1
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Wenhua S, Tsunematsu T, Umeda M, Tawara H, Fujiwara N, Mouri Y, Arakaki R, Ishimaru N, Kudo Y. Cancer cell-derived novel periostin isoform promotes invasion in head and neck squamous cell carcinoma. Cancer Med 2023; 12:8510-8525. [PMID: 36691359 PMCID: PMC10134278 DOI: 10.1002/cam4.5601] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2022] [Revised: 12/20/2022] [Accepted: 12/21/2022] [Indexed: 01/25/2023] Open
Abstract
It recently has been reported that partial-epithelial-mesenchymal transition (p-EMT) program is associated with metastasis in head and neck squamous cell carcinoma (HNSCC). We previously have identified POSTN (which encodes periostin) as an invasion-promoting molecule in HNSCC. Interestingly, POSTN expression is frequently observed in cancer cells with higher p-EMT score by using a previous single-cell transcriptomic data of HNSCC cases. Although it is known that POSTN has 11 splicing variants, the role of them has not been determined in HNSCC. Here, we found that HNSCC cells with EMT features expressed POSTN isoforms, Iso3 (lacking exon 17 and 21) and Iso5 (lacking exon 17), whereas fibroblast expressed Iso3 and Iso4 (lacking exon 17, 18, and 21). The expression of POSTN Iso3 and Iso4 are known to be widely observed in various cell types including stromal cells. Therefore, we focused on the role of novel cancer cell-derived POSTN isoform, Iso5, in HNSCC. Single overexpression of POSTN Iso5 as well as Iso3 promoted invasion. Surprisingly, Iso5 synergistically promoted invasion together with Iso3. Notably, Iso5 as well as Iso3 upregulated p-EMT-related genes. We suggest that a novel cancer-specific POSTN isoform lacking exon 17 (Iso5) can be a useful marker for detecting cancer cells undergoing p-EMT. Moreover, a POSTN Iso5 can be a novel target for diagnosis and therapy in HNSCC.
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Affiliation(s)
- Shao Wenhua
- Department of Oral Bioscience, Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan
| | - Takaaki Tsunematsu
- Department of Oral Molecular Pathology, Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan
| | - Masaaki Umeda
- Department of Oral Molecular Pathology, Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan
| | - Hiroaki Tawara
- Department of Oral Molecular Pathology, Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan
| | - Natsumi Fujiwara
- Department of Oral Healthcare Promotion, Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan
| | - Yasuhiro Mouri
- Department of Oral Bioscience, Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan
| | - Rieko Arakaki
- Department of Oral Molecular Pathology, Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan
| | - Naozumi Ishimaru
- Department of Oral Molecular Pathology, Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan
| | - Yasusei Kudo
- Department of Oral Bioscience, Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan
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2
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de Rutte J, Dimatteo R, Archang MM, van Zee M, Koo D, Lee S, Sharrow AC, Krohl PJ, Mellody M, Zhu S, Eichenbaum JV, Kizerwetter M, Udani S, Ha K, Willson RC, Bertozzi AL, Spangler J, Damoiseaux R, Di Carlo D. Suspendable Hydrogel Nanovials for Massively Parallel Single-Cell Functional Analysis and Sorting. ACS NANO 2022; 16:7242-7257. [PMID: 35324146 PMCID: PMC9869715 DOI: 10.1021/acsnano.1c11420] [Citation(s) in RCA: 39] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/26/2023]
Abstract
Techniques to analyze and sort single cells based on functional outputs, such as secreted products, have the potential to transform our understanding of cellular biology as well as accelerate the development of next-generation cell and antibody therapies. However, secreted molecules rapidly diffuse away from cells, and analysis of these products requires specialized equipment and expertise to compartmentalize individual cells and capture their secretions. Herein, we describe methods to fabricate hydrogel-based chemically functionalized microcontainers, which we call nanovials, and demonstrate their use for sorting single viable cells based on their secreted products at high-throughput using only commonly accessible laboratory infrastructure. These nanovials act as solid supports that facilitate attachment of a variety of adherent and suspension cell types, partition uniform aqueous compartments, and capture secreted proteins. Solutions can be exchanged around nanovials to perform fluorescence immunoassays on secreted proteins. Using this platform and commercial flow sorters, we demonstrate high-throughput screening of stably and transiently transfected producer cells based on relative IgG production. Chinese hamster ovary cells sorted based on IgG production regrew and maintained a high secretion phenotype over at least a week, yielding >40% increase in bulk IgG production rates. We also sorted hybridomas and B lymphocytes based on antigen-specific antibody production. Hybridoma cells secreting an antihen egg lysozyme antibody were recovered from background cells, enriching a population of ∼4% prevalence to >90% following sorting. Leveraging the high-speed sorting capabilities of standard sorters, we sorted >1 million events in <1 h. IgG secreting mouse B cells were also sorted and enriched based on antigen-specific binding. Successful sorting of antibody-secreting B cells combined with the ability to perform single-cell RT-PCR to recover sequence information suggests the potential to perform antibody discovery workflows. The reported nanovials can be easily stored and distributed among researchers, democratizing access to high-throughput functional cell screening.
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Affiliation(s)
- Joseph de Rutte
- Department of Bioengineering, University of California, Los Angeles, CA 90095, USA
- Partillion Bioscience Corporation, Los Angeles, CA 90095, USA
| | - Robert Dimatteo
- Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095, USA
| | - Maani M. Archang
- Department of Bioengineering, University of California, Los Angeles, CA 90095, USA
| | - Mark van Zee
- Department of Bioengineering, University of California, Los Angeles, CA 90095, USA
| | - Doyeon Koo
- Department of Bioengineering, University of California, Los Angeles, CA 90095, USA
| | - Sohyung Lee
- Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095, USA
| | - Allison C. Sharrow
- California NanoSystems Institute, University of California, Los Angeles, CA 90095, USA
| | - Patrick J. Krohl
- Department of Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21231, USA
| | - Michael Mellody
- Department of Bioengineering, University of California, Los Angeles, CA 90095, USA
- California NanoSystems Institute, University of California, Los Angeles, CA 90095, USA
| | - Sheldon Zhu
- Partillion Bioscience Corporation, Los Angeles, CA 90095, USA
| | - James V. Eichenbaum
- Department of Bioengineering, University of California, Los Angeles, CA 90095, USA
| | - Monika Kizerwetter
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21231, USA
| | - Shreya Udani
- Department of Bioengineering, University of California, Los Angeles, CA 90095, USA
| | - Kyung Ha
- Department of Mathematics, University of California, Los Angeles, CA 90095, USA
| | - Richard C. Willson
- Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX 77204, USA
| | - Andrea L. Bertozzi
- Department of Mechanical and Aerospace Engineering, University of California, Los Angeles, CA 90095, USA
- Department of Mathematics, University of California, Los Angeles, CA 90095, USA
- California NanoSystems Institute, University of California, Los Angeles, CA 90095, USA
| | - Jamie Spangler
- Department of Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21231, USA
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21231, USA
- Translational Tissue Engineering Center, Johns Hopkins University, Baltimore, MD 21231, USA
| | - Robert Damoiseaux
- Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA 90095, USA
- California NanoSystems Institute, University of California, Los Angeles, CA 90095, USA
- Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA 90095, USA
| | - Dino Di Carlo
- Department of Bioengineering, University of California, Los Angeles, CA 90095, USA
- Department of Mechanical and Aerospace Engineering, University of California, Los Angeles, CA 90095, USA
- California NanoSystems Institute, University of California, Los Angeles, CA 90095, USA
- Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA 90095, USA
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3
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Dimatteo R, Di Carlo D. IL-2 secretion-based sorting of single T cells using high-throughput microfluidic on-cell cytokine capture. LAB ON A CHIP 2022; 22:1576-1583. [PMID: 35293406 PMCID: PMC9013285 DOI: 10.1039/d1lc01098k] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/12/2023]
Abstract
Secreted proteins are critical for the coordination of potent immune defenses, such as in engineered T cell therapies, however, there are few widely accessible approaches to accurately analyze and sort large numbers of cells based on their secretory functions. We report a workflow for the rapid screening and sorting of single individual T cells based on IL-2 secretion accumulated at high concentrations in nanoliter droplets and encoded back onto the secreting cell's surface. In our method, droplets are used solely to partition cells, enabling rapid accumulation of signals onto cell surfaces, and eliminating diffusive crosstalk between neighbors. All downstream sorting leverages conventional high-throughput and readily accessible flow cytometry after the emulsion is disrupted. We achieve monodisperse droplet generation (CV < 10%) at flow rates up to 200 μL min-1 using step emulsification, enabling processing of entire libraries of cells within tens of minutes without significant secretion crosstalk. In comparison to our approach, strong mitogenic activation overwhelmed the conventional bulk on-cell cytokine assay, rendering labeled, non-activated cells indistinguishable from actively secreting neighbors within one hour. Processing of identical cell mixtures following droplet encapsulation yielded no apparent crosstalk even after three hours. Instead, IL-2 production spanning several orders of magnitude was observed from roughly 20% of analyzed activated lymphocytes, representing an at least 10-fold increase in dynamic range compared to unencapsulated cells. Secreting cells could also be sorted using fluorescence activated cell sorting (FACS). The approach can ultimately enable sorting of cells based on functional properties with higher accuracy in a more accessible format to life science researchers.
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Affiliation(s)
- Robert Dimatteo
- Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, 5531 Boelter Hall, P.O. Box 951592, Los Angeles, CA, 90095, USA
- Department of Bioengineering, University of California, Los Angeles, 420 Westwood Plaza, 5121 Engineering V, P.O. Box 951600, Los Angeles, CA 90095, USA.
| | - Dino Di Carlo
- Department of Bioengineering, University of California, Los Angeles, 420 Westwood Plaza, 5121 Engineering V, P.O. Box 951600, Los Angeles, CA 90095, USA.
- California Nano Systems Institute, 570 Westwood Plaza, Building 114, Los Angeles, CA 90095, USA
- Jonsson Comprehensive Cancer Center, Los Angeles, CA 90095, USA
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4
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Price MJ, Hicks SL, Bradley JE, Randall TD, Boss JM, Scharer CD. IgM, IgG, and IgA Influenza-Specific Plasma Cells Express Divergent Transcriptomes. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2019; 203:2121-2129. [PMID: 31501259 PMCID: PMC6783370 DOI: 10.4049/jimmunol.1900285] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/08/2019] [Accepted: 08/13/2019] [Indexed: 12/31/2022]
Abstract
Ab-secreting cells (ASC) or plasma cells are essential components of the humoral immune system. Although Abs of different isotypes have distinct functions, it is not known if the ASC that secrete each isotype are also distinct. ASC downregulate their surface BCR upon differentiation, hindering analyses that couple BCR information to other molecular characteristics. In this study, we developed a methodology using fixation, permeabilization, and intracellular staining coupled with cell sorting and reversal of the cross-links to allow RNA sequencing of isolated cell subsets. Using hemagglutinin and nucleoprotein Ag-specific B cell tetramers and intracellular staining for IgM, IgG, and IgA isotypes, we were able to derive and compare the gene expression programs of ASC subsets that were responding to the same Ags following influenza infection in mice. Intriguingly, whereas a shared ASC signature was identified, each ASC isotype-specific population expressed distinct transcriptional programs controlling cellular homing, metabolism, and potential effector functions. Additionally, we extracted and compared BCR clonotypes and found that each ASC isotype contained a unique, clonally related CDR3 repertoire. In summary, these data reveal specific complexities in the transcriptional programming of Ag-specific ASC populations.
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Affiliation(s)
- Madeline J Price
- Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322
| | - Sakeenah L Hicks
- Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322
| | - John E Bradley
- Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294; and
| | - Troy D Randall
- Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294; and
| | - Jeremy M Boss
- Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322
- Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322
| | - Christopher D Scharer
- Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322;
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5
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Luziga C. Potential role of cytotoxic T-lymphocyte antigen 2 alpha in secretory activity of endocrine cells in mouse adenohypophysis. Open Vet J 2019; 9:114-119. [PMID: 31360649 PMCID: PMC6626156 DOI: 10.4314/ovj.v9i2.4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2018] [Accepted: 03/10/2019] [Indexed: 11/18/2022] Open
Abstract
The peptide hormones of the adenohypophysis are produced by proteolytic processing of their prohormone precursors. Cathepsin L is known to function as a major proteolytic enzyme involved in the production of the peptide hormones. The structure of the propeptide region of cathepsin L is identical to cytotoxic T-lymphocyte antigen-2 alpha (CTLA-2α) which is also shown to exhibit selective inhibitory activities against cathepsin L. However, the specific cell types synthesizing CTLA-2α in mouse adenohypophysis and its functional implications as relevant in vivo have not been demonstrated. In this study, CTLA-2α expression in the adenohypophysis was evaluated by immunohistochemistry. In both male and female mice, strong immunoreactivity was specifically detected in folliculostellate (FS) cells surrounding endocrine cells which were delineated by CTLA-2α. These findings suggest that the CTLA-2α may be involved in the proteolytic processing and secretion of the hormones in the adenohypophysis through regulation of cathepsin L.
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Affiliation(s)
- Claudius Luziga
- Department of Veterinary Anatomy and Pathology, Sokoine University of Agriculture, Morogoro, Tanzania
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6
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Andersson J, Skansén-Saphir U, Sparrelid E, Andersson U. Intravenous immune globulin affects cytokine production in T lymphocytes and monocytesjmacrophages. Clin Exp Immunol 2019. [DOI: 10.1111/cei.1996.104.s1.10] [Citation(s) in RCA: 94] [Impact Index Per Article: 15.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022] Open
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7
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Stewart MP, Langer R, Jensen KF. Intracellular Delivery by Membrane Disruption: Mechanisms, Strategies, and Concepts. Chem Rev 2018; 118:7409-7531. [PMID: 30052023 PMCID: PMC6763210 DOI: 10.1021/acs.chemrev.7b00678] [Citation(s) in RCA: 436] [Impact Index Per Article: 62.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Intracellular delivery is a key step in biological research and has enabled decades of biomedical discoveries. It is also becoming increasingly important in industrial and medical applications ranging from biomanufacture to cell-based therapies. Here, we review techniques for membrane disruption-based intracellular delivery from 1911 until the present. These methods achieve rapid, direct, and universal delivery of almost any cargo molecule or material that can be dispersed in solution. We start by covering the motivations for intracellular delivery and the challenges associated with the different cargo types-small molecules, proteins/peptides, nucleic acids, synthetic nanomaterials, and large cargo. The review then presents a broad comparison of delivery strategies followed by an analysis of membrane disruption mechanisms and the biology of the cell response. We cover mechanical, electrical, thermal, optical, and chemical strategies of membrane disruption with a particular emphasis on their applications and challenges to implementation. Throughout, we highlight specific mechanisms of membrane disruption and suggest areas in need of further experimentation. We hope the concepts discussed in our review inspire scientists and engineers with further ideas to improve intracellular delivery.
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Affiliation(s)
- Martin P. Stewart
- Department of Chemical Engineering, Massachusetts Institute
of Technology, Cambridge, USA
- The Koch Institute for Integrative Cancer Research,
Massachusetts Institute of Technology, Cambridge, USA
| | - Robert Langer
- Department of Chemical Engineering, Massachusetts Institute
of Technology, Cambridge, USA
- The Koch Institute for Integrative Cancer Research,
Massachusetts Institute of Technology, Cambridge, USA
| | - Klavs F. Jensen
- Department of Chemical Engineering, Massachusetts Institute
of Technology, Cambridge, USA
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8
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Kupcova Skalnikova H, Cizkova J, Cervenka J, Vodicka P. Advances in Proteomic Techniques for Cytokine Analysis: Focus on Melanoma Research. Int J Mol Sci 2017; 18:E2697. [PMID: 29236046 PMCID: PMC5751298 DOI: 10.3390/ijms18122697] [Citation(s) in RCA: 51] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2017] [Revised: 12/07/2017] [Accepted: 12/08/2017] [Indexed: 12/16/2022] Open
Abstract
Melanoma is a skin cancer with permanently increasing incidence and resistance to therapies in advanced stages. Reports of spontaneous regression and tumour infiltration with T-lymphocytes makes melanoma candidate for immunotherapies. Cytokines are key factors regulating immune response and intercellular communication in tumour microenvironment. Cytokines may be used in therapy of melanoma to modulate immune response. Cytokines also possess diagnostic and prognostic potential and cytokine production may reflect effects of immunotherapies. The purpose of this review is to give an overview of recent advances in proteomic techniques for the detection and quantification of cytokines in melanoma research. Approaches covered span from mass spectrometry to immunoassays for single molecule detection (ELISA, western blot), multiplex assays (chemiluminescent, bead-based (Luminex) and planar antibody arrays), ultrasensitive techniques (Singulex, Simoa, immuno-PCR, proximity ligation/extension assay, immunomagnetic reduction assay), to analyses of single cells producing cytokines (ELISpot, flow cytometry, mass cytometry and emerging techniques for single cell secretomics). Although this review is focused mainly on cancer and particularly melanoma, the discussed techniques are in general applicable to broad research field of biology and medicine, including stem cells, development, aging, immunology and intercellular communication.
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Affiliation(s)
- Helena Kupcova Skalnikova
- Laboratory of Applied Proteome Analyses, Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Rumburska 89, 27721 Libechov, Czech Republic.
| | - Jana Cizkova
- Laboratory of Applied Proteome Analyses, Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Rumburska 89, 27721 Libechov, Czech Republic.
- Department of Veterinary Sciences, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences, Kamycka 129, 16500 Prague, Czech Republic.
| | - Jakub Cervenka
- Laboratory of Applied Proteome Analyses, Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Rumburska 89, 27721 Libechov, Czech Republic.
- Department of Cell Biology, Faculty of Science, Charles University, Vinicna 7, 12843 Prague 4, Czech Republic.
| | - Petr Vodicka
- Laboratory of Applied Proteome Analyses, Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Rumburska 89, 27721 Libechov, Czech Republic.
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9
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van der Loos CM, Houtkamp MA, de Boer OJ, Teeling P, van der Wal AC, Becker AE. Immunohistochemical Detection of Interferon-γ: Fake or Fact? J Histochem Cytochem 2016; 49:699-710. [PMID: 11373317 DOI: 10.1177/002215540104900604] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Immunohistochemistry is a widely accepted tool to investigate the presence and immunolocalization of cytokines in tissue sections at the protein level. We have tested the specificity and reproducibility of IFNγ immunohistochemistry on tissue sections with a large panel of anti-IFNγ antibodies. Thirteen different commercially available anti-IFNγ antibodies, including seven advertised and/or regularly applied for immunohistochemistry/-cytochemistry, were tested using a three-step streptavidin–biotin–peroxidase technique and a two-step immunofluorescence (FACS) analysis. Immunoenzyme double staining was used to identify the IFNγ-positive cells. Serial cryostat sections were used of human reactive hyperplastic tonsils, rheumatoid synovium, and inflammatory abdominal aortic aneurysms, known to possess a prominent Th1-type immune response. In vitro phorbol myristate acetate/ionomycin-stimulated T-cells served as positive control; unstimulated cells served as negative control. Cultured T-cells were used adhered to glass slides (immunocytochemistry), in suspension (FACS), or snap-frozen and sectioned (immunohistochemistry). Immunocytochemistry and FACS analysis on stimulated cultured T-cells showed positive staining results with 12 of 13 anti-IFNγ antibodies. However, immunohistochemistry of sectioned stimulated T-cells was negative with all. Unstimulated cells were consistently negative. IFNγ immunohistochemical single- and double staining analysis of the tissue sections showed huge variations in staining patterns, including positivity for smooth muscle cells ( n = 8), endothelial cells ( n = 4), extracellular matrix ( n = 4), and CD138+ plasma cells ( n = 12). Specific staining of T-cells, as the sole positive staining, was not achieved with any of the 13 antibodies. IFNIFNγ-immunohistochemistry appears unreliable because of lack of specificity to stain T-cells in situ. In fact, depending on the type of anti-IFNγ antibody used, a variety of different cell constituents were nonspecifically stained. Consequently, data based on IFNγ-immunohistochemistry must be interpreted with great caution.
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Affiliation(s)
- C M van der Loos
- Academic Medical Center, Department of Cardiovascular Pathology, Amsterdam, The Netherlands.
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10
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Sandstedt M, Jonsson M, Asp J, Dellgren G, Lindahl A, Jeppsson A, Sandstedt J. Intracellular flow cytometry may be combined with good quality and high sensitivity RT-qPCR analysis. Cytometry A 2015; 87:1079-89. [PMID: 26348124 DOI: 10.1002/cyto.a.22783] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2015] [Revised: 06/30/2015] [Accepted: 08/23/2015] [Indexed: 02/04/2023]
Abstract
Flow cytometry (FCM) has become a well-established method for analysis of both intracellular and cell-surface proteins, while quantitative RT-PCR (RT-qPCR) is used to determine gene expression with high sensitivity and specificity. Combining these two methods would be of great value. The effects of intracellular staining on RNA integrity and RT-qPCR sensitivity and quality have not, however, been fully examined. We, therefore, intended to assess these effects further. Cells from the human lung cancer cell line A549 were fixed, permeabilized and sorted by FCM. Sorted cells were analyzed using RT-qPCR. RNA integrity was determined by RNA quality indicator analysis. A549 cells were then mixed with cells of the mouse cardiomyocyte cell line HL-1. A549 cells were identified by the cell surface marker ABCG2, while HL-1 cells were identified by intracellular cTnT. Cells were sorted and analyzed by RT-qPCR. Finally, cell cultures from human atrial biopsies were used to evaluate the effects of fixation and permeabilization on RT-qPCR analysis of nonimmortalized cells stored prior to analysis by FCM. A large amount of RNA could be extracted even when cells had been fixed and permeabilized. Permeabilization resulted in increased RNA degradation and a moderate decrease in RT-qPCR sensitivity. Gene expression levels were also affected to a moderate extent. Sorted populations from the mixed A549 and HL-1 cell samples showed gene expression patterns that corresponded to FCM data. When samples were stored before FCM sorting, the RT-qPCR analysis could still be performed with high sensitivity and quality. In summary, our results show that intracellular FCM may be performed with only minor impairment of the RT-qPCR sensitivity and quality when analyzing sorted cells; however, these effects should be considered when comparing RT-qPCR data of not fixed samples with those of fixed and permeabilized samples.
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Affiliation(s)
- Mikael Sandstedt
- Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.,Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden
| | - Marianne Jonsson
- Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.,Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden
| | - Julia Asp
- Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.,Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden
| | - Göran Dellgren
- Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.,Department of Cardiothoracic Surgery, Sahlgrenska University Hospital, Gothenburg, Sweden
| | - Anders Lindahl
- Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.,Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden
| | - Anders Jeppsson
- Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.,Department of Cardiothoracic Surgery, Sahlgrenska University Hospital, Gothenburg, Sweden
| | - Joakim Sandstedt
- Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.,Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden
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11
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Ermann J, Rao DA, Teslovich NC, Brenner MB, Raychaudhuri S. Immune cell profiling to guide therapeutic decisions in rheumatic diseases. Nat Rev Rheumatol 2015; 11:541-51. [PMID: 26034835 PMCID: PMC4898649 DOI: 10.1038/nrrheum.2015.71] [Citation(s) in RCA: 54] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Biomarkers are needed to guide treatment decisions for patients with rheumatic diseases. Although the phenotypic and functional analysis of immune cells is an appealing strategy for understanding immune-mediated disease processes, immune cell profiling currently has no role in clinical rheumatology. New technologies, including mass cytometry, gene expression profiling by RNA sequencing (RNA-seq) and multiplexed functional assays, enable the analysis of immune cell function with unprecedented detail and promise not only a deeper understanding of pathogenesis, but also the discovery of novel biomarkers. The large and complex data sets generated by these technologies--big data--require specialized approaches for analysis and visualization of results. Standardization of assays and definition of the range of normal values are additional challenges when translating these novel approaches into clinical practice. In this Review, we discuss technological advances in the high-dimensional analysis of immune cells and consider how these developments might support the discovery of predictive biomarkers to benefit the practice of rheumatology and improve patient care.
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Affiliation(s)
- Joerg Ermann
- Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Smith Building, 1 Jimmy Fund Way, Boston, MA 02115, USA
| | - Deepak A Rao
- Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Smith Building, 1 Jimmy Fund Way, Boston, MA 02115, USA
| | - Nikola C Teslovich
- 'Division of Genetics, Brigham and Women's Hospital, New Research Building NRB, 77 Avenue Louis Pasteur, Boston, MA 02115, USA
| | - Michael B Brenner
- Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Smith Building, 1 Jimmy Fund Way, Boston, MA 02115, USA
| | - Soumya Raychaudhuri
- Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, New Research Building (NRB), 77 Avenue Louis Pasteur, Boston, MA 02115, USA
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12
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Intracellular staining for cytokines and transcription factors. Methods Mol Biol 2014. [PMID: 25150995 DOI: 10.1007/978-1-4939-1212-4_5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2023]
Abstract
Within the past years immune cells in general and T cells in particular have been categorized into a vast variety of subsets with different functional properties. One of the key technologies fueling this emerging complexity is intracellular staining for effector cytokines and/or lineage-defining transcription factors. Here we discuss the critical steps for performing successful multicolor immunophenotyping of mouse T cells in combination with analysis of intracellular molecules after ex vivo isolation.
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13
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Van Hoof D, Lomas W, Hanley MB, Park E. Simultaneous flow cytometric analysis of IFN-γ and CD4 mRNA and protein expression kinetics in human peripheral blood mononuclear cells during activation. Cytometry A 2014; 85:894-900. [DOI: 10.1002/cyto.a.22521] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2014] [Revised: 07/21/2014] [Accepted: 07/24/2014] [Indexed: 01/23/2023]
Affiliation(s)
| | | | | | - Emily Park
- BD Biosciences; San Jose California 95131
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14
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Toyooka K, Liu F, Ishii M, Saito S, Kirikae T, Asano Y, Shinomiya H. Generation and Characterization of Monoclonal Antibodies That Specifically Recognize p65/L-Plastin Isoform but Not T-Plastin Isoform. Biosci Biotechnol Biochem 2014; 70:1402-7. [PMID: 16794320 DOI: 10.1271/bbb.50659] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The 65-kDa protein (p65) was previously identified as a phosphorylated protein in activated macrophages, and has turned out to be a member of a plastin protein family characterized by a series of Ca(2+)-, calmodulin-, and beta-actin-binding domains. In mice, two isoforms, p65/L-plastin and T-plastin, have so far been identified; p65/L-plastin is expressed in hemopoietic cells and cancer cells, and T-plastin in solid tissue cells. We generated monoclonal antibodies to p65/L-plastin, examined the isoform-specificity by using recombinant (r) T-plastin, and found that the antibodies were specific for rp65/L-plastin, whereas immune sera to rp65/L-plastin showed cross-reactions to rT-plastin. One of the antibodies, p65-7B5, was demonstrated to react to native p65/L-plastin by Western blot, flow cytometric, and immunohistochemical analysis. Furthermore, p65-7B5 has made it possible to detect p65/L-plastin-expressing cells in tissues where T-plastin is abundantly expressed. These reagents and procedures should provide specific tools to investigate the role of p65/L-plastin in leukocytes.
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15
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Sylwester AW, Hansen SG, Picker LJ. Quantification of T cell Antigen-specific Memory Responses in Rhesus Macaques, Using Cytokine Flow Cytometry (CFC, also Known as ICS and ICCS): Analysis of Flow Data. Bio Protoc 2014; 4:e1109. [PMID: 28280751 DOI: 10.21769/bioprotoc.1109] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022] Open
Abstract
What was initially termed 'CFC' (Cytokine Flow Cytometry) is now more commonly known as 'ICS' (Intra Cellular Staining), or less commonly as 'ICCS' (Intra Cellular Cytokine Staining). The key innovations were use of an effective permeant (allowing intracellular staining), and a reagent to disrupt secretion (trapping cytokines, thereby enabling accumulation of detectable intracellular signal). Because not all researchers who use the technique are interested in cytokines, the 'ICS' term has gained favor, though 'CFC' will be used here. CFC is a test of cell function, exposing lymphocytes to antigen in culture, then measuring any cytokine responses elicited. Test cultures are processed so as to stain cells with monoclonal antibodies tagged with fluorescent markers, and to chemically fix the cells and decontaminate the samples, using paraformaldehyde. CFC provides the powers of flow cytometry, which includes bulk sampling and multi-parametric cross-correlation, to the analysis of antigen-specific memory responses. A researcher using CFC is able to phenotypically characterize cells cultured with test antigen, and for phenotypic subsets (e.g. CD4+ or CD8+ T cells) determine the % frequency producing cytokine above background level. In contrast to ELISPOT and Luminex methods, CFC can correlate production of multiple cytokines from particular, phenotypically-characterized cells. The CFC assay is useful for detecting that an individual has had an antigen exposure (as in population screenings), or for following the emergence and persistence of antigen memories (as in studies of vaccination, infections, or pathogenesis). In addition to quantifying the % frequency of antigen-responding cells, mean fluorescence intensity can be used to assess how much of a cytokine is generated within responding cells. With the technological advance of flow cytometry, a current user of CFC often has access to 11 fluorescent channels (or even 18), making it possible to either highly-characterize the phenotypes of antigen-responding cells, or else simultaneously quantify the responses according to many cytokines or activation markers. Powerful software like FlowJo (TreeStar) and SPICE (NIAID) can be used to analyse the data, and to do sophisticated multivariate analysis of cytokine responses. The method described here is customized for cells from Rhesus macaque monkeys, and the extensive annotating notes represent a decade of accumulated technical experience. The same scheme is readily applicable to other mammalian cells (e.g. human or mouse), though the exact antibody clones will differ according to host system. The basic method described here incubates 1 × 106 Lymphocytes in 1 ml tube culture with antigen and co-stimulatory antibodies in the presence of Brefeldin A, prior to staining and fixation. Note: This is the second part of a two-part procedure. Part one has the same initial title, but the subtitle "From Assay Set-up to Data Acquisition (Sylwester et al., 2014)". The Abstract and Historical Background is the same for both documents.
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Affiliation(s)
- Andrew W Sylwester
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, USA
| | - Scott G Hansen
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, USA
| | - Louis J Picker
- Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, USA
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16
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Abstract
Analysis of intracellular cytokines is extremely important in the clinical treatment of numerous diseases. Flow cytometry (FCM) is a highly effective technique that detects intracellular cytokines using specific fluorescence-labeled antibodies. The common steps of this assay include cell collection, fixation, permeabilization, blocking, intracellular staining and analysis by FCM. This technique also allows for analyzing the biological function of cytokines. In this chapter, we describe a modified method to detect the specific intracellular cytokine staining using FCM, with an emphasis on the effects of variables including samples, temperature, buffers, data acquisition, and analysis.
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Affiliation(s)
- Jian-Ge Qiu
- Department of Cell Biology and Institute of Biomedicine, College of Life Science and Technology, Jinan University, Guangzhou, 510632, China
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17
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Borchers S, Ogonek J, Varanasi PR, Tischer S, Bremm M, Eiz-Vesper B, Koehl U, Weissinger EM. Multimer monitoring of CMV-specific T cells in research and in clinical applications. Diagn Microbiol Infect Dis 2013; 78:201-12. [PMID: 24331953 DOI: 10.1016/j.diagmicrobio.2013.11.007] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2013] [Revised: 10/11/2013] [Accepted: 11/04/2013] [Indexed: 10/26/2022]
Abstract
Multimer monitoring has become a standard technique for detection of antigen-specific T cells. The term "multimer" refers to a group of reagents based on the multimerisation of molecules in order to raise avidity and thus stabilize binding to their ligand. Multimers for detection of antigen-specific T-cell responses are based on major histocompatibility complex class I peptide complexes. Multimer staining enables fast and direct visualization of antigen-specific T cells; thus, it is widely applied to assess antiviral immunity, e.g., monitor patients in vaccination trials or confirm purity of cell products for adoptive transfer. Assessment of T-cell immunity against persistent pathogens like cytomegalovirus (CMV) is of major importance in immunosuppressed patients. Recent advancements of multimers facilitate reversible labeling and allow isolation of epitope-specific T cells for adoptive transfer. Here, we give an overview on the different multimers and their applications, with an emphasis on CMV-specific T-cell responses.
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Affiliation(s)
- Sylvia Borchers
- Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School (MHH), Hannover, Germany; Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover, Germany; German Centre for Infection Research (DZIF), Partnerside Hannover-Braunschweig, Germany.
| | - Justyna Ogonek
- Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School (MHH), Hannover, Germany.
| | - Pavankumar R Varanasi
- Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School (MHH), Hannover, Germany; Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover, Germany; German Centre for Infection Research (DZIF), Partnerside Hannover-Braunschweig, Germany.
| | - Sabine Tischer
- Institute of Transfusion Medicine, MHH, Hannover, Germany.
| | - Melanie Bremm
- Pediatric Hematology and Oncology, Johann Wolfgang Goethe-University, Frankfurt, Germany.
| | - Britta Eiz-Vesper
- Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover, Germany; Institute of Transfusion Medicine, MHH, Hannover, Germany.
| | - Ulrike Koehl
- Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover, Germany; Institute for Cellular Therapeutics, MHH, Hannover, Germany.
| | - Eva M Weissinger
- Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School (MHH), Hannover, Germany; Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover, Germany; German Centre for Infection Research (DZIF), Partnerside Hannover-Braunschweig, Germany.
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18
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Gaber T, Tran CL, Schellmann S, Hahne M, Strehl C, Hoff P, Radbruch A, Burmester GR, Buttgereit F. Pathophysiological hypoxia affects the redox state and IL-2 signalling of human CD4+T cells and concomitantly impairs survival and proliferation. Eur J Immunol 2013; 43:1588-97. [DOI: 10.1002/eji.201242754] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2012] [Revised: 01/23/2013] [Accepted: 03/15/2013] [Indexed: 11/08/2022]
Affiliation(s)
| | | | | | | | | | | | | | - Gerd-Rüdiger Burmester
- Department of Rheumatology and Clinical Immunology; Charité University Hospital; Berlin; Germany
| | - Frank Buttgereit
- Department of Rheumatology and Clinical Immunology; Charité University Hospital; Berlin; Germany
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19
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Kang I. Analysis of T Cells Using Flow Cytometry. JOURNAL OF RHEUMATIC DISEASES 2013. [DOI: 10.4078/jrd.2013.20.2.83] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Affiliation(s)
- Insoo Kang
- Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520, USA
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20
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Kim JW, Bilusic M, Heery CJ, Madan RA. Therapeutic cancer vaccines in prostate cancer: the quest for intermediate markers of response. Cancers (Basel) 2012; 4:1229-46. [PMID: 24213505 PMCID: PMC3712729 DOI: 10.3390/cancers4041229] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2012] [Revised: 11/09/2012] [Accepted: 11/14/2012] [Indexed: 11/17/2022] Open
Abstract
Despite recent advances in cancer immunotherapy, no prospectively validated intermediate biomarkers exist to predict response. These biomarkers are highly desirable given modern immunotherapy's paradoxical pattern of clinical benefit; that is, improvement in overall survival without short-term change in progression. Immunotherapy clinical trials have evaluated biomarkers that may correlate with clinical outcomes. Many of them are performed on peripheral blood to evaluate the systemic response, such as tumor-targeted humoral and cellular immunity, and cytokine responses. Accumulating evidence suggests that immune infiltrates in tumors may suggest evidence for the therapy's mechanism of action, and have greater potential for providing prognostic and predictive information. In addition, a non-immunologic biomarker, such as tumor growth kinetics, may explain this paradoxical pattern of clinical benefit, and predict survival in patients treated with an immunotherapy. Prospective assessment and validation of these and other intermediate markers would be required to better understand their potential clinical role.
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Affiliation(s)
- Joseph W Kim
- Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
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21
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Abstract
The effect of FK506 and cyclosporin A (CsA) on the production of
interleukin 6 (IL-6) in adherent monocytes was studied at a
single-cell level by the avidinbiotin- peroxidase complex methods.
The percentage of IL-6-producing monocytes increased when stimulated
with lipopolysaccharide (LPS) at concentrations between 10 ng/ml and
10 μg/ml, in a dose dependent manner. Both FK506 and CsA
enhanced the percentage of IL-6- producing monocytes stimulated with
100 pg/ml-1 μg/ml of LPS up to values near those
obtained with 10 μg/ml of LPS. The enhancement by FK506 and
CsA was not seen when monocytes were stimulated with a high
concentration of LPS (10 μg/ml). When monocytes were
stimulated with a low concentration of LPS (10 ng/ml), FK506 and
CsA enhanced IL-6 production in a dose dependent manner, at a drug
concentration of 0.12 nM–1.2 μM (0.1–1 000 ng/ml)
for FK506 and 0.83 nM–8.3 μM (1–10 000 ng/ml) for
CsA. The optimal effect of FK506 was achieved at a concentration
7-fold lower than that of CsA. In contrast, production of turnout
necrosis factor-α (TNFα and interleukin 1β
(IL-1β) was slightly suppressed by FK506 and CsA at the
concentrations tested. Moreover, pretreatment of monocytes with
FK506 and CsA had a significant enhancing effect on LPS-induced IL-6
production, while treatment with FK506 or CsA after LPS stimulation
had no effects on IL-6 production, suggesting that the enhancing
effect of each drug is exerted before LPS stimulation or at an early
stage of the post-receptor pathway after LPS stimulation. These
experiments demonstrate that FK506 and CsA can selectively enhance
IL-6 production in monocytes under certain conditions in
vitro and, possibly, also in vivo.
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22
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Muris AH, Damoiseaux J, Smolders J, Cohen Tervaert JW, Hupperts R, Thewissen M. Intracellular IL-10 detection in T cells by flowcytometry: The use of protein transport inhibitors revisited. J Immunol Methods 2012; 381:59-65. [DOI: 10.1016/j.jim.2012.04.011] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2011] [Revised: 04/18/2012] [Accepted: 04/24/2012] [Indexed: 11/29/2022]
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23
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Flesch IEA, Hollett NA, Wong YC, Tscharke DC. Linear fidelity in quantification of anti-viral CD8+ T cells. PLoS One 2012; 7:e39533. [PMID: 22745779 PMCID: PMC3379996 DOI: 10.1371/journal.pone.0039533] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2012] [Accepted: 05/23/2012] [Indexed: 11/19/2022] Open
Abstract
Enumeration of anti-viral CD8(+) T cells to make comparisons between mice, viruses and vaccines is a frequently used approach, but controversy persists as to the most appropriate methods. Use of peptide-MHC tetramers (or variants) and intracellular staining for cytokines, in particular IFNγ, after a short ex vivo stimulation are now common, as are a variety of cytotoxicity assays, but few direct comparisons have been made. It has been argued that use of tetramers leads to the counting of non-functional T cells and that measurement of single cytokines will fail to identify cells with alternative functions. Further, the linear range of these methods has not been tested and this is required to give confidence that relative quantifications can be compared across samples. Here we show for two acute virus infections and CD8(+) T cells activated in vitro that DimerX (a tetramer variant) and intracellular staining for IFNγ, alone or in combination with CD107 to detect degranulation, gave comparable results at the peak of the response. Importantly, these methods were highly linear over nearly two orders of magnitude. In contrast, in vitro and in vivo assays for cytotoxicity were not linear, suffering from high background killing, plateaus in maximal killing and substantial underestimation of differences in magnitude of responses.
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Affiliation(s)
- Inge E. A. Flesch
- Division of Biomedical Science and Biochemistry, Research School of Biology, The Australian National University, Canberra, Australia
| | - Natasha A. Hollett
- Division of Biomedical Science and Biochemistry, Research School of Biology, The Australian National University, Canberra, Australia
| | - Yik Chun Wong
- Division of Biomedical Science and Biochemistry, Research School of Biology, The Australian National University, Canberra, Australia
| | - David C. Tscharke
- Division of Biomedical Science and Biochemistry, Research School of Biology, The Australian National University, Canberra, Australia
- * E-mail:
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24
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Li W, Wu WW, Lin XS, Hou SX, Zhong HB, Ruan DK. Changes in T lymphocyte subsets and intracellular cytokines after transfer of chemically extracted acellular nerve allografts. Mol Med Rep 2012; 5:1080-6. [PMID: 22245851 PMCID: PMC3493064 DOI: 10.3892/mmr.2012.747] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2011] [Accepted: 12/27/2011] [Indexed: 11/06/2022] Open
Abstract
The aim of the present study was to observe the immune mechanism underlying the rejection of chemically extracted acellular nerve allografts for use in clinical applications. A total of 128 BALB/c mice were randomly divided into a negative contrast group (NC, 32 mice), a fresh autograft group (AG, 32 mice), a fresh allogeneic nerve group (FN, 32 mice) and a chemically extracted acellular allogeneic nerve group (CEN, 32 mice). Various types of nerve grafts were implanted into the thigh muscle of BALB/C mice in the corresponding groups. At 3, 7, 14 and 28 days post-operation, the mice (8 cases from each group) were sacrificed and their spleens were extracted. The spleens were ground into paste. The erythrocytes and other cells were lysed using distilled water and the T lymphocytes were collected. Monoclonal antibodies (CD3, CD4, CD8, CD25, IL-2, IFN-γ and TNF-α) were then added to the solution. The Facial Action Coding System was used to determine the positive rates of the cells combined with the monoclonal antibodies above. No significant statistical differences were observed between the CEN, NC and AG groups. However, some data of the FN group were significantly higher than those of the other groups at the corresponding time. No obvious immune rejections were observed among the chemically extracted acellular nerve allografts compared with fresh nerve autograft.
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Affiliation(s)
- Wei Li
- Department of Orthopaedics, Navy General Hospital, Beijing 100037, PR China.
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25
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Adams WC, Loré K. In vitro stimulation and detection of IFNα production in human plasmacytoid dendritic cells. Methods Mol Biol 2012; 820:215-230. [PMID: 22131034 DOI: 10.1007/978-1-61779-439-1_13] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/31/2023]
Abstract
Type-1 interferons (IFNs), including IFNα/β, are a family of cytokines produced rapidly upon pathogen encounter and crucial for bridging innate and adaptive immunity. IFNα has been widely appreciated as a multifunctional cytokine involved particularly in early immune responses against viral, bacterial, and parasitic infections. Although most cells may be competent to produce IFNα during specific conditions, plasmacytoid dendritic cells (PDCs) are unique in their capacity to produce rapid and robust levels in response to various pathogens. PDCs to a great extent utilize toll-like receptor (TLR) 7 and 9, localized in early endosomes, to sense pathogen-associated nucleic acids, and initiate the signaling cascade leading to induction of IFNα. Here, we provide basic protocols for the detection of IFNα in individual immune cells, particularly PDCs, using flow cytometry. We discuss the key elements for successful isolation of PDCs, stimulation, immunostaining, and identification of IFNα producing cells.
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Affiliation(s)
- William C Adams
- Department of Medicine, Center for Infectious Medicine, Karolinska University Hospital Huddinge, Stockholm, Sweden
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26
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Abstract
Mouse models of immunology are frequently used to study host responses to poxviruses or poxvirus-based recombinant vaccines. In this context, the magnitude of CD8(+) T cell responses is often of interest. Methods to evaluate CD8(+) T cell responses extend from those that rely on indirect measurement of effector function only, such as cytotoxicity assays, to those that only measure antiviral CD8(+) T cell numbers and not function, like peptide MHC tetramers. In this chapter, five methods are provided that cover this range: DimerX staining (a variant of peptide-MHC tetramers), intracellular cytokine staining for interferon-γ, CD62L/Granzyme B staining, and in vitro and ex vivo cytotoxicity assays. We also include tables of vaccinia virus peptide epitopes for use in most of these assays.
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27
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Hammers CM, Bieber K, Kalies K, Banczyk D, Ellebrecht CT, Ibrahim SM, Zillikens D, Ludwig RJ, Westermann J. Complement-fixing anti-type VII collagen antibodies are induced in Th1-polarized lymph nodes of epidermolysis bullosa acquisita-susceptible mice. THE JOURNAL OF IMMUNOLOGY 2011; 187:5043-50. [PMID: 21967893 DOI: 10.4049/jimmunol.1100796] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The environment encountered in secondary lymphoid organs (e.g., lymph nodes) influences the outcome of immune responses. Immunization of mice with type VII collagen, an adhesion protein expressed at the cutaneous basement membrane, induces experimental epidermolysis bullosa acquisita (EBA). In this model, clinical disease is associated with the H2s haplotype of the MHC found in SJL/J mice. Most other strains (e.g., BALB/c, C57BL/6, NZM2410/J) are resistant to clinical disease, despite autoantibody production. Comparison of autoantibody response in EBA-resistant and -susceptible mice showed an IgG2-dominated response in the latter. We hypothesized that EBA susceptibility is due to specific cytokine gene expression in draining lymph nodes (dLN). To challenge this hypothesis, EBA-susceptible (SJL/J) and -resistant (BALB/c, C57BL/6) mice were immunized with type VII collagen, followed by analysis of clinical phenotype, subclasses of circulating and tissue-bound autoantibodies, complement activation, and cytokine gene expression in dLN. Disease manifestation was associated with induction of complement-fixing autoantibodies, confirming previous observations. Furthermore, however, IFN-γ/IL-4 ratio in dLN of EBA-susceptible mice was significantly increased compared with EBA-resistant strains, suggesting a Th1 polarization. Immunization of H2s-congenic C57BL/6 mice (B6.SJL-H2s) led to Th1 polarization in dLN and clinical disease. In addition to their cytokine milieu, EBA-susceptible and -resistant mice also differed regarding the expression of FcγR on peripheral leukocytes, in which a higher FcγRIV expression in SJL/J and B6.SJL-H2s mice, compared with C57BL/6, was associated with skin lesions. In summary, blistering in experimental EBA is regulated by both adaptive (divergent class switch recombination due to polarized cytokine expression) and innate (FcγR expression) immune mechanisms.
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28
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Gaber T, Schellmann S, Erekul KB, Fangradt M, Tykwinska K, Hahne M, Maschmeyer P, Wagegg M, Stahn C, Kolar P, Dziurla R, Löhning M, Burmester GR, Buttgereit F. Macrophage Migration Inhibitory Factor Counterregulates Dexamethasone-Mediated Suppression of Hypoxia-Inducible Factor-1α Function and Differentially Influences Human CD4+ T Cell Proliferation under Hypoxia. THE JOURNAL OF IMMUNOLOGY 2010; 186:764-74. [DOI: 10.4049/jimmunol.0903421] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
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29
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Sjögren F, Anderson CD. Are cutaneous microdialysis cytokine findings supported by end point biopsy immunohistochemistry findings? AAPS J 2010; 12:741-9. [PMID: 20967522 PMCID: PMC2976991 DOI: 10.1208/s12248-010-9235-8] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2010] [Accepted: 09/24/2010] [Indexed: 12/30/2022] Open
Abstract
Insertion of a cutaneous microdialysis catheter into normal dermis has been shown to induce the production of IL1b, IL6 and IL8 in an innate response to minimal trauma. In the present study, skin biopsy for immunohistochemistry has been performed at the site of the microdialysis catheter to compare the findings with that of the microdialysis findings 24 h after insertion. Of the three named cytokines, concordance between the two investigated technologies was highest for IL8 (100%) followed by IL6 (70%) and IL1b (50%). For seven other pro-inflammatory and T cell-relevant cytokines studied, concordance ranged between 50% and 80%. The total number of positive (microdialysis or immunofluorescence) findings was similar between the two methodologies. Technical and biological phenomenon can explain the differences. We conclude that both methodologies illustrate important features of tissue biology and that a combination of the two methods in clinical research can provide the chronology of soluble mediator participation and the more classic, but also more invasive, biopsy-based methodology at a point which constitutes the end of the observation period. We conclude further that at the 24-h time period here studied, microdialysis catheters are still functional and thus capable of producing relevant data which can be corroborated and extended by the "end point biopsy".
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Affiliation(s)
- Florence Sjögren
- />Department of Clinical and Experimental Medicine, Division of Dermatology, Linköping University, Linköping, Sweden
| | - Chris D. Anderson
- />Department of Clinical and Experimental Medicine, Division of Dermatology, Linköping University, Linköping, Sweden
- />Department of Dermatology, University Hospital, 581 85 Linköping, Sweden
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30
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Vereecke L, Sze M, Mc Guire C, Rogiers B, Chu Y, Schmidt-Supprian M, Pasparakis M, Beyaert R, van Loo G, Yang H, Tracey KJ. Enterocyte-specific A20 deficiency sensitizes to tumor necrosis factor-induced toxicity and experimental colitis. ACTA ACUST UNITED AC 2010. [PMID: 15557347 DOI: 10.1084/jem] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
A20 is a nuclear factor kappaB (NF-kappaB) target gene that encodes a ubiquitin-editing enzyme that is essential for the termination of NF-kappaB activation after tumor necrosis factor (TNF) or microbial product stimulation and for the prevention of TNF-induced apoptosis. Mice lacking A20 succumb to inflammation in several organs, including the intestine, and A20 mutations have been associated with Crohn's disease. However, ablation of NF-kappaB activity, specifically in intestinal epithelial cells (IECs), promotes intestinal inflammation. As A20 deficiency sensitizes cells to TNF-induced apoptosis yet also promotes NF-kappaB activity, it is not clear if A20 deficiency in IECs would exacerbate or ameliorate intestinal inflammation. We generated mice lacking A20 specifically in IECs. These mice did not show spontaneous intestinal inflammation but exhibited increased susceptibility to experimental colitis, and their IECs were hypersensitive to TNF-induced apoptosis. The resulting TNF-driven breakdown of the intestinal barrier permitted commensal bacterial infiltration and led to systemic inflammation. These studies define A20 as a major antiapoptotic protein in the intestinal epithelium and further indicate that defects in A20 might contribute to inflammatory bowel disease in humans.
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Affiliation(s)
- Lars Vereecke
- Department for Molecular Biomedical Research, VIB, B-9052 Ghent, Belgium
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31
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Hellqvist E, Kvarnström M, Söderberg A, Vrethem M, Ernerudh J, Rosén A. Myelin protein zero is naturally processed in the B cells of monoclonal gammopathy of undetermined significance of immunoglobulin M isotype: aberrant triggering of a patient's T cells. Haematologica 2009; 95:627-36. [PMID: 20015874 DOI: 10.3324/haematol.2009.015123] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
BACKGROUND Monoclonal gammopathy of undetermined significance of immunoglobulin M isotype is a condition with clonally expanded B cells, recently suggested to have an infectious origin. This monoclonal gammopathy is frequently associated with polyneuropathy and antibodies against myelin protein zero, whereas the role of the T cells remains largely unknown. We analyzed protein zero-specific B cells, as antigen-presenting cells, and their capacity to activate T helper cells. DESIGN AND METHODS We used a well-characterized monoclonal gammopathy of undetermined significance-derived B-cell line, TJ2, expressing anti-protein zero immunoglobulin M. The ability of TJ2 cells to bind, endocytose, process, and present protein zero was investigated by receptor-clustering and immunofluorescence. The activation of protein zero-specific autologous T cells was studied by measuring interleukin-2 and interferon-gamma with flow cytometry, immunobeads, and enzyme-linked immunospot assays. RESULTS Surface-receptor clustering and endocytosis of receptor-ligand (immunoglobulin M/protein zero) complexes were pronounced after exposure to protein zero. Naturally processed or synthetic protein zero peptide (194-208)-pulsed TJ2 cells significantly induced interleukin-2 secretion from autologous T cells compared to control antigen-pulsed cells (P<0.001). The numbers of interferon-gamma-producing T helper cells, including CD4(+)/CD8(+) cells, were also significantly increased (P=0.0152). Affinity-isolated naturally processed myelin peptides were potent interferon-gamma stimulators for autologous peripheral blood mononuclear cells, but not for control peripheral blood mononuclear cells. CONCLUSIONS We show for the first time that myelin protein zero is naturally processed in B cells from monoclonal gammopathy of undetermined significance of immunoglobulin M isotype, acting as aberrant antigen-presenting cells in activation of a patient's T helper cells. Our findings cast new light on the important role of autoreactive protein zero-specific B cells in the induction of the pathogenic T-cell responses found in nerve lesions of patients with monoclonal gammopathy of undetermined significance with peripheral neuropathy.
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Affiliation(s)
- Eva Hellqvist
- Department of Clinical and Experimental Medicine, Division of Cell Biology, Linköping University, SE-581 85 Linköping, Sweden
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Gervais A, Eymard JC, Toulmonde E, Bernard J. Selected allogeneic dendritic cells markedly enhance human tumour antigen-specific T cell response in vitro. Cancer Immunol Immunother 2009; 58:1831-41. [PMID: 19330330 PMCID: PMC11030287 DOI: 10.1007/s00262-009-0694-7] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2008] [Accepted: 03/07/2009] [Indexed: 12/11/2022]
Abstract
BACKGROUND Alloreaction is known to accumulate several theoretical advantages that can improve dendritic cell (DC)-based anti-infective or antitumour strategies. Allogeneic DC have already been tested in experimental and clinical studies, but their efficacy compared with their autologous counterparts was rarely investigated and conclusions diverge. OBJECTIVE This study compared antigen-specific T cell responses following priming with autologous versus allogeneic DC and examined the possibility of screening these responses in order to select allogeneic DC that lead to a great amplification. RESULTS Allogeneic DC obtained from donors matched with the single HLA-A2 allele were efficient in generating in vitro peptide-specific T cell responses. When randomly chosen, allogeneic DC generated a broad range of antigen-specific T cell responses in comparison with autologous DC. When screened and selected, allogeneic DC markedly enhanced peptide-specific T cell priming and allowed a more efficient boosting of resulting T cells. These selected allogeneic DC provided a favourable cytokinic and cellular environment that can help concurrent antigen-specific responses. CONCLUSION Ex vivo selected allogeneic DC provide adjuvant effects that lead to amplification of concomitant antigen-specific T cell responses.
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Affiliation(s)
- Alban Gervais
- Institut Jean Godinot, Unité de Thérapie Cellulaire, Reims, France.
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Bond E, Adams WC, Smed-Sörensen A, Sandgren KJ, Perbeck L, Hofmann A, Andersson J, Loré K. Techniques for time-efficient isolation of human skin dendritic cell subsets and assessment of their antigen uptake capacity. J Immunol Methods 2009; 348:42-56. [PMID: 19576898 DOI: 10.1016/j.jim.2009.06.012] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2008] [Revised: 06/22/2009] [Accepted: 06/24/2009] [Indexed: 12/16/2022]
Abstract
Dendritic cells (DCs) residing in skin are important sentinels for foreign antigens. Methods to facilitate studies of subsets of skin DCs are important to increase the understanding of various pathogens, allergens, topical treatments or vaccine components targeting the skin. In this study, we developed a new DC purification method using a skin graft mesher, clinically used for expansion of skin grafts, to accelerate processing of skin into nets that allowed efficient enzymatic disruption and single cell isolation. The reduction in processing time using the skin graft mesher enabled processing of larger skin samples and also limited the ex vivo handling of the specimens which is associated with maturation of DCs. In addition, a skin explant model to functionally monitor early events of antigen uptake by DC subsets in situ was developed. DCs isolated from epidermis represented a uniform CD1a(+) HLA-DR(+) CD11c(+) Langerin(+) DC-SIGN(-) DC-LAMP(int) DEC-205(int) Langerhans cell (LC) population whereas three subtypes of HLA-DR(+) CD11c(+) DCs were isolated from dermis based on their varying expression of CD1a. Epidermal LCs showed a significantly higher antigen uptake capacity of fluorescently-labelled ovalbumin (OVA) and dextran as compared to any of the dermal DC (dDC) subsets. In contrast, injection of antigen directly into skin explants followed by in situ imaging revealed that the majority of DCs with internalized antigen were localized in the dermis, likely as a consequence of the anatomical site for antigen delivery. These methods offer potency for various applications addressing antigen uptake, microbial DC interactions or other antigenic stimulation targeting the skin and can enhance our knowledge of basic DC biology in human skin.
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Affiliation(s)
- Emily Bond
- Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden
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Matsuura N, Uchio E, Nakazawa M, Yago T, Matsumoto S, Ohno S, Minami M. Predominance of infiltrating IL-4-producing T cells in conjunctiva of patients with allergic conjunctival disease. Curr Eye Res 2009; 29:235-43. [PMID: 15590468 DOI: 10.1080/02713680490516738] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
PURPOSE Previous reports have suggested that type 2 cytokine responses at the site of inflammation are important in the pathogenesis of allergic disease. In this study, we investigated the frequency of IFN-gamma- or IL-4-producing T cells from conjunctiva or peripheral blood mononuclear cells (PBMC) from patients with allergic conjunctival diseases. METHODS We obtained conjunctival samples using Cytobrush and peripheral blood samples from the patients with allergic conjunctival disease. Conjunctival samples and PBMC were stimulated with phorbol myristate acetate and calcium ionophore. Frequencies of cytokine-producing T cells were analyzed by flow cytometry based on the intracellular cytokine staining. RESULTS The frequency of IL-4-producing conjunctival T cells in patients with vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC) were significantly higher than that in patients with allergic conjunctivitis (AC). The frequencies of IL-4-producing conjunctival CD4+ T cells of the patients with AC, AKC, or VKC were significantly higher than those of peripheral blood CD4+ T cells of the same disease. Further, increased frequency of P-selectin ligand expressing IL-4-producing T cells was observed in conjunctiva compared to that in PBMC of the patients with allergic conjunctival diseases. The frequencies of IL-4-producing peripheral blood T cells in the patients with ACD were significantly higher than that in normal controls. CONCLUSION These results suggest that IL-4-producing T cells that have infiltrated into the conjunctiva play an important role in the immunopathogenesis of allergic conjunctival diseases, and P-selectin ligand is involved in the entry of T cells into allergic inflammatory site.
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Affiliation(s)
- Noriko Matsuura
- Department of Ophthalmology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan.
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Fuggetta MP, Lanzilli G, Fioretti D, Rinaldi M. In vitro end points for the assessment of cellular immune response-modulating drugs. Expert Opin Drug Discov 2009; 4:473-93. [PMID: 23485082 DOI: 10.1517/17460440902821632] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
BACKGROUND The concept of immunotoxicology and the development of a battery of immune-function assays to screen potential immunotoxic compounds have been increasingly used in the past. Immunotoxic outcome generally seems appropriate to evaluate the risk in drug development. Improving this approach is possible, by using methods now available, to study the effect of a chemical compound on the immune system. OBJECTIVE The goal of this review is to provide an overview of the current and recent methodologies for testing the immunological effect and immunotoxic risks in drug candidates. METHODS The methodological details here discussed include a synthetic description of the immunocompetent cells in cell-mediated immunity and the choice of the most appropriate assay (bioassays, immunoassays, molecular biology techniques, flow cytometry). CONCLUSION This review offers an assessment of in vitro models to study the toxic impact of (bio)pharmaceuticals on cellular immune system and aid drug scientists in understanding the significance and the methods to approach immunotoxicology.
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Affiliation(s)
- Maria Pia Fuggetta
- Institute of Neurobiology and Molecular Medicine, CNR, Via Fosso del Cavaliere 100, 00133 Rome, Italy +39 06 4993 4610 ; +39 06 4993 4257 ;
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Geiss A, Larsson K, Junevik K, Rydevik B, Olmarker K. Autologous nucleus pulposus primes T cells to develop into interleukin-4-producing effector cells: an experimental study on the autoimmune properties of nucleus pulposus. J Orthop Res 2009; 27:97-103. [PMID: 18634006 DOI: 10.1002/jor.20691] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
An autoimmune response to herniated nucleus pulposus has been proposed to constitute a pathophysiologic mechanism for inducing sciatica based on the fact that nucleus pulposus under normal conditions is excluded from the development of immunological tolerance. The manifestation of an autoimmune response comprises different steps starting with antigen capture, continuing with activation of T helper (T(H)) cells and ending with production of autoantibodies. Activated T(H) cells differentiate into either T(H)1 cells, predominately producing proinflammatory cytokines such as interferon gamma (IFNgamma) or a T(H)2 subset mainly producing anti-inflammatory cytokines such as interleukin-4 (IL-4). The aim of the present study was to examine if exposure of autologous nucleus pulposus (NP) to the immune system for 3 weeks is potent enough to prime T(H) cells to differentiate into T(H)2 cells. The study was performed in a pig model allowing the exposure of NP to the immune system. To assess the polarization of T(H) cells the intracellular production of IFNgamma and IL-4 was measured in T cells by using flow cytometry. The revealed predominant production of IL-4 together with low production of IFNgamma in T cells after NP exposure to the immune system indicates that nucleus pulposus may prime T(H) cells to develop into IL-4-producing T(H)2 cells after being exposed to the immune system, for example, in association with disc herniation.
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Affiliation(s)
- Andrea Geiss
- Department of Orthopaedics, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
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Rothaeusler K, Baumgarth N. Assessment of cell proliferation by 5-bromodeoxyuridine (BrdU) labeling for multicolor flow cytometry. ACTA ACUST UNITED AC 2008; Chapter 7:Unit7.31. [PMID: 18770853 DOI: 10.1002/0471142956.cy0731s40] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Cell proliferation assays are used for a large variety of applications in the life sciences. This unit describes a flow-cytometry-based method that uses BrdU labeling in conjunction with multiple fluorescently labeled cell surface markers, allowing extensive phenotypic characterization of dividing cells in addition to assessment of their frequency. BrdU labeling has the advantage of constituting a nonradioactive technique that, when combined with additional fluorescent-based reagents, avoids time-consuming and often costly cell isolation procedures. Moreover, because BrdU is stably integrated into the DNA, it can be measured in a cell for many months. The method presented in this unit is based on paraformaldehyde fixation and reversible saponin-based cell membrane permeabilization, which maintains cell integrity as well as fluorescent labeling with a large number of different fluorochromes, allowing 10- to 12-color flow cytometric analysis of proliferating cells.
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Hans-Jurgen Haussmann Birgit Gerste. 12-MONTH INHALATION STUDY ON ROOM-AGED CIGARETTE SIDESTREAM SMOKE IN RATS. Inhal Toxicol 2008. [DOI: 10.1080/089583798197501] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2022]
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Norgren M, Eriksson A. Streptococcal Superantigens and Their Role in the Pathogenesis of Severe Infections. ACTA ACUST UNITED AC 2008. [DOI: 10.3109/15569549709064091] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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Babcock GF. Intracellular cytokines. CURRENT PROTOCOLS IN CYTOMETRY 2008; Chapter 9:Unit 9.9. [PMID: 18770810 DOI: 10.1002/0471142956.cy0909s28] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The principal technique described in this unit was developed for detecting cytokines produced by T cells, specifically interferon gamma (IFN-gamma) and interleukin 4 (IL-4). In addition to providing information about which cytokines are being produced, it also helps to phenotypically identify the specific cells producing them. For example, CD4(+) or CD8(+) cells can be subdivided into T(H)1/T(H)2 helper cells or T(C)1/T(C)2 suppressor cells, respectively. This procedure can be useful in examining the response of cells to a variety of agonists, the immune function in various disease states, and the level of lymphocyte activation or suppression. In addition, it can be used to detect a variety of cytokines in many cell types, although the methodology must be carefully evaluated for each cytokine or cell type of interest. Additional methods are included for the stimulation of T cells with allogeneic cells and for the performance of controls for labeling specificity.
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Affiliation(s)
- George F Babcock
- University of Cincinnati College of Medicine and Shriners Burns Institute, Cincinnati, Ohio, USA
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Tripmacher R, Gaber T, Dziurla R, Häupl T, Erekul K, Grützkau A, Tschirschmann M, Scheffold A, Radbruch A, Burmester GR, Buttgereit F. Human CD4(+) T cells maintain specific functions even under conditions of extremely restricted ATP production. Eur J Immunol 2008; 38:1631-42. [PMID: 18493983 DOI: 10.1002/eji.200738047] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
We investigated the energy-adaptive potential of human CD4(+) T cells under conditions of impaired oxidative phosphorylation (OXPHOS) and/or low glucose (inhibiting glycolysis). These cells often encounter these conditions when executing their functions in injured/inflamed tissues, even though T cells themselves require constant and adequate energy supply via ATP. We assessed two specific functions, cytokine synthesis and proliferation, and addressed whether adaptive characteristics also emerged in vivo. In glucose-containing medium, both cytokine production and proliferation were unaffected, even under complete OXPHOS suppression. Only when glucose was also absent were these functions significantly decreased. Partial recovery of OXPHOS and induced glycolysis were crucial for the maintenance of cellular energy supply. Adaptive regulatory mechanisms are clinically relevant because hypoxia up-regulates glycolytic genes but down-regulates OXPHOS genes in vivo. Our data demonstrate an unexpectedly high, clinically relevant adaptive potential of human CD4(+) T cells to maintain specific functions even under severely impaired bioenergetic conditions.
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Affiliation(s)
- Robert Tripmacher
- Department of Rheumatology and Clinical Immunology, Charité University Medicine, Berlin, Germany
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Kleinsasser NH, Harréus UA, Gamarra F, Driemel O, Hagen R, Buehrlen M. Cytochrome P4502A6 stability in a mini organ culture model of human nasal mucosa for genotoxicology studies as detected by flow cytometry. Eur Arch Otorhinolaryngol 2008; 266:385-9. [PMID: 18648831 DOI: 10.1007/s00405-008-0774-x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2007] [Accepted: 07/02/2008] [Indexed: 11/25/2022]
Abstract
Three dimensional mini organ cultures (MOCs) of human nasal turbinate epithelia have been shown to be a relevant tool in genotoxicology studies. MOCs allow repetitive or chronic exposure of cells in an organ specific mucosal architecture for an extended period of time and monitoring of possible adverse effects with, e.g., the comet assay. It is the aim to demonstrate whether the proteins of key enzymes of xenobiotic metabolism, represented by cytochrome P450 2A6 (CYP2A6), remain on a stable level for a culture period that allows repetitive or chronic exposure to xenobiotics. Culture of mini organs was performed by cutting pieces of 1 mm(3) from fresh specimens of human nasal turbinates. MOCs of five tissue donors were incubated on multi-well plates with BEBM, on days 0, 4, 7, 9, and 11 aliquots were transmitted to flow cytometric quantification of the CYP2A6 protein. The CYP2A6 protein could be demonstrated on all days of culture investigated. Interindividual differences were more pronounced on day 0 than at later stages of culture. Although there appeared to be a slight decrease over the culture period, flow cytometric analysis did not reveal a significant loss of the signals up to day 11. The present data could show a pre-requisite of metabolic competence of MOCs that is in contrast to single cell cultures. Thus, this type of organ culture provides an in vitro model suitable for the assessment of genotoxic effects of environmental pollutants mimicking the in vivo situation with target cells of carcinogens in their functional organ specific architecture.
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Byström J, Tenno T, Håkansson L, Amin K, Trulson A, Högbom E, Venge P. Monocytes, but not macrophages, produce the eosinophil cationic protein. APMIS 2008. [DOI: 10.1111/j.1600-0463.2001.907804.x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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Differential Induction of Immunoregulatory Circuits of Phagocytic Cells by Gal/Gal NAc Lectin from Pathogenic and Nonpathogenic Entamoeba. J Clin Immunol 2008; 28:542-57. [DOI: 10.1007/s10875-008-9184-5] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2007] [Accepted: 01/23/2008] [Indexed: 10/22/2022]
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Finkelman F, Morris S, Orekhova T, Sehy D. The in vivo cytokine capture assay for measurement of cytokine production in the mouse. ACTA ACUST UNITED AC 2008; Chapter 6:Unit 6.28. [PMID: 18432911 DOI: 10.1002/0471142735.im0628s54] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
Because most cytokines are utilized, catabolized, or excreted shortly after they are produced, it has been difficult to directly measure in vivo cytokine production. Consequently, it has been necessary to infer in vivo cytokine secretion levels from the results of ex vivo assays of cytokine secretion, assays that measure tissue levels of cytokine mRNA, or assays that stain tissues for cytokine protein levels. Results of these assays provide important and useful information, but do not necessarily reflect in vivo cytokine secretion. To better determine in vivo cytokine production, the in vivo cytokine capture assay (IVCCA) was developed. IVCCA facilitates measurement of cytokines in serum by increasing their in vivo half-lives. This increases the sensitivity of measurement of in vivo cytokine production 30- to 1,000-fold. The first protocol described in this unit is for luminescence-based ELISA, while the second is for an absorbance-based method.
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Affiliation(s)
- Fred Finkelman
- University of Cincinnati College of Medicine and Cincinnati Veterans Administration Medical Center, Cincinnati, Ohio, USA
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Holmes K, Lantz LM, Fowlkes BJ, Schmid I, Giorgi JV. Preparation of cells and reagents for flow cytometry. ACTA ACUST UNITED AC 2008; Chapter 5:Unit 5.3. [PMID: 18432799 DOI: 10.1002/0471142735.im0503s44] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Flow cytometry is widely used for analyzing the expression of cell surface and intracellular molecules (on a per cell basis), characterizing and defining different cell types in heterogeneous populations, assessing the purity of isolated subpopulations, and analyzing cell size and volume. This technique is predominantly used to measure fluorescence intensity produced by fluorescent-labeled antibodies or ligands that bind to specific cell-associated molecules. A procedure for direct and indirect staining of single-cell suspensions of lymphoid tissue or peripheral blood lymphocytes to detect cell surface membrane antigens is presented. In addition, support protocols present methods for fluorescence labeling of purified antibodies. A protocol for flow cytometric analysis of intracellular antigens in single-cell suspensions is also included. Alternate protocols describe intracellular staining of unfixed cells in the presence of a detergent and staining of nonviable cells to facilitate discrimination of dead cells in fixed or permeabilized cell preparations.
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Affiliation(s)
- K Holmes
- National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA
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Baigent SJ, Ross LJN, Davison TF. A flow cytometric method for identifying Marek's disease virus pp38 expression in lymphocyte subpopulations. Avian Pathol 2007; 25:255-67. [DOI: 10.1080/03079459608419140] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
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Sangrouber D, Marcou C, Le Discorde M, Chang CC, Carosella ED, Moreau P. Cellular co-localization of intron-4 containing mRNA and HLA-G soluble protein in melanoma analyzed by fluorescence in situ hybridization. J Immunol Methods 2007; 326:54-62. [PMID: 17689555 DOI: 10.1016/j.jim.2007.07.001] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2006] [Revised: 05/30/2007] [Accepted: 07/02/2007] [Indexed: 11/22/2022]
Abstract
HLA-G5, -G6, and -G7 soluble isoforms of the immunosuppressive HLA-G molecule are produced from the splice variants of the primary HLA-G mRNA transcript containing intron-4 that encodes a specific 21 amino acids tail. In particular, HLA-G5 interacts with the inhibitory ILT2/4 and KIR2DL4 receptors that are expressed on immune cells. Acquisition of soluble HLA-G in the microenvironment may turn a HLA-G non-expressing cell into a HLA-G-bearing one. To address the question of how to distinguish cells that express soluble HLA-G generated by alternative splicing from those that have acquired HLA-G, we have developed a method capable of detecting intron-4 containing mRNA and protein in situ simultaneously. M8 melanoma cell line either transfected or not with HLA-G5 cDNA was analyzed by indirect immunofluorescence confocal microscopy using double staining with a HLA-G intron-4 digoxygenin labeled probe along with a monoclonal antibody directed against the 21 amino acid tail. The combined fluorescence in situ hybridization was also used on the HLA-G-positive choricarcinoma cell line JEG-3. This method would be helpful to follow-up bona fide HLA-G expression in a heterogeneous cell population and to elucidate the mechanisms underlying soluble HLA-G mediated immune modulation in physiological conditions such as pregnancy and pathophysiological situations such as cancer.
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Affiliation(s)
- Déborah Sangrouber
- Service de Recherches en Hémato-Immunologie, Commissariat à L'Energie Atomique- DSV- I(2)BM, Institut Universitaire d'Hématologie, Hôpital Saint-Louis, Paris, France
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Lee JW, Bajwa PJ, Carson MJ, Jeske DR, Cong Y, Elson CO, Lytle C, Straus DS. Fenofibrate represses interleukin-17 and interferon-gamma expression and improves colitis in interleukin-10-deficient mice. Gastroenterology 2007; 133:108-23. [PMID: 17631136 DOI: 10.1053/j.gastro.2007.03.113] [Citation(s) in RCA: 97] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/15/2006] [Accepted: 03/22/2007] [Indexed: 12/02/2022]
Abstract
BACKGROUND & AIMS Interleukin-10 knockout (IL-10(-/-)) mice spontaneously develop colitis characterized by T-helper cell type 1-polarized inflammation. We tested the possible therapeutic activity of the peroxisome proliferator-activated receptor alpha (PPARalpha) ligand fenofibrate, and the PPARdelta ligand GW0742, in IL-10(-/-) mice and investigated the cellular/molecular mechanisms for fenofibrate action. METHODS The effect of fenofibrate or GW0742 on the progression of colitis in C3H.IL-10(-/-) mice was evaluated. Effects of fenofibrate on cytokine and chemokine gene expression were studied in cultured splenocytes, pathogenic T cells isolated from C3H/HeJBir mice, and HT-29 colorectal cancer cells. RESULTS Treatment of C3H.IL-10(-/-) mice with fenofibrate delayed the onset of colitis, decreased the colonic histopathology score, and decreased colonic expression of genes encoding the inflammatory cytokines interferon-gamma and interleukin (IL)-17. The target for fenofibrate, PPARalpha, was expressed in lymphocytes, macrophages, and crypt and surface epithelial cells of the colon. The mean number of lymphocytes was decreased by more than 75% in colonic sections of fenofibrate-treated as compared with control IL-10(-/-) mice, and fenofibrate repressed interferon-gamma and IL-17 expression in isolated T cells. Fenofibrate also repressed the expression of the genes encoding 3 chemokines, CXCL10, CCL2, and CCL20, and repressed CXCL10 gene promoter activity in tumor necrosis factor-alpha-treated HT-29 cells. In contrast to the beneficial effect of fenofibrate, the PPARdelta ligand GW0742 accelerated the onset of colitis in IL-10(-/-) mice. CONCLUSIONS The immunopathology observed in IL-10(-/-) mice resembles that seen in Crohn's disease. The novel therapeutic activity of fenofibrate in this mouse model suggests that it may also have activity in Crohn's disease.
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Affiliation(s)
- Jimmy W Lee
- Biomedical Sciences Division, University of California, Riverside, California 92521-0121, USA
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