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1.
Expert Opin Drug Discov ; 14(2): 115-125, 2019 02.
Article in English | MEDLINE | ID: mdl-30583700

ABSTRACT

INTRODUCTION: Single-cell imaging-based assays are an area of active and growing investment in drug discovery and development. This approach offers researchers the capability to interrogate rare subpopulations of cells with minimal sample consumption and multiplexed readouts. Recent technological advances in the optical interrogation and manipulation of single cells have substantially increased the throughput and sensitivity of these assays. Areas covered: In this review, the authors focus on three classes of single-cell imaging-based analyses: single-cell microscopy combined with microfluidics, mass spectrometric imaging for subcellular compound localization, and imaging mass cytometry (IMC). They provide an overview of each technology and recent examples of their utility in advancing drug discovery, based on the potential for scalability, multiplexing, and capability to generate definitive data on cellular heterogeneity and target engagement. Expert opinion: Understanding target engagement and heterogeneity at the single-cell level will enable the development of safer and more effective therapies, particularly for new modalities like CAR-T cell therapies and gene editing approaches (AAV, CRISPR). Successful adoption of new single-cell imaging-based approaches in drug discovery will require tandem investment in advanced computational analysis and bioinformatic approaches, due to the complexity and multivariate nature of single-cell imaging data.


Subject(s)
Drug Development/methods , Drug Discovery/methods , Single-Cell Analysis/methods , Animals , Computational Biology/methods , Humans , Image Cytometry/methods , Mass Spectrometry/methods , Microfluidics/methods , Microscopy/methods
2.
SLAS Discov ; 23(7): 697-707, 2018 08.
Article in English | MEDLINE | ID: mdl-29843542

ABSTRACT

The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.


Subject(s)
Drug Discovery , Flow Cytometry , High-Throughput Screening Assays , Automation, Laboratory , Blood Platelets/drug effects , Cell Line , Computational Biology/methods , Data Analysis , Drug Discovery/instrumentation , Drug Discovery/methods , Drug Evaluation, Preclinical , Flow Cytometry/instrumentation , Flow Cytometry/methods , High-Throughput Screening Assays/instrumentation , High-Throughput Screening Assays/methods , Humans , Hybridomas , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism
3.
Stem Cells Transl Med ; 7(6): 468-476, 2018 06.
Article in English | MEDLINE | ID: mdl-29543389

ABSTRACT

Hematopoietic stem cells (HSCs) are the self-renewing multipotent progenitors to all blood cell types. Identification and isolation of HSCs for study has depended on the expression of combinations of surface markers on HSCs that reliably distinguish them from other cell types. However, the increasing number of markers required to isolate HSCs has made it tedious, expensive, and difficult for newcomers, suggesting the need for a simpler panel of HSC markers. We previously showed that phenotypic HSCs could be separated based on expression of CD11a and that only the CD11a negative fraction contained true HSCs. Here, we show that CD11a and another HSC marker, endothelial protein C receptor (EPCR), can be used to effectively identify and purify HSCs. We introduce a new two-color HSC sorting method that can highly enrich for HSCs with efficiencies comparable to the gold standard combination of CD150 and CD48. Our results demonstrate that adding CD11a and EPCR to the HSC biologist's toolkit improves the purity of and simplifies isolation of HSCs. Stem Cells Translational Medicine 2018;7:468-476.


Subject(s)
Biomarkers/metabolism , CD11a Antigen/metabolism , Endothelial Protein C Receptor/metabolism , Hematopoietic Stem Cells/metabolism , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Flow Cytometry/methods , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Lipopolysaccharides/toxicity , Mice , Mice, Inbred C57BL , Poly I-C/toxicity
4.
Stem Cell Reports ; 3(5): 707-15, 2014 Nov 11.
Article in English | MEDLINE | ID: mdl-25418718

ABSTRACT

Small numbers of hematopoietic stem cells (HSCs) generate large numbers of mature effector cells through the successive amplification of transiently proliferating progenitor cells. HSCs and their downstream progenitors have been extensively characterized based on their cell-surface phenotype and functional activities during transplantation assays. These cells dynamically lose and acquire specific sets of surface markers during differentiation, leading to the identification of markers that allow for more refined separation of HSCs from early hematopoietic progenitors. Here, we describe a marker, CD11A, which allows for the enhanced purification of mouse HSCs. We show through in vivo transplantations that upregulation of CD11A on HSCs denotes the loss of their long-term reconstitution potential. Surprisingly, nearly half of phenotypic HSCs (defined as Lin-KIT(+)SCA-1(+)CD150(+)CD34-) are CD11A(+) and lack long-term self-renewal potential. We propose that CD11A(+)Lin-KIT(+)SCA-1(+)CD150(+)CD34- cells are multipotent progenitors and CD11A-Lin-KIT(+)SCA-1(+)CD150(+)CD34- cells are true HSCs.


Subject(s)
CD11a Antigen/metabolism , Cell Differentiation , Cell Proliferation , Hematopoietic Stem Cells/metabolism , Up-Regulation , Animals , Antigens, CD/metabolism , Antigens, CD34/metabolism , Antigens, Ly/metabolism , CD11a Antigen/genetics , Flow Cytometry , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Membrane Proteins/metabolism , Mice, Inbred C57BL , Proto-Oncogene Proteins c-kit/metabolism , Receptors, Cell Surface/metabolism , Signaling Lymphocytic Activation Molecule Family Member 1
5.
Stem Cell Reports ; 2(4): 457-72, 2014 Apr 08.
Article in English | MEDLINE | ID: mdl-24749071

ABSTRACT

Hematopoiesis in the embryo proceeds in a series of waves, with primitive erythroid-biased waves succeeded by definitive waves, within which the properties of hematopoietic stem cells (multilineage potential, self-renewal, and engraftability) gradually arise. Whereas self-renewal and engraftability have previously been examined in the embryo, multipotency has not been thoroughly addressed, especially at the single-cell level or within well-defined populations. To identify when and where clonal multilineage potential arises during embryogenesis, we developed a single-cell multipotency assay. We find that, during the initiation of definitive hematopoiesis in the embryo, a defined population of multipotent, engraftable progenitors emerges that is much more abundant within the yolk sac (YS) than the aorta-gonad-mesonephros (AGM) or fetal liver. These experiments indicate that multipotent cells appear in concert within both the YS and AGM and strongly implicate YS-derived progenitors as contributors to definitive hematopoiesis.


Subject(s)
Embryonic Development , Hematopoietic Stem Cells/metabolism , Multipotent Stem Cells/metabolism , Animals , Antigens, Surface/metabolism , CD11a Antigen/genetics , Cell Differentiation , Cell Lineage , Colony-Forming Units Assay , Embryonic Development/genetics , Female , Hematopoietic Stem Cells/cytology , Immunophenotyping , Leukosialin/genetics , Leukosialin/metabolism , Mice , Multipotent Stem Cells/cytology , Phenotype , Proto-Oncogene Proteins c-kit/metabolism , Yolk Sac/embryology
6.
Proc Natl Acad Sci U S A ; 110(27): 11103-8, 2013 Jul 02.
Article in English | MEDLINE | ID: mdl-23690610

ABSTRACT

Mobilization of the T-cell response against cancer has the potential to achieve long-lasting cures. However, it is not known how to harness antigen-presenting cells optimally to achieve an effective antitumor T-cell response. In this study, we show that anti-CD47 antibody-mediated phagocytosis of cancer by macrophages can initiate an antitumor T-cell immune response. Using the ovalbumin model antigen system, anti-CD47 antibody-mediated phagocytosis of cancer cells by macrophages resulted in increased priming of OT-I T cells [cluster of differentiation 8-positive (CD8(+))] but decreased priming of OT-II T cells (CD4(+)). The CD4(+) T-cell response was characterized by a reduction in forkhead box P3-positive (Foxp3(+)) regulatory T cells. Macrophages following anti-CD47-mediated phagocytosis primed CD8(+) T cells to exhibit cytotoxic function in vivo. This response protected animals from tumor challenge. We conclude that anti-CD47 antibody treatment not only enables macrophage phagocytosis of cancer but also can initiate an antitumor cytotoxic T-cell immune response.


Subject(s)
Antibodies, Monoclonal/therapeutic use , CD47 Antigen/immunology , Colonic Neoplasms/immunology , Colonic Neoplasms/therapy , Macrophages/immunology , T-Lymphocytes/immunology , Animals , Antibodies, Blocking/therapeutic use , CD8-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Colonic Neoplasms/pathology , Cytotoxicity, Immunologic/genetics , Down-Regulation/genetics , Down-Regulation/immunology , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phagocytosis/immunology , Up-Regulation/genetics , Up-Regulation/immunology , Xenograft Model Antitumor Assays
7.
PLoS One ; 7(7): e40321, 2012.
Article in English | MEDLINE | ID: mdl-22815738

ABSTRACT

Gene expression profiling using microarrays has been limited to comparisons of gene expression between small numbers of samples within individual experiments. However, the unknown and variable sensitivities of each probeset have rendered the absolute expression of any given gene nearly impossible to estimate. We have overcome this limitation by using a very large number (>10,000) of varied microarray data as a common reference, so that statistical attributes of each probeset, such as the dynamic range and threshold between low and high expression, can be reliably discovered through meta-analysis. This strategy is implemented in a web-based platform named "Gene Expression Commons" (https://gexc.stanford.edu/) which contains data of 39 distinct highly purified mouse hematopoietic stem/progenitor/differentiated cell populations covering almost the entire hematopoietic system. Since the Gene Expression Commons is designed as an open platform, investigators can explore the expression level of any gene, search by expression patterns of interest, submit their own microarray data, and design their own working models representing biological relationship among samples.


Subject(s)
Gene Expression Profiling/standards , Search Engine , Animals , Databases, Genetic , Gene Expression Profiling/methods , Hematopoiesis/genetics , Humans , Mice , Oligonucleotide Array Sequence Analysis , Reference Standards
8.
Blood ; 118(20): 5439-47, 2011 Nov 17.
Article in English | MEDLINE | ID: mdl-21931117

ABSTRACT

Natural killer (NK) cells develop in the bone marrow and are known to gradually acquire the ability to eliminate infected and malignant cells, yet the cellular stages of NK lineage commitment and maturation are incompletely understood. Using 12-color flow cytometry, we identified a novel NK-committed progenitor (pre-NKP) that is a developmental intermediate between the upstream common lymphoid progenitor and the downstream NKP, previously assumed to represent the first stage of NK lineage commitment. Our analysis also refined the purity of NKPs (rNKP) by 6-fold such that 50% of both pre-NKP and rNKP cells gave rise to NKp46+ NK cells at the single-cell level. On transplantation into unconditioned Rag2-/-Il2rγc-/- recipients, both pre-NKPs and rNKPs generated mature NK cells expressing a repertoire of Ly49 family members that degranulated on stimulation ex vivo. Intrathymic injection of these progenitors, however, yielded no NK cells, suggesting a separate origin of thymic NK cells. Unlike the rNKP, the pre-NKP does not express IL-2Rß (CD122), yet it is lineage committed toward the NK cell fate, adding support to the theory that IL-15 signaling is not required for NK commitment. Taken together, our data provide a high-resolution in vivo analysis of the earliest steps of NK cell commitment and maturation.


Subject(s)
Bone Marrow Cells/cytology , Cell Lineage/immunology , Killer Cells, Natural/cytology , Lymphoid Progenitor Cells/cytology , Thymocytes/cytology , Animals , Biomarkers , Cell Differentiation/immunology , Cell Separation/methods , Cells, Cultured , Flow Cytometry/methods , Immunophenotyping , Mice , Mice, Inbred C57BL , Mice, Mutant Strains
9.
Proc Natl Acad Sci U S A ; 107(8): 3663-8, 2010 Feb 23.
Article in English | MEDLINE | ID: mdl-20133688

ABSTRACT

Nuclear factor of activated T cell (NFAT) transcription factors are key regulators of gene transcription within immune cells. The NFAT-interacting protein, (NIP45), augments NFAT-driven IL-4 expression by a mechanism that relies on arginine methylation. To establish the function of NIP45 in vivo, we generated mice with a targeted deletion of the gene encoding this cofactor. NIP45-deficient T helper cells displayed profound defects in the expression of NFAT-regulated cytokine genes, including IL-4. Whereas NIP45 deficiency does not interfere with T helper cell NFAT activation or lineage-specific transcription-factor expression, NIP45 acts as an enhancer for the assembly of protein arginine methyltransferase 1 and the protein arginine methyltransferase 1-linked histone 4 arginine 3 methylation with the IL-4 promoter. Our study reveals an essential role for NIP45 in promoting robust cytokine expression in vivo, which is required for the efficient handling of parasites. We propose that NIP45 acts as a molecular rheostat serving to amplify the type-2 immune response.


Subject(s)
Cytokines/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Proteins/metabolism , Th2 Cells/immunology , Animals , Arginine/metabolism , Chromatin Assembly and Disassembly , Gene Deletion , Gene Expression Regulation , Histones/metabolism , Interleukin-4/genetics , Intracellular Signaling Peptides and Proteins/genetics , Methylation , Mice , Mice, Mutant Strains , NFATC Transcription Factors/metabolism , Nuclear Proteins/genetics , Promoter Regions, Genetic , Protein-Arginine N-Methyltransferases/metabolism , Trichinella spiralis , Trichinellosis/immunology
10.
Proc Natl Acad Sci U S A ; 104(8): 2827-30, 2007 Feb 20.
Article in English | MEDLINE | ID: mdl-17307869

ABSTRACT

Tissue remodeling with fibrosis is a predominant pathophysiological mechanism of many human diseases. Systemic sclerosis is a rare, often lethal, disorder of unknown etiology manifested by dermal fibrosis (scleroderma) and excessive connective tissue deposition in internal organs. Currently, there are no available antifibrotic therapeutics, a reflection of our lack of understanding of this process. Animal models of scleroderma are useful tools to dissect the transcription factors and cytokines that govern fibrosis. A disproportionate increase of type 2 cytokines, like TGF-beta and IL-4, more than type 1 cytokines, like IFN-gamma, is thought to underlie the pathogenesis of scleroderma. In this study, we show that mice deficient in the transcription factor T-box expressed in T cells (T-bet), a master regulator of type 1 immunity, display increased sensitivity to bleomycin-induced dermal sclerosis. Despite the well-established role of T-bet in adaptive immunity, we also show that RAG2(-/-) mice, which lack T and B cells, are vulnerable to bleomycin-induced scleroderma and that RAG2/T-bet double-deficient mice maintain the increased sensitivity to bleomycin observed in T-bet(-/-) mice. Furthermore, overexpression of T-bet in T cells does not affect the induction of skin sclerosis in this model. Lastly, we show that IL-13 is the profibrotic cytokine regulated by T-bet in this model. Together, we conclude that T-bet serves as a repressor of dermal sclerosis through an IL-13-dependent pathway in innate immune cells. T-bet, and its transcriptional network, represent an attractive target for the treatment of systemic sclerosis and other fibrosing disorders.


Subject(s)
Immunity, Innate/immunology , Interleukin-13/metabolism , Skin/pathology , T-Box Domain Proteins/metabolism , Animals , Bleomycin , Cytokines/metabolism , Fibrosis , Immune System , Interleukin-13/deficiency , Mice , Mice, Inbred BALB C , Mice, Knockout , Scleroderma, Systemic/chemically induced , Scleroderma, Systemic/pathology , Sclerosis , T-Box Domain Proteins/deficiency
11.
J Clin Invest ; 116(2): 414-21, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16410834

ABSTRACT

The transcription factor T-bet (Tbx21) plays a major role in adaptive immunity and is required for optimal IFN-gamma production by DCs. Here we demonstrate an essential function for T-bet in DCs in controlling inflammatory arthritis. We show that collagen antibody-induced arthritis (CAIA), a model of human RA, is a bipartite disease characterized by an early innate immune system component intact in RAG2 mice and a later adaptive immune system phase. Mice lacking T-bet had markedly reduced joint inflammation at both early and late time points and RAG2T-bet double-deficient mice were essentially resistant to disease. Remarkably, adoptive transfer of T-bet-expressing DCs reconstituted inflammation in a T-bet deficient and T-bet/RAG2-deficient milieu. T-bet regulates the production of proinflammatory cytokine IL-1alpha and chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and thymus- and activation-related chemokine (TARC) by DCs. Further, T-bet expression in DCs is required for T helper cell activation. We conclude that T-bet plays a vital function in DCs that links innate and adaptive immunity to regulate inflammatory responses. T-bet provides an attractive new target for the development of novel therapeutics for inflammatory arthritis.


Subject(s)
Arthritis, Experimental/immunology , Dendritic Cells/immunology , Transcription Factors/immunology , Adoptive Transfer , Animals , Arthritis, Experimental/pathology , Cell Movement , Chemokines/immunology , Cytokines/immunology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dendritic Cells/cytology , Dendritic Cells/transplantation , Female , Humans , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Mice, Knockout , T-Box Domain Proteins , T-Lymphocytes/immunology , Transcription Factors/genetics
12.
Mol Cell ; 15(4): 559-71, 2004 Aug 27.
Article in English | MEDLINE | ID: mdl-15327772

ABSTRACT

Posttranslational modification of proteins within T cell receptor signaling cascades allows T lymphocytes to rapidly initiate an appropriate immune response. Here we report a role for arginine methylation in regulating cytokine gene transcription in the T helper lymphocyte. Inhibition of arginine methylation impaired the expression of several cytokine genes, including the signature type 1 and type 2 helper cytokines, interferon gamma, and interleukin-4. T cell receptor signaling increased expression of the protein arginine methyltransferase PRMT1, which in turn methylated the nuclear factor of activated T cells (NFAT) cofactor protein, NIP45. Arginine methylation of the amino terminus of NIP45 modulated its interaction with NFAT and resulted in augmented cytokine production, while T cells from mice lacking NIP45 had impaired expression of IFNgamma and IL-4. Covalent modification of NIP45 by arginine methylation is an important mechanism of regulating the expression of NFAT-dependent cytokine genes.


Subject(s)
Arginine/metabolism , Carrier Proteins/metabolism , Cytokines/metabolism , Gene Expression Regulation , Intracellular Signaling Peptides and Proteins , Nuclear Proteins/metabolism , Protein-Arginine N-Methyltransferases/metabolism , T-Lymphocytes, Helper-Inducer/physiology , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Cells, Cultured , Cytokines/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Interferon-gamma/metabolism , Interleukin-4/metabolism , Methylation , Mice , Mice, Inbred BALB C , Molecular Sequence Data , NFATC Transcription Factors , Nuclear Proteins/genetics , Protein-Arginine N-Methyltransferases/genetics , Receptors, Antigen, T-Cell/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction/physiology , T-Lymphocytes, Helper-Inducer/immunology , Transcription Factors/genetics , Transcription Factors/metabolism
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