Measurement of Interleukin‐17

Bhanu P. Pappu1, Chen Dong1

1 M.D. Anderson Cancer Center, Houston, Texas
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.25
DOI:  10.1002/0471142735.im0625s79
Online Posting Date:  November, 2007
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Abstract

Upon antigenic stimulation, naive CD4+ T cells undergo proliferation and differentiate into cytokine‐producing T helper (TH) effector cells. TH1 cells secrete effector cytokine IFN‐γ and regulate cell‐mediated immunity, whereas TH2 cells produce IL‐4, IL‐5, and IL‐13 cytokines, and mediate immunity against extracellular pathogens and allergic reactions. Recent studies have identified a novel TH subset, called TH17, THIL‐17, or inflammatory TH (THi) cells, characterized by the production of a proinflammatory cytokine, IL‐17, and regulating inflammatory responses. In this unit, we describe the protocols for the differentiation of mouse IL‐17‐expressing T cells in vitro, detection of IL‐17‐expressing T cells by intracellular cytokine staining, and measurement of IL‐17 secretion in culture supernatants by ELISA. Generation of IL‐17‐expressing T cells in vitro under defined culture conditions allows investigation of their differentiation regulation. Detection of IL‐17 in cell culture and tissue samples helps in monitoring inflammatory diseases and determining efficacy of therapeutic interventions. Curr. Protoc. Immunol. 79:6.25.1‐6.25.8. © 2007 by John Wiley & Sons, Inc.

Keywords: IL‐17; helper T cells; autoimmunity; T cell differentiation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: In Vitro Differentiation of Murine IL‐17‐Producing Cells
  • Basic Protocol 2: Quantitation of Human and Murine IL‐17 Using ELISA
  • Basic Protocol 3: Intracellular Cytokine Staining Analysis of Mouse IL‐17‐Producing Cells
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: In Vitro Differentiation of Murine IL‐17‐Producing Cells

  Materials
  • Mice on desirable genetic background as a source of naive CD4+ T cells (C57BL/6 or OT‐II TCR transgenic mice on C57BL/6 genetic background are routinely used)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 0.1 mM sodium carbonate buffer, pH 9.5
  • Anti‐CD3ɛ (2C11, BD Pharmingen) and anti‐CD28 (37.51, BD Pharmingen)
  • Complete RPMI‐1640 medium containing 10% FBS ( appendix 2A)
  • Anti‐IFN‐γ antibody, clone XMG1.1 (BD Pharmingen)
  • Anti‐IL‐4 antibody, clone 11B11 (BD Pharmingen)
  • Murine TGF‐β and IL‐6 (PeproTech)
  • Murine recombinant IL‐23 (R&D systems)
  • 6‐ or 24‐well tissue culture plates
  • Additional reagents and equipment for harvesting lymphoid organs of mice (unit 1.9) preparing single‐cell suspensions from mouse lymphoid organs (unit 3.1), depletion of red blood cells from cell suspensions (unit 7.2), purification of T cells (unit 3.5), counting of viable cells by trypan blue exclusion ( appendix 3B), preparation of APC by pulsing BMDC with cognate peptide (Lanzavecchia and Sallusto, ) or irradiation of T cell–depleted splenocytes (unit 3.12), and stimulation of naive CD4+ T cells using APC or antibody‐coated plates (unit 3.13)

Basic Protocol 2: Quantitation of Human and Murine IL‐17 Using ELISA

  Materials
  • Capture antibody: anti–mouse IL‐17, clone TC11‐18H10 (BD Pharmingen) or anti–human IL‐17, clone eBio64CAP17 (eBioscience)
  • 0.1 mM sodium carbonate buffer, pH 9.5
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • PBS ( appendix 2A) containing 5% FBS or BSA
  • Washing solution: PBS ( appendix 2A) containing 0.05% (v/v) Tween 20
  • Human or murine recombinant IL‐17 (Peprotech)
  • Assay diluent: 1% (v/v) FBS in PBS
  • Samples to be assayed for IL‐17
  • Detection antibody: biotinylated anti–mouse IL‐17, clone TC11‐8H4.1 (BD Pharmingen) or biotinylated anti–human IL‐17, clone eBio64CAP17 (eBioscience)
  • Avidin‐conjugated horseradish peroxidase (avidin‐HRP; Vector Laboratories)
  • ELISA substrate: SigmaFAST OPD (o‐phenylenediamine dihydrochloride; Sigma)
  • ELISA stop solution: 2 M sulfuric acid
  • 96‐well flat‐bottom MaxiSorp plates (Nunc)
  • Microtiter plate sealers (R&D systems)
  • ELISA plate reader with 450 and 570 nm filters
NOTE: It is recommended that the samples be run in triplicate and standards in duplicate.

Basic Protocol 3: Intracellular Cytokine Staining Analysis of Mouse IL‐17‐Producing Cells

  Materials
  • In vitro–differentiated mouse IL‐17‐producing cells ( protocol 1)
  • Complete RPMI‐1640 medium containing 10% FBS ( appendix 2A)
  • Phorbol myristate acetate (PMA; Sigma)
  • Ionomycin (Sigma)
  • Staining buffer: PBS ( appendix 2A) containing 1% (v/v) FBS and 0.05% (w/v) sodium azide
  • Fixation/Permeabilization kit containing:
    • GolgiPlug (BD Biosciences)
    • Cytofix/Cytoperm solution
    • 10× Perm/Wash solution
  • PerCP/Cy5.5‐conjugated anti‐CD4 antibody, clone GK1.5 (eBioscience)
  • Anti–mouse CD16/CD32 antibody, clone 2.4G2 (also referred to as Fc‐block; BD Pharmingen)
  • APC‐conjugated anti–mouse IFN‐γ antibody, clone XMG1.2 (BD Pharmingen)
  • PE‐conjugated anti–mouse IL‐17 antibody, clone TC11‐18H10 (BD Pharmingen)
  • Additional reagents and equipment for flow cytometry (Chapter 5)
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Literature Cited

Literature Cited
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