa B CURRENT BIOLOGY Yu, P. W., Huang, B. C., Shen, M., Quast, J., Chan, E., Xu, X., Nolan, G. P., Payan, D. G., Luo, Y. 1999; 9 (10): 539-542

  Abstract

  The tumor necrosis factor receptor 1 (TNFR1) and the Fas receptor recruit complexes formed by the interactions between RIP kinase, TRADD, FADD and RAIDD - adaptor proteins that contain death domains - which in turn recruit other proteins to initiate signaling [1][2][3][4][5]. To identify proteins associated with the TNF signaling pathway, we performed a yeast two-hybrid interaction screen using RIP as bait. We isolated a kinase, RIP3, which shares homology with the kinase domain of RIP and RIP2 (also known as Rick or CARDIAK). RIP3 could be co-immunoprecipitated with RIP, TRAF2 and TNFR1 in mammalian cells. The carboxy-terminal domain of RIP3, like that of RIP, could activate the transcription factor NFkappaB and induce apoptosis when expressed in mammalian cells. Interestingly, this region shares no significant sequence homology to the death domain of RIP, the caspase-recruiting domain (CARD) of RIP2 [6][7][8] or any other apoptosis-inducing domain. As with RIP and RIP2, the kinase domain of RIP3 was not required for either NFkappaB activation or apoptosis induction. Overexpression of a dominant-negative mutant of RIP3 strongly inhibited the caspase activation but not the NFkappaB activation induced by TNFalpha. Therefore, RIP3 appears to function as an intermediary in TNFalpha-induced apoptosis.

  View details for Web of Science ID 000080465900020

  View details for PubMedID 10339433

• Analysis of genomic integrity and p53-dependent G(1) checkpoint in telomerase-induced extended-life-span human fibroblasts MOLECULAR AND CELLULAR BIOLOGY Vaziri, H., Squire, J. A., Pandita, T. K., Bradley, G., Kuba, R. M., Zhang, H. H., Gulyas, S., Hill, R. P., Nolan, G. P., Benchimol, S. 1999; 19 (3): 2373-2379

  Abstract

  Life span determination in normal human cells may be regulated by nucleoprotein structures called telomeres, the physical ends of eukaryotic chromosomes. Telomeres have been shown to be essential for chromosome stability and function and to shorten with each cell division in normal human cells in culture and with age in vivo. Reversal of telomere shortening by the forced expression of telomerase in normal cells has been shown to elongate telomeres and extend the replicative life span (H. Vaziri and S. Benchimol, Curr. Biol. 8:279-282, 1998; A. G. Bodnar et al., Science 279:349-352, 1998). Extension of the life span as a consequence of the functional inactivation of p53 is frequently associated with loss of genomic stability. Analysis of telomerase-induced extended-life-span fibroblast (TIELF) cells by G banding and spectral karyotyping indicated that forced extension of the life span by telomerase led to the transient formation of aberrant structures, which were subsequently resolved in higher passages. However, the p53-dependent G1 checkpoint was intact as assessed by functional activation of p53 protein in response to ionizing radiation and subsequent p53-mediated induction of p21(Waf1/Cip1/Sdi1). TIELF cells were not tumorigenic and had a normal DNA strand break rejoining activity and normal radiosensitivity in response to ionizing radiation.

  View details for Web of Science ID 000078697900074

  View details for PubMedID 10022923

• Growth inhibition and apoptosis due to restoration of E2A activity in T cell acute lymphoblastic leukemia cells JOURNAL OF EXPERIMENTAL MEDICINE Park, S. T., Nolan, G. P., Sun, X. H. 1999; 189 (3): 501-508

  Abstract

  Two models have been proposed for the molecular mechanism by which the Tal1 oncogene causes T cell acute lymphoblastic leukemia (T-ALL). The activation model suggests that Tal1 as heterodimers with the E2A transcription factor activates the expression of oncogenes. The inhibition model postulates that Tal1 interferes with the tumor-suppressing function of E2A. In the Jurkat T cell line, originally derived from a patient with T-ALL, Tal1 is complexed with E2A proteins and the transcriptional activity of E2A is very low. When E2A activity was restored by expressing an E2A-Tal1 fusion protein, E-T/2, the Jurkat cells underwent growth arrest and subsequently apoptosis, thus supporting the inhibition model and suggesting that E2A loss may contribute to leukemic progression.

  View details for Web of Science ID 000078470300007

  View details for PubMedID 9927512

• Single cell analysis and selection of living retrovirus vector-corrected mucopolysaccharidosis VII cells using a fluorescence-activated cell sorting-based assay for mammalian beta-glucuronidase enzymatic activity JOURNAL OF BIOLOGICAL CHEMISTRY Lorincz, M. C., Parente, M. K., Roederer, M., Nolan, G. P., Diwu, Z. J., Martin, D. I., Herzenberg, L. A., Wolfe, J. H. 1999; 274 (2): 657-665

  Abstract

  Mutations in the acid beta-glucuronidase gene lead to systemic accumulation of undegraded glycosaminoglycans in lysosomes and ultimately to clinical manifestations of mucopolysaccharidosis VII (Sly disease). Gene transfer by retrovirus vectors into murine mucopolysaccharidosis VII hematopoietic stem cells or fibroblasts ameliorates glycosaminoglycan accumulation in some affected tissues. The efficacy of gene therapy for mucopolysaccharidosis VII depends on the levels of beta-glucuronidase secreted by gene-corrected cells; therefore, enrichment of transduced cells expressing high levels of enzyme prior to transplantation is desirable. We describe the development of a fluorescence-activated cell sorter-based assay for the quantitative analysis of beta-glucuronidase activity in viable cells. Murine mucopolysaccharidosis VII cells transduced with a beta-glucuronidase retroviral vector can be isolated by cell sorting on the basis of beta-glucuronidase activity and cultured for further use. In vitro analysis revealed that sorted cells have elevated levels of beta-glucuronidase activity and secrete higher levels of cross-correcting enzyme than the population from which they were sorted. Transduced fibroblasts stably expressing beta-glucuronidase after subcutaneous passage in the mucopolysaccharidosis VII mouse can be isolated by cell sorting and expanded ex vivo. A relatively high percentage of these cells maintain stable expression after secondary transplantation, yielding significantly higher levels of enzymatic activity than that generated in the primary transplant.

  View details for Web of Science ID 000077968500014

  View details for PubMedID 9872999

• Generation of retroviral vector for clinical studies using transient transfection HUMAN GENE THERAPY Yang, S. L., Delgado, R., King, S. R., Woffendin, C., Barker, C. S., Yang, Z. Y., Xu, L., Nolan, G. P., Nabel, G. J. 1999; 10 (1): 123-132

  Abstract

  Transient transfection of 293T cells was utilized to produce high-titer murine recombinant retroviral vectors for clinical studies. This system was initially optimized by gene transfer using different retroviral envelope proteins into activated human CD4+ T lymphocytes in vitro. Higher titer and infectivity were obtained than with stable murine producer lines; titers of 0.3-1 x 10(7) infectious units per milliliter for vectors encoding the green fluorescent protein (GFP) were achieved. Virions pseudotyped with envelope proteins from gibbon ape leukemia virus or amphotropic murine leukemia virus resulted in gene transfer of > or = 50% in CD4+ human T lymphocytes with this marker. Gene transfer of Rev M10 with this vector conferred resistance to HIV infection compared with negative controls in the absence of drug selection. Thus, the efficiency of transduction achieved under these conditions obviated the need to include selection to detect biologic effects in T cells. Finally, a protocol for the production of large-scale supernatants using transient transfection was optimized up to titers of 1.9 x 10(7) IU/ml. These packaging cells can be used to generate high-titer virus in sufficient quantities for clinical studies and will facilitate the rapid, cost-effective generation of improved retroviral, lentiviral, or other viral vectors for human gene therapy.

  View details for Web of Science ID 000077960800013

  View details for PubMedID 10022537

• Local delivery of TNF by retrovirus-transduced T lymphocytes exacerbates experimental autoimmune encephalomyelitis CLINICAL IMMUNOLOGY Dal Canto, R. A., Shaw, M. K., Nolan, G. P., Steinman, L., Fathman, C. G. 1999; 90 (1): 10-14

  Abstract

  Experimental autoimmune encephalomyelitis (EAE) is an inflammatory autoimmune disease of the central nervous system that serves as a model for the human disease multiple sclerosis. Paralysis is "induced" by CD4+ T cells of the Th1 phenotype. Tumor necrosis factor (TNF), a Th1 type cytokine, has been shown to be upregulated in the CNS during the onset of EAE, and systemic manipulations of TNF have had substantial effects on disease progression. However, the precise role of TNF in EAE has been called into question by recent experiments utilizing TNF and lymphotoxin knockout mice. We demonstrate here that the local delivery of TNF by myelin basic protein (MBP)-specific T cells, retrovirally transduced to express TNF, exacerbated MBP-induced disease following adoptive transfer into syngeneic mice.

  View details for Web of Science ID 000079158600002

  View details for PubMedID 9884347

• Evolution of peptides that modulate the spectral qualities of bound, small-molecule fluorophores CHEMISTRY & BIOLOGY Rozinov, M. N., Nolan, G. P. 1998; 5 (12): 713-728

  Abstract

  Fluorophore dyes are used extensively in biomedical research to sensitively assay cellular constituents and physiology. We have created, as proof of principle, fluorophore dye binding peptides that could have applications in fluorescent dye-based approaches in vitro and in vivo.A panel of Texas red, Rhodamine red, Oregon green 514 and fluorescein binding peptides, termed here 'fluorettes', was selected via biopanning of a combinatorial library of 12-mer peptides fused to a minor coat pIII protein of the filamentous bacteriophage M13. The 'best' fluorette sequences from each of the groups were subjected to further mutagenesis, followed by a second biopanning to select a new generation of improved fluorettes. Phage were selected that had higher avidity for each fluorophore except Rhodamine red. Of these, peptides were characterized that could specifically and with high affinity bind at least one dye, Texas red, in solution. In addition, the binding of certain peptides to Texas red shifted the peak excitation and/or the emission spectra of the bound dye.Peptides in the context of phage display could readily be selected that could bind to small-molecule fluorophores. The affinities of selected mutant fluorettes could be increased by mutation and further selection. Only a subset of the free peptides could bind free dyes in solution, suggesting that phage context contributed to the selection and ability of certain peptidic regions to independently bind the dyes. Future screens might lead to the creation of other dye-binding peptides with novel characteristics or Texas red derivatives with cross-linking substituents might be designed to increase the utility of the system.

  View details for Web of Science ID 000077383600007

  View details for PubMedID 9862799

• Host control of HIV-1 parasitism in T cells by the nuclear factor of activated T cells CELL Kinoshita, S., Chen, B. K., Kaneshima, H., Nolan, G. P. 1998; 95 (5): 595-604

  Abstract

  Post HIV-1 entry, productive HIV-1 infection of primary T cells requires overcoming several cellular blocks to provirus establishment and replication. Activation of unknown host intracellular events overcomes such inhibitory steps and is concomitant with HIV-1 replication. We show that the transcription factor NFATc was sufficient as a cellular factor to induce a highly permissive state for HIV-1 replication in primary CD4+ T cells. NFATc overcame a blockade at reverse transcription and permitted active HIV-1 replication. Pharmacologic blockade of endogenous NFAT activity by FK506 or CsA inhibited synthesis of reverse transcription and also potently blocked HIV-1 replication. T cells therefore can become competent for HIV-1 replication by control of regulated host factors such as the NFATc transcription factor. The host mechanisms regulated by such permissivity factors are potential targets for anti-HIV-1 therapy.

  View details for Web of Science ID 000077253700007

  View details for PubMedID 9845362

• The ICAM-2 alpha-actinin binding site mediates a survival signal Lorens, J. B., Hitoshi, Y., Caldwell, J. S., Nolan, G. P. AMER SOC CELL BIOLOGY. 1998: 367A–367A

  View details for Web of Science ID 000076906702132

• Expression vectors and delivery systems - Editorial overview CURRENT OPINION IN BIOTECHNOLOGY Nolan, G. P., Shatzman, A. R. 1998; 9 (5): 447-450

  View details for Web of Science ID 000076409800001

  View details for PubMedID 9821270

• Local delivery of cytokines by retrovirally transduced antigen-specific TCR+ hybridoma cells in experimental autoimmune encephalomyelitis Vth International Workshop on Cytokines at the Joint Meeting of the Ares-Sereno Foundation / European-Cytokine-Society Dal Canto, R. A., Costa, G., Shaw, M. D., Seroogy, C., Nolan, G. P., Fathman, C. G. JOHN LIBBEY EUROTEXT LTD. 1998: 83–91

  Abstract

  Autoimmune diseases in humans represent an immune attack on self tissue. Current therapies for almost all autoimmune diseases utilize potent and nonspecific immunosuppressive regimens. These therapies are complicated by their side effects and also place the patient at increased risk for opportunistic infections and malignancies. Our current understanding of immune mechanisms underlying autoimmune diseases remains limited. Ongoing studies include identifying genes that predispose an individual to developing autoimmunity, identification of autoantigens that trigger or perpetuate autoimmunity, and studies of immune cell interactions that lead to immune response. Although it may be many years before a full understanding of autoimmunity is obtained, treatment in animal models of autoimmune disease and some human clinical trials have begun to study alternative treatment approaches to therapy of autoimmune disease. Future therapies for autoimmune diseases should target the inappropriate autoimmune response. This article will describe the use of gene therapy in the treatment of autoimmune disease. We believe that autoimmunity can be ameliorated by delivering trans-acting immunoregulatory molecules by retrovirally transduced autoantigen specific T cells that home to lesions of autoimmunity. Until recently, there has not been a practical alternative to systemic delivery of immunoregulatory molecules, however systemic delivery suffers from toxic side effects and dangerous global immunosuppression. In order to study immune regulation using retroviral transduction for local delivery of immunoregulatory products, we used myelin basic protein (MBP) reactive T cell hybridomas in the murine model of multiple sclerosis (MS), experimental allergic encephalomyelitis (EAE). In this report, we show that MBP reactive T cell hybridomas transduced to express IL-4 or TNF, ameliorated or exacerbated disease, respectively. Additionally, the effects of these cells were dependent on T cell receptor (TCR) expression, indicating that the effects were due to homing of the T cells and the local delivery of cytokines. We believe that gene therapy, allowing local delivery of immunoregulatory proteins by autoantigen specific T cells, represents an interesting potential therapy for autoimmune disease.

  View details for Web of Science ID 000076885900015

  View details for PubMedID 9831193

• Toso, a cell surface, specific regulator of Fas-induced apoptosis in T cells IMMUNITY Hitoshi, Y., Lorens, J., Kitada, S. I., Fisher, J., Labarge, M., Ring, H. Z., FRANCKE, U., Reed, J. C., Kinoshita, S., Nolan, G. P. 1998; 8 (4): 461-471

  Abstract

  Fas is a surface receptor that can transmit signals for apoptosis. Using retroviral cDNA library-based functional cloning we identified a gene, toso, that blocks Fas-mediated apoptosis. Toso expression was confined to lymphoid cells and was enhanced after cell-specific activation processes in T cells. Toso appeared limited to inhibition of apoptosis mediated by members of the TNF receptor family and was capable of inhibiting T cell self-killing induced by TCR activation processes that up-regulate Fas ligand. We mapped the effect of Toso to inhibition of caspase-8 processing, the most upstream caspase activity in Fas-mediated signaling, potentially through activation of cFLIP. Toso therefore serves as a novel regulator of Fas-mediated apoptosis and may act as a regulator of cell fate in T cells and other hematopoietic lineages.

  View details for Web of Science ID 000073348000008

  View details for PubMedID 9586636

• Effects of locally delivered cytokines on EAE Dal Canto, R. A., Shaw, M. K., Steinman, L., Nolan, G. P., Fathman, C. G. FEDERATION AMER SOC EXP BIOL. 1998: A308–A308

  View details for Web of Science ID 000076006401793

• Cardiac development - Transcription and the broken heart NATURE Nolan, G. P. 1998; 392 (6672): 129-130

  View details for Web of Science ID 000072462700030

  View details for PubMedID 9515954

• High-efficiency gene transfer and selection of human hematopoietic progenitor cells with a hybrid EBV/retroviral vector expressing the green fluorescence protein CANCER RESEARCH Grignani, F., Kinsella, T., Mencarelli, A., Valtieri, M., Riganelli, D., Grignani, F., Lanfrancone, L., Peschle, C., Nolan, G. P., Pelicci, P. G. 1998; 58 (1): 14-19

  Abstract

  We report a retroviral expression vector (PINCO) that allows high-efficiency gene transfer and selection of hemopoietic progenitor cells (HPCs). The main characteristics of this vector are the presence outside the two long terminal repeats of the EBV origin of replication and the EBNA-1 gene and the presence in the retrovirus of the cDNA that encodes for the enhanced green fluorescence protein (GFP), controlled by a cytomegalovirus promoter. Transient transfection of PINCO in Phoenix packaging cells results in episomal propagation of the plasmid and generates viral titers as high as 10(7) colony-forming units/ml. Infection of established cell lines with the PINCO retrovirus yields more than 95% GFP-expressing cells. GFP expression remains stable for months in infected cell cultures and can easily be monitored by fluorescent microscopy or fluorescence-activated cell-sorting (FACS) analysis of living cells. The PINCO vector allows efficient expression of a second gene (thymidine kinase, Shc, and PML), and there is strict correlation between GFP and second gene expression levels in the infected cells. PINCO was used to infect human HPCs; infection efficiency was about 50%. GFP-positive cells can be FACS sorted to yield a homogeneous population of infected cells. FACS-sorted GFP-positive HPC cells have, with respect to unfractionated HPC cells, the same frequency of long-term culture initiating cells and an identical capacity to undergo multilineage and unilineage differentiation. The entire gene transfer procedure, from the transfection of the packaging cell line to the infection of target cells, requires less than a week. The high viral titer and the easy obtainment of homogeneously infected cell populations without drug selection procedures make PINCO an ideal vector for gene transfer of human primary hemopoietic cells.

  View details for Web of Science ID 000071229000004

  View details for PubMedID 9426049

• Inhibition of T cell and promotion of natural killer cell development by the dominant negative helix loop helix factor Id3 JOURNAL OF EXPERIMENTAL MEDICINE Heemskerk, M. H., Blom, B., Nolan, G., Stegmann, A. P., Bakker, A. Q., Weijer, K., Res, P. C., Spits, H. 1997; 186 (9): 1597-1602

  Abstract

  Bipotential T/natural killer (NK) progenitor cells are present in the human thymus. Despite their bipotential capacity, these progenitors develop predominantly to T cells in the thymus. The mechanisms controlling this developmental choice are unknown. Here we present evidence that a member(s) of the family of basic helix loop helix (bHLH) transcription factors determines lineage specification of NK/T cell progenitors. The natural dominant negative HLH factor Id3, which blocks transcriptional activity of a number of known bHLH factors, was expressed in CD34+ progenitor cells by retrovirus-mediated gene transfer. Constitutive expression of Id3 completely blocks development of CD34+ cells into T cells in a fetal thymic organ culture (FTOC). In contrast, development into NK cells in an FTOC is enhanced. Thus, the activity of a bHLH transcription factor is necessary for T lineage differentiation of bipotential precursors, in the absence of which a default pathway leading to NK cell development is chosen. Our results identify a molecular switch for lineage specification in early lymphoid precursors of humans.

  View details for Web of Science ID A1997YE94200021

  View details for PubMedID 9348318

• Engineered virus targets HIV-infected cells: interview with Garry Nolan, Ph.D. Interview by John S. James. AIDS treatment news Nolan, G. 1997: 1-?

  View details for PubMedID 11364688

• Harnessing viral devices as pharmaceuticals: Fighting HIV-1's fire with fire CELL Nolan, G. P. 1997; 90 (5): 821-824

  View details for Web of Science ID A1997XV56300001

  View details for PubMedID 9298892

• Introduction of soluble proteins into the MHC class I pathway by conjugation to an HIV tat peptide JOURNAL OF IMMUNOLOGY Kim, D. T., Mitchell, D. J., Brockstedt, D. G., Fong, L., Nolan, G. P., Fathman, C. G., Engleman, E. G., Rothbard, J. B. 1997; 159 (4): 1666-1668

  Abstract

  Protection against most intracellular pathogens requires T cells that recognize pathogen-derived peptides in association with MHC class I molecules on the surface of infected cells. However, because exogenous proteins do not ordinarily enter the cytosol and access the MHC class I-processing pathway, protein-based vaccines that induce class I-restricted CTL responses have proved difficult to design. We have addressed this problem by conjugating proteins, such as OVA, to a short cationic peptide derived from HIV-1 tat (residues 49-57). When APC were exposed in vitro to such protein conjugates, they processed and presented the peptides in association with MHC class I molecules and stimulated CD8+ Ag-specific T cells. Moreover, Ag-specific CTLs were generated in vivo by immunizing mice with histocompatible dendritic cells that had been exposed to protein-tat conjugates.

  View details for Web of Science ID A1997XP43800012

  View details for PubMedID 9257826

• Local delivery of interleukin 4 by retrovirus-transduced T lymphocytes ameliorates experimental autoimmune encephalomyelitis JOURNAL OF EXPERIMENTAL MEDICINE Shaw, M. K., Lorens, J. B., Dhawan, A., DalCanto, R., Tse, H. Y., Tran, A. B., Bonpane, C., Eswaran, S. L., Brocke, S., Sarvetnick, N., Steinman, L., Nolan, G. P., Fathman, C. G. 1997; 185 (9): 1711-1714

  Abstract

  Experimental autoimmune encephalomyelitis (EAE) is an inflammatory autoimmune disease of the central nervous system which serves as a model for the human disease multiple sclerosis. We demonstrate here that encephalitogenic T cells, transduced with a retroviral gene, construct to express interleukin 4, and can delay the onset and reduce the severity of EAE when adoptively transferred to myelin basic protein-immunized mice. Thus, T lymphocytes transduced with retroviral vectors can deliver "regulatory cytokines" in a site-specific manner and may represent a viable therapeutic strategy for the treatment of autoimmune disease.

  View details for Web of Science ID A1997WY11700020

  View details for PubMedID 9151908

• The T cell activation factor NF-ATc positively regulates HIV-1 replication and gene expression in T cells IMMUNITY Kinoshita, S., Su, L. S., Amano, M., Timmerman, L. A., Kaneshima, H., Nolan, G. P. 1997; 6 (3): 235-244

  Abstract

  Clinical deterioration in human immunodeficiency virus type 1 (HIV-1) infection is associated with increased levels of viral replication and burden in the peripheral blood and lymphoid organs. T cell activation and ensuing cellular gene activation can be critical for HIV-1 replication. The hypothesis that the nuclear factor of activated T cells (NF-AT) may influence HIV-1 replication is therefore compelling given the tight correlation of HIV-1 transcriptional induction to T cell activation. We report that certain NF-AT(Rel) family members productively bind the kappaB regulatory elements, synergize with NF-kappaB and Tat in transcriptional activation of HIV-1, and enhance HIV-1 replication in T cells. These results link regulatory factors critical to T cell commitment directly to HIV-1 replication.

  View details for Web of Science ID A1997XC61800003

  View details for PubMedID 9075924

• Local delivery of interleukin-4 by retrovirus-transduced lymphocytes ameliorates experimental autoimmune encephalomyelitis. Shaw, M. K., Lorens, J. B., Dhawan, A., DalCanto, R., Tse, H. Y., Tran, A. B., Bonpane, C., Eswaran, S. L., Brocke, S., Sarvetnick, N., Steinman, L., Nolan, G. P., Fathman, C. G. MOSBY-ELSEVIER. 1997: 1976–76

  View details for Web of Science ID A1997WH14201970

• Generation of high-titer, helper-free retroviruses by transient transfection. Methods in molecular medicine Pear, W. S., Scott, M. L., Nolan, G. P. 1997; 7: 41-57

  Abstract

  Retroviral gene transfer is presently one of the most powerful techniques for introducing stably heritable genetic material into mammalian cells (reviewed in ref. 1). One serious drawback of this technique, however, has been the difficulty in readily producing high-titer recombinant retroviruses. For many applications, such as infecting rare target cells or the majority of cells in tissue culture, the recombinant virus titer must be at least 10(6) infectious units/mL. Although one can usually obtain high-titer mixtures of recombinant and replication-competent retroviruses in a relatively short time, many applications such as cell marking studies or studying genes in vivo demand freedom from replication-competent virus.

  View details for DOI 10.1385/0-89603-484-4:41

  View details for PubMedID 24493417

• Transport of immunogens into the MHC class I and II pathways by a peptide from HIV tat 40th Symposium of the Alfred-Benzon-Foundation on HLA and Disease - the Molecular Basis Rothbard, J., Kim, D., Mitchell, D., Bockstedt, D., Fong, L., Nolan, G., Fathman, C. G., Engleman, E. MUNKSGAARD. 1997: 161–175

  View details for Web of Science ID A1997BJ08M00012

• Transglutaminase 1 delivery to lamellar ichthyosis keratinocytes HUMAN GENE THERAPY Choate, K. A., Kinsella, T. M., WILLIAMS, M. L., Nolan, G. P., Khavari, P. A. 1996; 7 (18): 2247-2253

  Abstract

  Therapeutic gene delivery in severe genetic skin disease may require production of a uniformly corrected population of cells capable of regeneration of normal skin elements when returned to the host. To achieve this, we have used lamellar ichthyosis (LI), a disorder of epidermal differentiation recently associated with defects in keratinocyte transglutaminase (TGase1), as a prototype. We have used a high-efficiency retroviral delivery approach to uniformly restore normal levels of TGase1 expression to primary keratinocytes from severely affected LI patients previously lacking TGase1. Delivered TGase1 was correctly targeted to membrane association and restored patient cell transglutaminase activity levels to normal. Corrected primary LI patient keratinocytes also demonstrated restoration of previously defective involucrin cross-linking and in vitro measures of cornification to levels found in normal cells. These results indicate that efficient TGase1 delivery to early passage keratinocytes can produce a population of corrected LI patient cells. The capability to produce such cells may provide a basis for future efforts at gene therapy for genetic skin disease.

  View details for Web of Science ID A1996WD32900007

  View details for PubMedID 8953315

• NF-kappa B homodimer binding within the HIV-1 initiator region and interactions with TFII-I PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Montano, M. A., Kripke, K., Norina, C. D., Achacoso, P., Herzenberg, L. A., Roy, A. L., Nolan, G. P. 1996; 93 (22): 12376-12381

  Abstract

  We show that the binding of Rel p50 and p52 homodimers at sites within the transcriptional initiation region of HIV-1 provides for their ability to interact with other proteins that bind the initiator. The binding of one such protein, the initiator protein TFII-I, to the initiation region of HIV-1 is augmented in the presence of Rel p50 and Rel p52 homodimers. Consistent with this, in vitro Rel homodimers potentiate HIV-1 transcription in a manner dependent upon TFII-I. The findings suggest that Rel dimers may regulate HIV-1 transcription in two ways. First, through binding at the kappa B enhancer sites at (-104 to -80), NF-kappa B p50:p65 participates in classical transcriptional activation. Second, Rel dimers such as p50 or p52 might bind at initiator sequences to regulate the de novo binding of components of certain preinitiation complexes. These findings, and the existence of Rel binding sites at the initiators of other genes, suggest roles for Rel proteins in early events determining transcriptional control.

  View details for Web of Science ID A1996VP93700060

  View details for PubMedID 8901589

• Identification of an oncogenic form of the thrombopoietin receptor MPL using retrovirus-mediated gene transfer BLOOD Onishi, M., Mui, A. L., Morikawa, Y., Cho, L., Kinoshita, S., Nolan, G. P., Gorman, D. M., Miyajima, A., Kitamura, T. 1996; 88 (4): 1399-1406

  Abstract

  Thrombopoietin and its receptor (MPL) are important regulators of megakaryopoiesis. We have identified an activating mutation of MPL using a combination of a retrovirus-mediated gene transfer and polymerase chain reaction-driven random mutagenesis. This point mutation causes a single amino acid substitution from Ser498 to Asn498 in the transmembrane region and abrogates factor-dependency of all interleukin-3-dependent cell lines tested. Murine interleukin-3-dependent Ba/F3 cells expressing the mutated but not the normal form of MPL were tumorigenic when transduced into syngeneic mice. Analysis of intracellular signaling pathways indicated that the mutant MPL protein constitutively activated two distinct signaling pathways, SHC-Raf-MAPK and JAK2-STAT3/STAT5.

  View details for Web of Science ID A1996VC85400030

  View details for PubMedID 8695859

• Simultaneous fluorescence-activated cell sorter analysis of two distinct transcriptional elements within a single cell using engineered green fluorescent proteins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Anderson, M. T., Tjioe, I. M., Lorincz, M. C., Parks, D. R., Herzenberg, L. A., Nolan, G. P., Herzenberg, L. A. 1996; 93 (16): 8508-8511

  Abstract

  Green fluorescent protein (GFP) is widely used as a reporter gene in both prokaryotes and eukaryotes. However, the fluorescence levels of wild-type GFP (wtGFP) are not bright enough for fluorescence-activated cell sorting or flow cytometry. Several GFP variants were generated that are brighter or have altered excitation spectra when expressed in prokaryotic cells. We engineered two GFP genes with different combinations of these mutations, GFP(S65T,V163A) termed GFP-Bex1, and GFP(S202F,T203I,V163A) termed GFP-Vex1. Both show enhanced brightness and improved signal-to-noise ratios when expressed in mammalian cells and appropriately excited, compared with wtGFP. Each mutant retains only one of the two excitation peaks of the wild-type protein. GFP-Bex1 excites at 488 nm (blue) and GFP-Vex1 excites at 406 nm (violet), both of which are available laser lines. Excitation at these wavelengths allows for the independent analyses of these mutants by fluorescence-activated cell sorting, permitting simultaneous, quantitative detection of expression from two different genes within single mammalian cells.

  View details for Web of Science ID A1996VB32500065

  View details for PubMedID 8710900

• Episomal vectors rapidly and stably produce high-titer recombinant retrovirus HUMAN GENE THERAPY Kinsella, T. M., Nolan, G. P. 1996; 7 (12): 1405-1413

  Abstract

  The nuclear replication and retention functions of the Epstein-Barr virus (EBV) have been utilized here to maintain retroviral constructs episomally within human cell-based retroviral packaging lines. These hybrid EBV/retroviral constructs are capable of producing helper-free recombinant retrovirus as soon as 48 hr and for at least 30 days after transfection into 293T-based ecotropic and/or amphotropic retroviral packaging cells. Viral titers greater than 10(7) TU/ml were obtained after puromycin selection of transfected retroviral packaging cells. This episomal approach to retroviral production circumvents some limitations inherent in transient and chromosomally stable retroviral producer systems, affording reproducibly rapid, large-scale, stable, and high-titer retrovirus production.

  View details for Web of Science ID A1996WD32100002

  View details for PubMedID 8844199

• Enzyme-generated intracellular fluorescence for single-cell reporter gene analysis utilizing Escherichia coli beta-glucuronidase CYTOMETRY Lorincz, M., Roederer, M., Diwu, Z., Herzenberg, L. A., Nolan, G. P. 1996; 24 (4): 321-329

  Abstract

  We report the development of a new fluorescence-activated cell sorter (FACS)-based reporter gene system utilizing the enzymatic activity of the E. coli beta-glucuronidase (gus) gene. When loaded with the Gus substrate fluorescein-di-beta-D-glucuronide (FDGlcu), individual mammalian cells expressing and translating gus mRNA liberate sufficient levels of intracellular fluorescein for quantitative analysis by flow cytometry. This assay can be used to FACS sort viable cells based on Gus enzymatic activity, and the efficacy of the assay can be measured independently by using a fluorometric lysate assay. Furthermore, both the beta-glucuronidase and the previously described E. coli beta-galactosidase enzymes have high specificities for their cognate substrates, allowing each reporter gene to be measured by FACS independently.

  View details for Web of Science ID A1996VD78900004

  View details for PubMedID 8866216

• Rapid retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hofmann, A., Nolan, G. P., Blau, H. M. 1996; 93 (11): 5185-5190

  Abstract

  We describe a single autoregulatory cassette that allows reversible induction of transgene expression in response to tetracycline (tet). This cassette contains all of the necessary components previously described by others on two separate plasmids that are introduced sequentially over a period of months [Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. The cassette is introduced using a retrovirus, allowing transfer into cell types that are difficult to transfect. Thus, populations of thousands of cells, rather than a few clones, can be isolated and characterized within weeks. To avoid potential interference of the strong retroviral long terminal repeat enhancer and promoter elements with the function of the tet-regulated cytomegalovirus minimal promoter, the vector is self-inactivating, eliminating transcription from the long terminal repeat after infection of target cells. Tandem tet operator sequences and the cytomegalovirus minimal promoter drive expression of a bicistronic mRNA, leading to transcription of the gene of interest (lacZ) and the internal ribosome entry site controlled transactivator (Tet repressor-VP16 fusion protein). In the absence of tet, there is a progressive increase in transactivator by means of an autoregulatory loop, whereas in the presence of tet, gene expression is prevented. Northern blot, biochemical, and single cell analyses have all shown that the construct yields low basal levels of gene expression and induction of one to two orders of magnitude. Thus, the current cassette of the retroviral construct (SIN-RetroTet vector) allows rapid delivery of inducible genes and should have broad applications to cultured cells, transgenic animals, and gene therapy.

  View details for Web of Science ID A1996UN25300003

  View details for PubMedID 8643550

• Applications of retrovirus-mediated expression cloning 1995 Annual Meeting of the ISEH Onishi, M., Kinoshita, S., Morikawa, Y., Shibuya, A., Phillips, J., Lanier, L. L., Gorman, D. M., Nolan, G. P., Miyajima, A., Kitamura, T. ELSEVIER SCIENCE INC. 1996: 324–29

  Abstract

  We have recently established a novel expression cloning system using retroviral vectors. The system is based on a high-efficiency packaging cell line, BOSC23, and a simplified retroviral vector, pBabeX, carrying no selection marker. cDNA libraries, constructed in the pBabeX vector, are transiently transfected into BOSC23 cells. The supernatant contains more than 3X10(6)/mL, which would cover large complexities of cDNA libraries. The retrovirus stock gave 100% infection efficiency in NIH3T3 cells and 5-40% infection efficiency in various hematopoietic cell lines. In contrast to the conventional expression cloning system, in which it is necessary to transfect cDNA libraries transiently into particular cell types such as COS cells, retrovirus-mediated expression cloning allows us to transduce cDNAs into a wide variety of cell types. This method therefore makes it possible to select cells expressing a cDNA of interest by various functional assays. When combined with polymerase chain reaction (PCR)-driven random mutagenesis, this system is also useful in searching for mutations of various molecules that will result in alterations of their functions.

  View details for Web of Science ID A1996TX75300035

  View details for PubMedID 8641361

• EFFICIENT SCREENING OF RETROVIRAL CDNA EXPRESSION LIBRARIES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kitamura, T., Onishi, M., Kinoshita, S., Shibuya, A., Miyajima, A., Nolan, G. P. 1995; 92 (20): 9146-9150

  Abstract

  Expression cloning of cDNAs was first described a decade ago and was based on transient expression of cDNA libraries in COS cells. In contrast to transient transfection of plasmids, retroviral gene transfer delivers genes stably into a wide range of target cells. We utilize a simple packaging system for production of high-titer retrovirus stock from cDNA libraries to establish a cDNA expression cloning system. In two model experiments, murine interleukin (IL)-3-dependent Ba/F3 cells were infected with libraries of retrovirally expressed cDNA derived from human T-cell mRNA or human IL-3-dependent TF-1 cell line mRNA. These infected Ba/F3 cells were selected for the expression of CD2 by flow cytometry or for the alpha subunit of the human IL-3 receptor (hIL-3R alpha) by factor-dependent growth. CD2 (frequency, 1 in 10(4)) and hIL-3R alpha (frequency, 1 in 1.5 x 10(5)) cDNAs were readily detected in small-scale experiments, indicating this retroviral expression cloning system is efficient enough to clone low-abundance cDNAs by their expression or function.

  View details for Web of Science ID A1995RX20000029

  View details for PubMedID 7568090

• EXPANDED ROLES FOR REL PROTEINS AT THE INITIATOR AND ENHANCER OF HIV-1 Nolan, G. P., Kinoshita, S., Achacoso, P., Roy, A., Montano, M. MARY ANN LIEBERT INC. 1995: S114–S114

  View details for Web of Science ID A1995RQ68900198

• ACTIVATION OF THE TRANSCRIPTION FACTOR NF-KB IN GH(3) PITUITARY-CELLS MOLECULAR AND CELLULAR ENDOCRINOLOGY Grandison, L., Nolan, G. P., Pfaff, D. W. 1994; 106 (1-2): 9-15

  Abstract

  Since several genes expressed in the pituitary can bind the transcription factor NF-KB, its presence and regulation was examined in the GH3 pituitary cell line. An electrophoretic mobility shift assay using nuclear extracts and an oligonucleotide probe corresponding to the Ig KB binding site was employed to identify activated NF-KB. One complex possessed properties characteristic of NF-KB: co-migration with an NF-KB complex and binding specificity restricted to NF-KB binding DNA sequences. Antibodies to the NF-KB subunits NFKB1p50 (p50) and RelA (p65) interacted with the extract-DNA complex. Activation of NF-KB in GH3 cells was increased by PMA or the cytokine tumor necrosis factor alpha. A synergy between PMA and TNF or a calcium mobilizing agent was seen in NF-KB activation. Further TNF activation was enhanced by TRH. These observations indicate the presence of NF-KB in GH3 cells and demonstrate its activation by hormones/second messengers that act on pituitary cells.

  View details for Web of Science ID A1994PZ34100002

  View details for PubMedID 7895918

• GENE-TRANSFER INTO HIGHLY-PURIFIED CD34+THY-1+LIN-HUMAN PERIPHERAL-BLOOD HEMATOPOIETIC STEM-CELLS Kerr, W., Fraser, C., Chen, B., Kaneshima, H., Hoffman, R., Lorens, J., Caldwell, J., Nolan, G., Cohen, L., Mulligan, R. C., Mule, J. AMER SOC HEMATOLOGY. 1994: A402–A402

  View details for Web of Science ID A1994PR75401591

• NF-AT-AP-1 AND REL-BZIP - HYBRID VIGOR AND BINDING UNDER THE INFLUENCE CELL Nolan, G. P. 1994; 77 (6): 795-798

  View details for Web of Science ID A1994NT33100004

  View details for PubMedID 8004669

• NF-AT COMPONENTS DEFINE A FAMILY OF TRANSCRIPTION FACTORS TARGETED IN T-CELL ACTIVATION NATURE Northrop, J. P., Ho, S. N., Chen, L., Thomas, D. J., Timmerman, L. A., Nolan, G. P., Admon, A., Crabtree, G. R. 1994; 369 (6480): 497-502

  Abstract

  The NF-AT transcription complex is required for the expression of a group of proteins that collectively coordinate the immune response. Here we purify two proteins encoded by separate genes that represent the pre-existing (p) and cytosolic (c) components of NF-AT. Expression of the full-length complementary DNA encoding NF-ATc activates the interleukin (IL-2) promoter in non-T lymphocytes, whereas a dominant negative of NF-ATc specifically blocks activation of the IL-2 promoter in T lymphocytes, indicating that NF-ATc is required for IL-2 gene expression. NF-ATc RNA expression is largely restricted to lymphoid tissues and is induced upon T-cell activation. The other protein, NF-ATp, is highly homologous to NF-ATc over a limited domain which shows similarity to the Dorsal/Rel family, but has a wider tissue distribution. Agents that increase intracellular Ca2+ or activate protein kinase C independently modify NF-ATc, indicating that distinct signalling pathways converge on NF-ATc to regulate its function.

  View details for Web of Science ID A1994NQ28600061

  View details for PubMedID 8202141

• PRODUCTION OF HIGH-TITER HELPER-FREE RETROVIRUSES BY TRANSIENT TRANSFECTION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pear, W. S., Nolan, G. P., Scott, M. L., Baltimore, D. 1993; 90 (18): 8392-8396

  Abstract

  The generation of high-titer, helper-free retroviruses by transient transfection has been achieved by using the highly transfectable 293T cell line into which are stably introduced constructs that express retroviral packaging functions. The resulting ecotropic virus packaging cell line BOSC 23 produces infectious retrovirus at > 10(6) infectious units/ml of supernatant within 72 hr after CaPO4-mediated transfection. A stringent assay for replication-competent virus showed that no helper virus was present. The system can produce high titers of retroviral vectors expressing genes that are extremely difficult to propagate at high titer in stable producer lines. This method should facilitate and extend the use of helper-free retroviral gene transfer, as well as be useful for gene therapy.

  View details for Web of Science ID A1993LX75000020

  View details for PubMedID 7690960

• THE P65-SUBUNIT OF NF-KAPPA-B REGULATES I-KAPPA-B BY 2 DISTINCT MECHANISMS GENES & DEVELOPMENT Scott, M. L., Fujita, T., Liou, H. C., Nolan, G. P., Baltimore, D. 1993; 7 (7A): 1266-1276

  Abstract

  Transcription factor NF-kappa B (p50/p65) is generally localized to the cytoplasm by its inhibitor I kappa B. Overproduced I kappa B, free from NF-kappa B, is rapidly degraded. Overexpression of p65 increases endogenous I kappa B protein in both carcinoma and lymphoid cells by two mechanisms: protein stabilization and increased transcription of I kappa B mRNA. In contrast, p65 delta, a naturally occurring splice variant, fails to markedly augment I kappa B protein levels. Both overexpressed p65 and coexpressed p50 are cytoplasmic, whereas p65 delta is partly nuclear, indicating that the I kappa B induced by p65 can maintain NF-kappa B in the cytoplasm. Thus, p65 and I kappa B are linked in an autoregulatory loop, ensuring that NF-kappa B is held in the cytoplasm until cells are specifically induced to translocate it to the nucleus.

  View details for Web of Science ID A1993LL40300013

  View details for PubMedID 8319912

• THE CANDIDATE PROTOONCOGENE BCL-3 ENCODES A TRANSCRIPTIONAL COACTIVATOR THAT ACTIVATES THROUGH NF-KAPPA-B P50 HOMODIMERS GENES & DEVELOPMENT Fujita, T., Nolan, G. P., Liou, H. C., Scott, M. L., Baltimore, D. 1993; 7 (7B): 1354-1363

  Abstract

  The candidate proto-oncogene bcl-3 encodes a protein that shares structural features with I kappa B-alpha and other proteins that bind to members of the Rel protein family. Here, we show that in contrast to the inhibitory activity of I kappa B-alpha, the bcl-3 gene product superactivates NF-kappa B p50 homodimer-mediated gene expression both in vivo and in vitro. BCL-3 protein can, as well, selectively associate with p50 homodimers in the presence of DNA containing a kappa B motif. These results strongly suggest that BCL-3 can act as a transcriptional coactivator, acting through DNA-bound p50 homodimers.

  View details for Web of Science ID A1993LM68000006

  View details for PubMedID 8330739

• THE BCL-3 PROTOONCOGENE ENCODES A NUCLEAR I-KAPPA-B-LIKE MOLECULE THAT PREFERENTIALLY INTERACTS WITH NF-KAPPA-B P50 AND P52 IN A PHOSPHORYLATION-DEPENDENT MANNER MOLECULAR AND CELLULAR BIOLOGY Nolan, G. P., Fujita, T., Bhatia, K., HUPPI, C., Liou, H. C., Scott, M. L., Baltimore, D. 1993; 13 (6): 3557-3566

  Abstract

  The product of the putative proto-oncogene bcl-3 is an I kappa B-like molecule with novel binding properties specific for a subset of the rel family of transcriptional regulators. In vitro, Bcl-3 protein specifically inhibited the DNA binding of both the homodimeric NF-kappa B p50 subunit and a closely related homolog, p52 (previously p49), to immunoglobulin kappa NF-kappa B DNA motifs. Bcl-3 could catalyze the removal of these proteins from DNA. At concentrations that significantly inhibited DNA binding by homodimeric p50, Bcl-3 did not inhibit binding of reconstituted heterodimeric NF-kappa B (p50:p65), a DNA-binding homodimeric form of p65, or homodimers of c-Rel. Phosphatase treatment of Bcl-3 partially inactivated its inhibitory properties, implicating a role for phosphorylation in the regulation of Bcl-3 activity. Bcl-3, like p50, localizes to the cell nucleus. In cells cotransduced with Bcl-3 and p50, both molecules could be found in the nucleus of the same cells. Interestingly, coexpression of Bcl-3 with a p50 mutant deleted for its nuclear-localizing signal resulted in the relocalization of Bcl-3 to the cytoplasm, showing that the proteins interact in the cell. These properties contrast Bcl-3 to classically defined I kappa B, which maintains heterodimeric NF-kappa B p50:p65 in the cytoplasm through specific interactions with the p65 subunit. Bcl-3 appears to be a nuclear, I kappa B-related molecule that regulates the activity of homodimeric nuclear p50 and its homolog p52.

  View details for Web of Science ID A1993LD73300043

  View details for PubMedID 8497270

• THE NF-KAPPA-B P50 PRECURSOR, P105, CONTAINS AN INTERNAL I-KAPPA-B-LIKE INHIBITOR THAT PREFERENTIALLY INHIBITS P50 EMBO JOURNAL Liou, H. C., Nolan, G. P., Ghosh, S., Fujita, T., Baltimore, D. 1992; 11 (8): 3003-3009

  Abstract

  The p50 subunit of NF-kappa B is apparently synthesized as a precursor molecule of 105 kDa (p105); subsequent processing releases the amino-terminal p50 polypeptide with rel homology, DNA binding activity and transcriptional activation potential. The carboxy-terminal region of p105 contains seven copies of an ankyrin-related sequence previously found in several genes involved in differentiation and cell cycle control. Two proteins with I kappa B activity, MAD-3 and pp40, have been cloned and found to contain five obvious ankyrin repeats that align with those in the carboxy-terminus of p105. Both proteins target their inhibitory activity to the p65 subunit of NF-kappa B and to c-rel. Here we show that the bacterially expressed and purified carboxy-terminal region (CTR) of p105 abolishes the binding of p50 homodimers to a kappa B motif but minimally affects the binding of p65 homodimers and NF-kappa B. By contrast, MAD-3 inhibits the binding of p65 and NF-kappa B but not p50. Both the CTR and MAD-3 interact with their respective targets through physical association both in vitro and in vivo. The CTR can be expressed as an independent entity and thus may play two roles, as a cis inhibitor built into the p105 molecule and as a trans regulator of p50.

  View details for Web of Science ID A1992JE53700027

  View details for PubMedID 1639070

• INDEPENDENT MODES OF TRANSCRIPTIONAL ACTIVATION BY THE P50-SUBUNIT AND P65-SUBUNIT OF NF-KAPPA-B GENES & DEVELOPMENT Fujita, T., Nolan, G. P., Ghosh, S., Baltimore, D. 1992; 6 (5): 775-787

  Abstract

  Recombinant subunits of the transcription factor NF-kappa B, p50 and p65, were analyzed both for binding to various kappa B motifs and in vitro activation. The subunits preferentially form a heterodimer that activates transcription. Although p50 and p65 bind DNA individually as homodimers and are structurally related, their activation mechanisms are distinct. p65 activates transcription by its unique carboxy-terminal activation domain. (p50)2 displays higher affinity DNA binding than (p65)2 for many distinct kappa B motifs and provides strong transcriptional activation only when adopting a chymotrypsin-resistant conformation induced by certain kappa B motifs but not others. Thus, (p50)2 acts as a positive regulator in vitro, consistent with its isolation as a putative constitutive regulator of MHC class I genes. Both subunits of NF-kappa B, therefore, contribute independently to provide regulation at given kappa B motifs.

  View details for Web of Science ID A1992HT80400007

  View details for PubMedID 1577272

• The inhibitory ankyrin and activator Rel proteins. Current opinion in genetics & development Nolan, G. P., Baltimore, D. 1992; 2 (2): 211-220

  Abstract

  The gene families encoding the proteins NF-kappa B, c-Rel and Dorsal, in conjunction with their respective inhibitors l kappa B, pp40, and Cactus, achieve specificity in gene regulation by means of common principles. The related activities of NF-kappa B and Dorsal are mediated by heterodimeric or homodimeric complexes of proteins containing the conserved dimerization and DNA-binding domain termed Rel. The l kappa Bs and Cactus, which share a core series of structural repeats termed ankyrin, inhibit cognate activators through differential interactions with the Rel-homology domain. Together, the inhibitory ankyrin proteins and their cognate Rel dimers probably define specific signalling pathways able to activate specific gene expression. Both gene families include proto-oncogenes, thus broadly implicating Rel/l kappa B in the control of both normal gene expression and the aberrant gene expression that makes cells cancerous.

  View details for PubMedID 1386268

• DNA-BINDING AND I-KAPPA-B INHIBITION OF THE CLONED P65 SUBUNIT OF NF-KAPPA-B, A REL-RELATED POLYPEPTIDE CELL Nolan, G. P., Ghosh, S., Liou, H. C., Tempst, P., Baltimore, D. 1991; 64 (5): 961-969

  Abstract

  The sequence and biochemical properties of the product of the cloned cDNA for the p65 subunit of nuclear factor kappa B (NF-kappa B) have been determined. The cDNA has an open reading frame of 549 amino acids capable of encoding a 60 kd protein. NF-kappa B p65 contains an amino-terminal region of 320 amino acids with extensive similarity to the oncogene c-rel and lesser similarity to NF-kappa B p50. In vitro translated p65 forms a DNA-binding complex with NF-kappa B p50, and the binding of this complex can be specifically inhibited by purified I kappa B. Progressive carboxy-terminal deletions of p65 show that, contrary to previous assumptions, p65 does include a DNA-binding domain that in vivo might become activated only through hetero-oligomerization with p50. DNA binding by truncated p65 is inhibited by I kappa B, thus mapping the I kappa B interaction domain to the rel-homologous region and suggesting that I kappa B exerts its inhibitory effect upon NF-kappa B primarily through interaction with p65.

  View details for Web of Science ID A1991FA94000013

  View details for PubMedID 2001591

• WHOLE ANIMAL-CELL SORTING OF DROSOPHILA EMBRYOS SCIENCE Krasnow, M. A., Cumberledge, S., Manning, G., Herzenberg, L. A., Nolan, G. P. 1991; 251 (4989): 81-85

  Abstract

  Use of primary culture cells has been limited by the inability to purify most types of cells, particularly cells from early developmental stages. In whole animal cell sorting (WACS), live cells derived from animals harboring a lacZ transgene are purified according to their level of beta-galactosidase expression with a fluorogenic beta-galactosidase substrate and fluorescence-activated cell sorting. With WACS, incipient posterior compartment cells that express the engrailed gene were purified from early Drosophila embryos. Neuronal precursor cells were also purified, and they differentiated into neurons with high efficiency in culture. Because there are many lacZ strains, it may be possible to purify most types of Drosophila cells. The same approach is also applicable to other organisms for which germ-line transformation is possible.

  View details for Web of Science ID A1991EQ60300032

  View details for PubMedID 1898782

• Use of Escherichiu coli (E. coli) lacZ (ß-Galactosidase) as a Reporter Gene. Methods in molecular biology (Clifton, N.J.) Macgregor, G. R., Nolan, G. P., Fiering, S., Roederer, M., Herzenberg, L. A. 1991; 7: 217-235

  Abstract

  Our understanding of the molecular mechanisms that govern gene expression has been facilitated by the ability to introduce recombinant DNA molecules into heterologous cellular systems both in vitro and in vivo. One approach to defining DNA sequences important in the regulation of gene expression is to place controlling elements (e.g., promoter/enhancer sequences) upstream of a DNA coding sequence, introduce these constructs into transgenic animals or cells in culture, and analyze the levels of gene product produced by the introduced construct. Ideally, such a reporter gene should encode a product that is stable, innocuous to the cell or organism in which it is being expressed, and should be readily detectable, even when present in small quantities.

  View details for DOI 10.1385/0-89603-178-0:217

  View details for PubMedID 21416358

• IMPROVED FACS-GAL - FLOW CYTOMETRIC ANALYSIS AND SORTING OF VIABLE EUKARYOTIC CELLS EXPRESSING REPORTER GENE CONSTRUCTS CYTOMETRY Fiering, S. N., Roederer, M., Nolan, G. P., Micklem, D. R., Parks, D. R., Herzenberg, L. A. 1991; 12 (4): 291-301

  Abstract

  The previously reported FACS-Gal assay (Nolan et al., Proc Natl Acad Sci USA 85:2603-2607, 1988) measures E. coli lacZ-encoded beta-galactosidase activity in individual viable eukaryotic cells for a variety of molecular and cellular biological applications. Enzyme activity is measured by flow cytometry, using a fluorogenic substrate, which is hydrolyzed and retained intracellularly. In this system, lacZ serves both as a reporter gene to quantitate gene expression and as a selectable marker for the fluorescence-activated sorting of cells based on their lacZ expression level. This report details the following improvements of the original assay: 1) use of phenylethyl-beta-D-thiogalactoside, a competitive inhibitor, to inhibit beta-galactosidase activity; 2) reduction of false positives by two-color measurements; and 3) inhibition of interfering mammalian beta-galactosidases by the weak base chloroquine. We found an exponential relationship between fluorescence generated by beta-galactosidase in this assay and the intracellular concentration of beta-galactosidase molecules. Finally, we report conditions for optimal loading of the substrate (FDG) and retention of the product, fluorescein. Under these conditions, we found uniform loading of FDG in all cells of a clone in individual experiments. Together, these improvements make FACS-Gal an extremely powerful tool for investigation of gene expression in eukaryotic cells.

  View details for Web of Science ID A1991FK38600001

  View details for PubMedID 1905992

• INSITU DETECTION OF STAGE-SPECIFIC GENES AND ENHANCERS IN B-CELL DIFFERENTIATION VIA GENE-SEARCH RETROVIRUSES 3RD INTERNATIONAL CONF ON LYMPHOCYTE ACTIVATION IMMUNE REGULATION Kerr, W. G., Nolan, G. P., JOHNSEN, J. B., Herzenberg, L. A. PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1991: 187–200

  Abstract

  We demonstrate that infection of an LPS-responsive pre-B cell line with transcriptionally-defective retroviruses containing a reporter gene (lacZ) can result in viral integrations where expression of lacZ is differentiation stage-dependent. Because expression of lacZ is dependent upon flanking cellular sequences these retroviral integrations represent in situ gene fusions with cellular enhancers (Enhsr1) and genes (Gensr1) which are either induced or repressed during LPS-stimulated differentiation. One of the well-documented effects of LPS upon pre-B cells is the induction of kappa light chain transcription via NF-kappa B. The identification of LPS-stimulated gene repression during B cell differentiation indicates that LPS has multiple effects upon gene expression during the pre-B to B cell transition. The identification of cellular enhancers and genes which are downregulated during the transition from the pre-B to the B cell stage indicates that other transcription factors, in addition to NF-kappa B, are required for this step in differentiation. Finally, we present some initial experiments which indicate the gene-search retroviruses can introduce expression of lacZ into normal hematopoietic cells in vitro and in vivo.

  View details for Web of Science ID A1991BT82R00021

  View details for PubMedID 1950769

• SINGLE CELL ASSAY OF A TRANSCRIPTION FACTOR REVEALS A THRESHOLD IN TRANSCRIPTION ACTIVATED BY SIGNALS EMANATING FROM THE T-CELL ANTIGEN RECEPTOR GENES & DEVELOPMENT Fiering, S., Northrop, J. P., Nolan, G. P., Mattila, P. S., Crabtree, G. R., Herzenberg, L. A. 1990; 4 (10): 1823-1834

  Abstract

  Stimulation of T lymphocytes through their antigen receptor leads to the appearance of several transcription factors, including NF-AT and NF-kappa B, which are involved in regulating genes required for immunologic activation. To investigate the activity of a single transcription factor in individual viable cells, we have applied an assay that uses the fluorescence-activated cell sorter to quantitate beta-galactosidase (beta-gal). We have analyzed the distribution of NF-AT transcriptional activity among T cells undergoing activation by using a construct in which three tandem copies of the NF-AT-binding site directs transcription of the lacZ gene. Unexpectedly, stimulation of cloned stably transfected Jurkat T cells leads to a bimodal pattern of beta-gal expression in which some cells express no beta-gal and others express high levels. This expression pattern cannot be accounted for by cell-cycle position or heritable variation. Further results, in which beta-gal activity is correlated with NF-AT-binding activity, indicate that the concentration of NF-AT must exceed a critical threshold before transcription initiates. This threshold likely reflects the NF-AT concentration-dependent assembly of transcription complexes at the promoter. Similar constructs controlled by NF-kappa B or the entire interleukin-2 enhancer show bimodal expression patterns during induction, suggesting that thresholds set by the concentration of transcription factors may be a common property of inducible genes.

  View details for Web of Science ID A1990EC63000016

  View details for PubMedID 2123468

• CLONING OF THE P50 DNA-BINDING SUBUNIT OF NF-KAPPA-B - HOMOLOGY TO REL AND DORSAL CELL Ghosh, S., Gifford, A. M., RIVIERE, L. R., Tempst, P., Nolan, G. P., Baltimore, D. 1990; 62 (5): 1019-1029

  Abstract

  The DNA binding subunit of the transcription factor NF-kappa B, p50, has been cloned. p50 appears to be synthesized as a larger protein that is then processed to its functional size. Sequence analysis reveals remarkable homology for over 300 amino acids at the amino-terminal end to the oncogene v-rel, its cellular homolog c-rel, and the Drosophila maternal effect gene dorsal. This establishes NF-kappa B as a member of the rel family of proteins, all of which display nuclear-cytosolic translocation. Protein sequence from the p65 polypeptide has established that it is not encoded in the same mRNA as p50. However, p65 appears homologous to c-rel, suggesting that c-rel may form heterodimers with p50 and rel may include a homodimerization motif.

  View details for Web of Science ID A1990DY10000020

  View details for PubMedID 2203532

• A NOVEL FLUORESCENCE-BASED SYSTEM FOR ASSAYING AND SEPARATING LIVE CELLS ACCORDING TO VDJ RECOMBINASE ACTIVITY MOLECULAR AND CELLULAR BIOLOGY Yancopoulos, G. D., Nolan, G. P., Pollock, R., Prockop, S., LI, S. C., Herzenberg, L. A., Alt, F. W. 1990; 10 (4): 1697-1704

  Abstract

  We describe two retroviral vector-based recombination substrate systems designed to assay for lymphoid VDJ recombinase activity in cultured cells. Both substrates incorporate a constitutive dominant marker gene (the simian virus promoter-driven neo gene) to allow selection of cells that stably integrate the substrate. Both substrates also include a second marker gene that becomes transcriptionally active only when inverted by a site-specific recombination event between flanking immunoglobulin variable-region gene segments. The first vector, similar in structure to previous retrovirus-based recombination substrates, utilizes the bacterial guanine-xanthine phosphoribosyltransferase gene (gpt) as its activatable marker; detection of inversion (VDJ recombinase activity) involves drug selection and Southern blotting analyses. We have used this vector to make a more extensive and quantitative survey of VDJ recombinase activity in B-lineage cell lines than has previously been performed with stable substrates, and we have compared our results with those of other studies that use transient recombination substrates. In the second vector, the activatable gene is the bacterial beta-galactosidase gene (lacZ). Detection for inversional activation of this gene is achieved by a fluorogenic assay, termed FACS-Gal, that detects beta-galactosidase activity in viable cells. The latter assay has the unique advantage of rapidly detecting cells that undergo recombination and also allows viable sorting of cells on the basis of the presence or absence of VDJ recombinase activity. We have used the lacZ vector to rapidly quantitate VDJ recombinase activity in B-lineage cell lines and compared the results with those obtained with the gpt vector. We have also used the lacZ vector to isolate variant pre-B-cell lines with low and high levels of VDJ recombinase activity.

  View details for Web of Science ID A1990CW99600046

  View details for PubMedID 2320007

• INSITU DETECTION OF TRANSCRIPTIONALLY ACTIVE CHROMATIN AND GENETIC REGULATORY ELEMENTS IN INDIVIDUAL VIABLE MAMMALIAN-CELLS IMMUNOLOGY Kerr, W. G., Nolan, G. P., Herzenberg, L. A. 1989: 74-79

  Abstract

  Using a newly developed FACS method for quantifying the expression of the Escherischia coli lacZ reporter gene in viable mammalian cells, we have obtained cloned cell lines in which the expression of lacZ is under the control of native endogenous transcription elements. We infected the murine pre-B cell 70Z/3 with transcriptionally disabled retroviruses containing lacZ and employed the FACS-FDG technique to detect and sort rare lacZ+ cells in which we expect integration is near such endogenous transcription elements. After two rounds of enrichment we obtained a population of cells that was 80-90% positive for lacZ activity. Clones derived from the lacZ+ pool differ from each other with respect to their overall level of lacZ activity as well as in the pattern of lacZ expression among cells within an individual clone. Treatment of these lacZ+ 70Z/3 clones with lipopolysaccharide (LPS; which is known to stimulate differentiation of 70Z/3 from a pre-B cell to an IgM-expressing B cell) greatly decreased lacZ expression in one clone, 7e17. lacZ expression in this clone was 50-100 times lower within 24 hr of LPS addition and coincided with the acquisition of IgM kappa on the surface of 7e17. This suggests that a transcriptionally active domain of chromatin that harbors the lacZ construct is down-regulated during the transition induced by LPS stimulation.

  View details for Web of Science ID A1989AR92700015

  View details for PubMedID 2807403

• TRANSCRIPTIONALLY DEFECTIVE RETROVIRUSES CONTAINING LACZ FOR THE INSITU DETECTION OF ENDOGENOUS GENES AND DEVELOPMENTALLY REGULATED CHROMATIN 3RD SYMP ON IMMUNOLOGICAL RECOGNITION Kerr, W. G., Nolan, G. P., SERAFINI, A. T., Herzenberg, L. A. COLD SPRING HARBOR LABORATORY PRESS. 1989: 767–776

  View details for Web of Science ID A1989BR13A00088

• TRANSCRIPTIONALLY DEFECTIVE RETROVIRUSES CONTAINING LACZ FOR THE INSITU DETECTION OF ENDOGENOUS GENES AND DEVELOPMENTALLY REGULATED CHROMATIN COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Kerr, W. G., Nolan, G. P., SERAFINI, A. T., Herzenberg, L. A. 1989; 54: 767-776

  View details for Web of Science ID A1989JX74600019

  View details for PubMedID 2518010

• FLUORESCENCE-ACTIVATED CELL ANALYSIS AND SORTING OF VIABLE MAMMALIAN-CELLS BASED ON BETA-D-GALACTOSIDASE ACTIVITY AFTER TRANSDUCTION OF ESCHERICHIA-COLI LACZ PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Nolan, G. P., Fiering, S., Nicolas, J. F., Herzenberg, L. A. 1988; 85 (8): 2603-2607

  Abstract

  We demonstrate that individual cells infected with and expressing a recombinant retrovirus carrying the Escherichia coli beta-galactosidase gene (lacZ) can be viably stained, analyzed, sorted, and cloned by fluorescence-activated cell sorting based on the levels of lacZ expressed. To accomplish this we have devised a method to enzymatically generate and maintain fluorescence in live mammalian cells. Accumulation of fluorescent products in cells is linear with time, with a direct correlation of fluorescence to enzymatic activity. This technology for beta-galactosidase detection is more sensitive than other available cytochemical or biochemical methods. We have used this procedure to show that the expression of psi-2-MMuLVSVnlsLacZ in the T-cell lymphoma BW5147 and the B-cell hybridoma SP2/0 is not completely stable and that subclones selected by the fluorescence-activated cell sorter for low lacZ activity demonstrate distinctly lower average expression of LacZ. These findings indicate the utility of beta-galactosidase as a reporter molecule at the single-cell level for studies of gene regulation, including studies of promoter efficacy, enhancer activity, trans-acting factors, and other regulatory elements.

  View details for Web of Science ID A1988N023400040

  View details for PubMedID 3128790

• ISOLATION AND CHARACTERIZATION OF THE GENE FOR THE MURINE T-CELL DIFFERENTIATION ANTIGEN AND IMMUNOGLOBULIN-RELATED MOLECULE, LYT-2 NUCLEIC ACIDS RESEARCH Nakauchi, H., Tagawa, M., Nolan, G. P., Herzenberg, L. A. 1987; 15 (10): 4337-4347

  Abstract

  We present here the sequence of the 5310 base pair Hind III-cleaved genomic DNA segment that includes the gene for the Lyt-2, a murine differentiation antigen expressed on most immature T lymphocytes as well as the cytotoxic suppressor T cell subset. We also present the complete intron/exon structure of Lyt-2. There are five exons: a fused leader and immunoglobulin variable region like exon, a hinge region exon, a transmembrane exon and two alternatively spliced intracytoplasmic exons (alternative splicing of these exons yields the 38 kDa alpha and 34 kDa alpha' Lyt-2 polypeptides). The promotor region contains a "TATA" box and sequences homologous to the putative immunoglobulin transcriptional control elements cd/pd. S1 protection analysis reveals that thymocytes, T cells from lymph nodes, and a Lyt-2 transfectant obtained by introduction of total genomic DNA have the same initiation site. In the 3' region, there is a polyadenylation signal sequence after a 700 bp long 3' untranslated region.

  View details for Web of Science ID A1987H489600029

  View details for PubMedID 3495785

• FORMAL PROOF THAT DIFFERENT-SIZE LYT-2 POLYPEPTIDES ARISE FROM DIFFERENTIAL SPLICING AND POSTTRANSCRIPTIONAL REGULATION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tagawa, M., Nakauchi, H., Herzenberg, L. A., Nolan, G. P. 1986; 83 (10): 3422-3426

  Abstract

  We recently isolated the gene and a cDNA clone for the mouse T-cell surface antigen Lyt-2 and showed that Lyt-2 is homologous to the human Leu-2 (T8) antigen and that the gene encoding it is a member of the immunoglobulin gene superfamily. By screening a mouse thymus cDNA library with the Lyt-2 cDNA clone, we isolated two classes of cDNA clones, alpha and alpha', which differ by 31 base pairs. Comparison of the alpha cDNA with genomic sequence data indicates that there are five exons encoding Lyt-2: a fused leader/immunoglobulin variable region-like exon, a spacer region exon, a transmembrane exon, and two cytoplasmic exons. The alpha' cDNA clones lack the first of the two cytoplasmic exons and have a direct splice from the donor splice site of the transmembrane exon to the acceptor of the second cytoplasmic exon. This splice changes the reading frame for the second cytoplasmic exon, causing a stop codon shortly after the splice so that the alpha' cDNA clone codes for a peptide 25 residues shorter than the alpha cDNA-encoded peptide. We have constructed expression vectors with alpha and alpha' cDNAs and have shown that L-cell transfectants of these produce Lyt-2 polypeptides of the predicted sizes and that these associate as homodimers on the cell membranes. We found the two species of mRNA corresponding to alpha and alpha' cDNAs at equal levels in thymus RNA by using S1 nuclease analysis. Although lymph node T cells have only the alpha form of Lyt-2 protein, S1 nuclease analysis shows that lymph nodes have about 20% alpha' mRNA relative to alpha. Thus, Lyt-2 is regulated at RNA processing, translational, and/or post-translational steps.

  View details for Web of Science ID A1986C379200078

  View details for PubMedID 3085089

• MOLECULAR-CLONING OF LYT-2, A MEMBRANE GLYCOPROTEIN MARKING A SUBSET OF MOUSE LYMPHOCYTES-T - MOLECULAR HOMOLOGY TO ITS HUMAN COUNTERPART, LEU-2/T8, AND TO IMMUNOGLOBULIN VARIABLE REGIONS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Nakauchi, H., Nolan, G. P., Hsu, C., Huang, H. S., Kavathas, P., Herzenberg, L. A. 1985; 82 (15): 5126-5130

  Abstract

  The sequence of Lyt-2 cDNA shows that it is a new member of the immunoglobulin super gene family. Analysis of the predicted amino acid sequence indicates that the Lyt-2 polypeptide is synthesized with a 27-amino acid leader, and that the mature protein has an immunoglobulin variable region (Ig V)-related sequence of approximately 100 amino acids, an extracellular spacer of 43, a transmembrane region of 38, and an intracytoplasmic region of 27 amino acids. Lyt-2 and its human analogue Leu-2 are 56% homologous; analysis indicates that the Ig V-related domains of the two molecules have evolved away from each other faster than the carboxyl-terminal half of the proteins.

  View details for Web of Science ID A1985ANN3100056

  View details for PubMedID 3927298

• MOLECULAR-WEIGHT DETERMINATION PROGRAM NUCLEIC ACIDS RESEARCH Maina, C. V., Nolan, G. P., Szalay, A. A. 1984; 12 (1): 695-702

  Abstract

  A computer program is described that will determine the molecular weight of DNA, RNA or protein molecules separated according to size by gel electrophoresis. It uses the sizes and migration distances of known molecules in a reference lane to compute a second or third order equation whose curve best fits the data points. It then computes the sizes of all molecules from this equation. Migration distances are measured and entered using an analog tablet. The program is written in Apple Pascal and designed to run on an Apple II Plus computer.

  View details for Web of Science ID A1984SA44800028

  View details for PubMedID 6198629

• PLASMID MAPPING COMPUTER-PROGRAM NUCLEIC ACIDS RESEARCH Nolan, G. P., Maina, C. V., Szalay, A. A. 1984; 12 (1): 717-729

  Abstract

  Three new computer algorithms are described which rapidly order the restriction fragments of a plasmid DNA which has been cleaved with two restriction endonucleases in single and double digestions. Two of the algorithms are contained within a single computer program (called MPCIRC). The Rule-Oriented algorithm, constructs all logical circular map solutions within sixty seconds (14 double-digestion fragments) when used in conjunction with the Permutation method. The program is written in Apple Pascal and runs on an Apple II Plus Microcomputer with 64K of memory. A third algorithm is described which rapidly maps double digests and uses the above two algorithms as adducts. Modifications of the algorithms for linear mapping are also presented.

  View details for Web of Science ID A1984SA44800030

  View details for PubMedID 6320105