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Surface IL-17A Expression Th17 Cells Unambiguously Identified by Phenotypical Characterization of Human

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Surface IL-17A Expression Th17 Cells Unambiguously Identified by Phenotypical Characterization of Human Phenotypical Characterization of Human Th17 Cells Unambiguously Identified by Surface IL-17A Expression This information is current as Verena Brucklacher-Waldert, Karin Steinbach, Michael of September 24, 2021. Lioznov, Manuela Kolster, Christoph Hölscher and Eva Tolosa J Immunol 2009; 183:5494-5501; ; doi: 10.4049/jimmunol.0901000 http://www.jimmunol.org/content/183/9/5494 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2009/10/19/183.9.5494.DC1 Material http://www.jimmunol.org/ References This article cites 44 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/183/9/5494.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 24, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Phenotypical Characterization of Human Th17 Cells Unambiguously Identified by Surface IL-17A Expression1 Verena Brucklacher-Waldert,*†‡ Karin Steinbach,* Michael Lioznov,§ Manuela Kolster,* Christoph Ho¨lscher,¶ʈ and Eva Tolosa2*† Th17 cells are involved in the defense against bacteria and fungi and play a prominent role in the pathogenesis of autoimmune diseases, but research on human Th17 cells is hindered due to the lack of a surface marker. In this study, we report that a subset .of human and mouse CD4؉ T cells as well as human Th17 T cell clones express IL-17A on their surface upon stimulation Correlation of surface IL-17A expression with intracellular IL-17A production and with ROR␥t mRNA expression identified surface IL-17A as a specific marker for human and mouse Th17 cells. Phenotype characterization of ex vivo CD4؉ IL-17A؉ cells showed that the chemokines CCR6 and CCR4, costimulatory molecules, as well as CD2 and CD49d were more prominently ؊ expressed on these cells than in surface IL-17A cells, supporting the concept of Th17 cells as a potent inflammatory effector Downloaded from subtype. In addition, we generated human Th1, Th1/17 (producing both IFN-␥ and IL-17A), and Th17 T cell clones based on single cell sorting of surface IL-17A؊, IL-17Aint, and IL-17Ahigh CD4؉ T cells, respectively, and showed the plasticity of the double producing clones to the cytokine milieu. The identification of surface IL-17A as a marker for Th17 cells should facilitate research on this subset. The Journal of Immunology, 2009, 183: 5494–5501. L-17 is a potent inducer of inflammation by triggering the 17A and IFN-␥, suggests that commitment to a specific effector http://www.jimmunol.org/ release of cytokines and chemokines from a broad range of type might be more flexible than previously assumed. I stromal cells (1). Through its regulation of proinflammatory Th17 cells have been linked to the pathogenesis of several inflam- gene expression, IL-17A has the capability to recruit neutrophils matory (22, 23) and autoimmune diseases (24–26) and therefore they (2) and to promote antimicrobial protein synthesis (3). Until now, are under intense investigation. Although reporter lines allow tracking six structurally related isoforms for IL-17 (IL-17A, B, C, D, E, and of Th17 cells in mice, human Th17 cells are currently identified ex F) have been identified and grouped into the IL-17 cytokine family vivo by intracellular IL-17 expression, which does not allow further (4–6). Among them, IL-17F has the highest homology with IL- functional analysis. Alternatively, Th17 cells can be differentiated in 17A. Both IL-17A and IL-17F are active as homodimers and can vitro from naive CD4ϩ T cells using combinations of cytokines, but form biologically functional IL-17A/IL-17F heterodimers (7). IL- culture conditions may change their properties. by guest on September 24, 2021 17A and IL-17F are produced by CD4ϩ T cells, ␥␦ T cells, NKT We report in this study the unambiguous identification of Th17 cells, NK cells, CD8 cells, neutrophils, and eosinophils (1, 8–13). cells by the expression of surface IL-17A. Using surface IL-17A as IL-17A-producing CD4ϩ T cells, defined as Th17 cells, have been a marker, we could phenotypically characterize ex vivo human recently identified as a distinct Th cell lineage (14), which differ- Th17 cells and generate Th17 and Th1/17 T cell clones (TCC).3 entiates upon stimulation from naive CD4ϩ T cells in presence of Furthermore, we show for the first time in human cells the plas- TGF-␤ and IL-6 (15–17). Maintenance of Th17 cells requires in ticity of the Th1/17 cells in response to the cytokine milieu. addition IL-23 (18), and they express the lineage-specific tran- scription factor ROR␥t (19). In contrast, Th1 cell differentiation Materials and Methods requires IL-12 and the expression of the transcription factor T-bet Cells (20), while Th2 cells are dependent on IL-4 and the expression of Peripheral blood was taken from healthy donors after informed consent GATA-3 (21). The existence of Th1/17 cells, producing both IL- under a protocol approved by the local ethics committee. CD4ϩ cells were isolated (Ͼ95% purity) from PBMC by negative selection using the CD4ϩ T Cell Isolation Kit II (Miltenyi Biotec) according to manufacturer’s in- *Institute for Neuroimmunology and Clinical Multiple Sclerosis Research, Center for structions. Th1 and Th17 TCC were generated from PBMC by limiting Molecular Neurobiology Hamburg and †Department of Immunology, University ‡ dilution (27). Clonality was assessed by TCR V␤-chain staining (28). For Medical Center Hamburg-Eppendorf, Hamburg, Germany; Graduate School of Cel- ϩ ϩ lular and Molecular Neuroscience, University of Tu¨bingen, Germany; §Interdiscipli- the isolation of surface IL-17A cells, CD4 T cells were stimulated with nary Clinic for Stem Cell Transplantation, University Medical Center Hamburg-Ep- 50 ng/ml PMA and 1 ␮g/ml ionomycin for the indicated time period, pendorf, Hamburg, Germany; ¶Division of Infection Immunology, Research Center stained with anti-IL-17A Alexa647 (eBio64DEC17, eBioscience), and ʈ Borstel, Borstel, Germany; and Cluster of Excellence Inflammation at Interfaces, FACS sorted or isolated using anti-Cy5/Alexa Fluor 647 Microbeads Borstel-Kiel-Lu¨beck-Plo¨n, Germany (Miltenyi Biotec). To induce a Th17 response, C57BL/6 wild-type or IL- Received for publication March 27, 2009. Accepted for publication August 21, 2009. 17R-deficient mice (29) (provided by Amgen) were immunized with 200 ␮g MOG (NeoMPS) in CFA supplemented with 4 mg/ml Mycobac- The costs of publication of this article were defrayed in part by the payment of page 35–55 charges. This article must therefore be hereby marked advertisement in accordance terium tuberculosis H37RA (Difco Laboratories), followed by injection of with 18 U.S.C. Section 1734 solely to indicate this fact. 300 ng pertussis toxin (Sigma-Aldrich) at the time point of immunization 1 The Institute for Neuroimmunology and Clinical Multiple Sclerosis Research and this work are supported by the Hertie Foundation. V.B.-W. was partly supported by 3 Abbreviations used in this paper: TCC, T cell clone; MFI, mean fluorescence intensity; the German Research Council (SFB 685) and C.H. by the Research Focus Autoim- ICS, intracellular cytokine staining; PD-1, programmed cell death 1; TMHMM, trans- munity at the University of Lu¨beck. membrane prediction using hidden Markov models; MINNOU, membrane protein iden- 2 Address correspondence and reprint requests to Eva Tolosa, Department of Immu- tification without explicit use of hydropathy profiles and alignments. nology, University Medical Center Hamburg-Eppendorf (UKE), Martinistr. 52, 20251 Hamburg, Germany. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901000 The Journal of Immunology 5495 and repeated 2 days later. After 12 or 25 days, CD4ϩ T cells were Surface biotinylation and Western blot isolated from spleen and from the spinal cord and brain of the immu- ϩ nized mice as described before (30) by magnetic isolation (Miltenyi Purified CD4 T cells were stimulated overnight with PMA/ionomycin. Biotec) and used for staining. All experiments involving animals were Cells were then washed with PBS and surface molecules were first biotin- performed according to the German animal protection act and have been ylated and subsequently isolated using the cell surface protein isolation kit approved by local authorities. (Pierce) according to the manufacturer’s protocol. Collected fractions, el- uate (biotinylated proteins), and flow-through (intracellular proteins) were Flow cytometry separated by SDS gel electrophoresis under reducing conditions and blot- ted onto polyvinylidine difuloride membrane. Membranes were incubated The following anti-human Abs were used: anti-CD4 Pacific Blue (clone with polyclonal rabbit anti-IL-17A and anti-IL-17F (both PeproTech), MT310, DakoCytomation), anti-IFN-␥ PE, (clone 4S.B3), anti-IL-17A mouse anti-GAPDH (6C5, Abcam) overnight at 4°C, followed by anti- Alexa647 (clone eBio64DEC17), anti-IL-17F PE (clone SHLR17), anti- rabbit or anti-mouse HRP-coupled secondary Ab (Sigma-Aldrich), respec- ICOS FITC (clone ISA-3), anti-CD25 PE (clone 12–1278-73), anti- tively. Nonbiotinylated cell lysates and human rIL-17A and rIL-17F (both CD28 PE-Cy7 (clone CD28.2), anti-CD39 PE-Cy7 (clone 25–0399), PeproTech) were used as controls.
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