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Effector Memory T Helper Cells Secrete IFN-Γ Upon Stimulation with Cytokines

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Effector Memory T Helper Cells Secrete IFN-Γ Upon Stimulation with Cytokines Excerpt from MACS&more Vol 13 – 1/2011 Effector memory T helper cells secrete IFN-γ upon stimulation with cytokines: a role in chronic inflammation Arne Sattler1*, Ulf Wagner2, Manuela Rossol2, Joachim Sieper3, Peihua Wu3, Andreas Krause4, Wolfgang A. Schmidt4, Sebastian Radmer5, Siegfried Kohler6, Chiara Romagnani7, and Andreas Thiel8 1 Berlin-Brandenburg Center for Regenerative Therapies, Immunology Department, Charité, Berlin, 2 Department of Medicine IV, University of Leipzig, Leipzig, 3 Department of Rheumatology, Charité Campus Benjamin Franklin, Berlin; 4 Rheumaklinik Berlin-Buch, Berlin, 5 Immanuel Hospital, Berlin, 6 Klinik und Poliklinik für Neurologie, Charité, Berlin, 7 Deutsches Rheumaforschungszentrum Berlin, 8Berlin-Brandenburg Center for Regenerative Therapies, Regenerative Immunology and Aging, Charité, Berlin; *to whom correspondence should be addressed, e-mail: [email protected] Hier ist ein Überhang Miltenyi Biotec provides products and services worldwide. Visit www.miltenyibiotec.com/local to find your nearest Miltenyi Biotec contact. Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. autoMACS, gentleMACS, and MACS are registered trademarks or trademarks of Miltenyi Biotec GmbH. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Copyright © 2012 Miltenyi Biotec GmbH. All rights reserved. REPORT autOIMMUNITY Effector memory T helper cells secrete IFN-g upon stimulation with cytokines: a role in chronic inflammation Arne Sattler1*, Ulf Wagner2, Manuela Rossol2, Joachim Sieper3, Peihua Wu3, Andreas Krause4, Wolfgang A. Schmidt4, Sebastian Radmer5, Siegfried Kohler6, Chiara Romagnani7, and Andreas Thiel8 1Berlin-Brandenburg Center for Regenerative Therapies, Immunology Department, Charité, Berlin, 2Department of Medicine IV, University of Leipzig, Leipzig, 3Department of Rheumatology, Charité Campus Benjamin Franklin, Berlin; 4Rheumaklinik Berlin-Buch, Berlin, 5Immanuel Hospital, Berlin, 6 Klinik und Poliklinik für Neurologie, Charité, Berlin, 7 Deutsches Rheumaforschungszentrum Berlin, 8Berlin-Brandenburg Center for Regenerative Therapies, Regenerative Immunology and Aging, Charité, Berlin; *to whom correspondence should be addressed, e-mail: [email protected] Introduction Interferon g (IFN-g) produced by T helper We here show that a subset of human resting (Miltenyi Biotec). Memory T cells were cells plays a prominent role in both host- effector memory Th cells, expressing IL-12R, subsequently isolated by depletion of naive protective and pathologic immune responses. IL-18Ra, and CCR5 ex vivo, secrete IFN-g Th cells (CD45RA MicroBeads, human; Antigen-specific IFN-g–producing T cells upon stimulation via the IL-2R common Miltenyi Biotec) and monocytes (CD14 (Th1 cells) are prerequisite for the control and g chain in combination with IL-12 and MicroBeads, human; Miltenyi Biotec). Cell clearance of infections.¹,² Effector memory IL-18. CD137 (4-1BB) was identified as a separation was performed using LS Columns Th1 cells are also abundant in chronically discrimination marker that was only detectable or the autoMACS® Separator (Miltenyi Biotec). inflamed tissues, for example, in rheumatoid on TCR- but not on cytokine-induced IFN-g+ Purities were higher than 98%. arthritis (RA)³–⁵; it is, however, not clear how Th cells. We were able to detect a significant Viable IFN-g–secreting cells were detected these cells get activated at the site of chronic fraction of Th cells in RA patients’ synovial and isolated using the IFN-g Secretion Assay inflammation. Recently, it was shown that fluid and membrane that spontaneously – Cell Enrichment and Detection Kit, human the proinflammatory cytokines interleukin secreted IFN-g directly ex vivo but lacked (Miltenyi Biotec). (IL-)12 and IL-18 synergistically induce IFN-g CD137 expression, indicating that cytokine- production in in vitro generated murine Th1 induced IFN-g+ Th cells operate in chronic Preparation of single-cell suspensions from cells in a TCR-independent manner.⁶ As the autoimmune inflammation. synovial fluid and synovial membrane severity of autoimmune diseases such as RA Mononuclear cells from synovial fluid (SF- correlates with elevated levels of IL-12 and MNCs) were washed twice with PBS/BSA IL-18⁷,⁸, we hypothesized that the cytokine Material and methods containing 2 mM EDTA. Cell debris was milieu might be sufficient to induce IFN-g Cell isolation removed by using Pre-Separation Filters secretion in Th cells, potentially contributing CD4+ Th cells were separated from PBMCs (Miltenyi Biotec). For the preparation to inflammation. using the CD4 MultiSort Kit, human of single-cell suspensions from synovial 18 MACS & more Vol 13 • 1/2011 www.miltenyibiotec.com REPORT autOIMMUNITY membrane (SM) the tissue was minced into Cell culture and stimulation stimulation was performed by incubation of pieces of 1 to 5 mm3, dissociated using the Cells were cultured in RPMI 1640 with cells in polysterene tubes coated with anti-CD3 gentleMACS™ Dissociator (Miltenyi Biotec, glutamine (Invitrogen) and 10% human AB and anti-CD28 antibodies (BD Biosciences) at program spleen _04, followed by brain_03) serum (PAA Laboratories). Recombinant 0.5 µg/mL and 2.5 µg/mL, respectively. and digested for 1 h with collagenase IA, cytokines (R&D Systems) were used at 25 ng/ CMV pp65–specific Th1 cells were generated hyaluronidase, and DNAse I (Sigma-Aldrich). mL unless otherwise indicated. rIL-2 (Roche by culturing PBMCs with 5 µg/mL CMV Diagnostics) was used at 20 U/mL. TCR pp65 Recombinant Protein (Miltenyi Biotec) A B 5000 20 IL-12 + IL-18 IL-2 cytokine cocktail 4000 IL-4 15 αCD3 + αCD28 IL-5 cells 3000 IL-10 H T + 10 IFN-γ (pg/mL) 2000 % IFN-γ 5 1000 0 0 no stimulation cytokine cocktail 0 12 24 36 48 60 72 84 96 C Time (h) αCD3 + cytokine αCD28 w/o IL-12 + IL-18 cocktail TNF-α 0.09 0.13 4.0 IL-4 IL-2 IL-7 IL-15 0.08 0.10 0.11 0.09 IL-4 + IL-2 + IL-7 + IL-15 + IL-12 + IL18 IL-12 + IL-18 IL12 + IL-18 IL-12 + IL-18 0.08 0.77 0.67 1.46 TNF-α + IL-2 + TNF-α + IL-7 + TNF-α + IL-15 + TNF-α + IL-12 + IL-18 IL-12 + IL-18 IL-12 + IL-18 IL-12 + IL-18 0.12 1.23 0.88 1.99 IFN-γ Figure 1 Induction of IFN-γ secretion in resting human Th cells by inflammatory cytokines. (A) Th cells were stimulated with the cytokine cocktail containing IL-1β, IL-6, IL-7, IL-8, IL-12, IL-15, IL-17, IL-18, TNF-a, and MIP-1a. After 72 h, supernatants were analyzed for secreted IL-2, IL-4, IL-5, IL-10, and IFN-γ by cytometric bead array (CBA, BD Biosciences). (B) Th cells were stimulated as indicated. Frequencies of IFN-γ–expressing cells were analyzed at different time points intracellularly by flow cytometry. (C) Th cells were stimulated for 36 h with different cytokines or for 12 h with aCD3 + aCD28 and assessed for intracellular IFN-g and TNF-a production. 20 MACS & more Vol 13 • 1/2011 www.miltenyibiotec.com REPORT autOIMMUNITY and 1 µg/mL anti-CD28 (BD Biosciences) for Results induced by the g-chain cytokines IL-2, IL-7, 6 h. Viable antigen-specific IFN-g+ cells were Resting human Th cells secrete IFN-g upon IL-15, but not IL-4, synergistically with IL-12 stained by using the IFN-g Secretion Assay stimulation with inflammatory cytokines and IL-18. IL-15 together with IL-12 and IL-18 (Miltenyi Biotec) and isolated by flow sorting. To determine whether resting human Th proved to be the most effective combination, Th1 cells were expanded for 10 to 14 days in cells are able to secrete IFN-g in response to resulting in frequencies of IFNg–producing the presence of IL-7 and IL-15 (10 ng/mL each). cytokines that are present at sites of chronic cells of 1.68%+/–0.40% (mean+/–SEM) in Th Antigen-specific restimulation was achieved inflammation, cells were stimulated with a cells from healthy donors (fig. 1C). by culturing 105 CMV p65–specific Th1 cells cytokine cocktail comprising IL-1β, IL-6, in the presence of 5×105 irradiated autologous IL-7, IL-8, IL-12, IL-15, IL-17, IL-18, TNF-a, Cytokine induced IFN-g–secreting cells PBMCs, 5 µg/mL CMV pp65 protein and 1 µg/ and MIP-1a for 72 h. Besides IFN-g, we exhibit a differentiated effector memory mL anti-CD28. analyzed secretion of IL-2, IL-4, IL-5, and phenotype IL-10. The cells secreted large amounts of We isolated human CD45RA– memory Th cell Analysis of intracellular cytokines IFN-g; other cytokines were not detectable subsets according to CCR7 and found that the Stimulated cells were cultured in the presence (fig. 1A). Cytokine-induced IFN-g production CCR7– effector memory fraction was highly of brefeldin A for the last 4 to 12 h. Cells were peaked after 36 h of stimulation, whereas responsive to cytokine stimulation unlike fixed in 2% formalin, permeabilized, stained TCR-mediated production of IFN-g showed a the CCR7+ central memory subset (data not for 30 min with fluorochrome-conjugated maximum between 6 h and 12 h (fig. 1B). After shown). We then tested whether resting anti-IFN-g and/or anti-TNF-a and analyzed testing all cytokines alone and in combinations effector memory Th cells express receptors by flow cytometry. to identify the essential components of the for the cytokines being essential for the IFN-g cocktail, we found that IFN-g secretion was response. The IL-18 receptor alpha chain A CCR7 CCR7 CD45RA CD45RA 3.5 3.5 84 84 IL-18Rα CCR5 IL-18Rα CCR5 B αCD3 + cytokine re-analysis w/o αCD3αCD28 + IL-12 + IL-18 cytokinecocktail re-analysis w/o αCD28 IL-12 + IL-18 cocktail CD45RA no stain CD45RA no stain 0.01 15 0.01 0.9 99 0.01 15 0.01 0.9 99 0.01 36 0.88 39 99 0.01 36 0.88 39 99 IL-18Rα IFN-γ IL-18Rα IFN-γ Figure 2 Cytokine-induced IFN-γ secretion is restricted to CCR7–CCR5+IL-18Rα+ effector memory Th cells.
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