Measurement of Interleukin‐21

Rong Zeng1, Rosanne Spolski1, Warren J. Leonard1

1 National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.30
DOI:  10.1002/0471142735.im0630s78
Online Posting Date:  August, 2007
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Abstract

This unit describes three procedures for measurement of interleukin‐21 (IL‐21). The first employs the use of an antibody sandwich ELISA. An alternative procedure measures proliferative responses of T cells to a combination of IL‐21 and IL‐15 using CFSE. Finally, a method to assess IL‐21‐induced tyrosine phosphorylation of Stat3 in splenic CD8+ T cells using a flow cytometry–based analysis is described. Curr. Protoc. Immunol. 78:6.30.1‐6.30.8. © 2007 by John Wiley & Sons, Inc.

Keywords: cytokine; ELISA; CFSE; tyrosine phosphorylation; flow cytometry; IL‐21; IL‐15

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Detection of IL‐21 Using ELISA
  • Alternate Protocol 1: Detection of IL‐21 Activity Using CD8+ T Cells
  • Alternate Protocol 2: Detection of IL‐21 Signaling Using Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Detection of IL‐21 Using ELISA

  Materials
  • 0.5 mg/ml coating (capture) antibody: purified rat anti–mouse IL‐21 MAb (e.g., 149215.111, R&D Systems)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • PBS/Tween: 0.05% (v/v) Tween 20 in PBS
  • Blocking buffer: 10% fetal bovine serum (FBS) in PBS
  • 1 ng/ml recombinant mouse IL‐21 standard (e.g., R&D Systems)
  • Culture supernatant or serum samples to be tested
  • Secondary antibody: biotin‐labeled goat polyclonal anti–mouse IL‐21 (e.g., R&D Systems)
  • Avidin‐horseradish peroxidase conjugate (avidin‐HRP; BD PharMingen, cat. no. 554058)
  • TMB Substrate Reagent Set (e.g., BD PharMingen) containing substrate reagents A and B
  • Stop solution: 8.5% H 3PO 4 (dilute 85% phosphoric acid 1:10)
  • 96‐well flat‐bottom Immulon ELISA microtiter plates (Thermo Lab Systems)
  • Microtiter plate washer (e.g., Beckman Coulter)
  • Microtiter plate reader (e.g., Perkin Elmer/Wallac)

Alternate Protocol 1: Detection of IL‐21 Activity Using CD8+ T Cells

  Materials
  • Mice (C57BL/6 or BALB/c, 6‐ to 12‐weeks old)
  • Complete RPMI medium containing 10% FBS ( appendix 2A)
  • PB buffer: PBS ( appendix 2A) containing 0.5% (w/v) BSA
  • Interleukin 15 (IL‐15; PeproTech)
  • Interleukin 21 (IL‐21; R&D Systems)
  • Test samples
  • 12‐well flat‐bottom tissue culture plates
  • Flow cytometry software (e.g., FlowJo; http://www.flowjo.com)
  • Additional reagents and equipment for removal of mouse lymphoid organs (unit 1.10), preparation of single‐cell suspensions (unit 3.1), counting cells using a hemacytometer ( appendix 3A), isolation of CD8+ T cells by immunomagnetic selection (unit 3.5), CFSE labeling of lymphocytes and flow cytometric analysis of CFSE‐labeled cells (unit 4.9), culturing mouse T lymphocytes (unit 3.12), and flow cytometric analysis (units 5.3& 5.4; also see unit 4.9 for flow cytometry of CFSE‐labeled cells)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All incubations should be carried out in a humidified 37°C, 6% CO 2 incubator.

Alternate Protocol 2: Detection of IL‐21 Signaling Using Flow Cytometry

  Materials
  • 10 ng/µl IL‐21 (R&D Systems)
  • 2% (v/v) formaldehyde (methanol‐free) in PBS (see appendix 2A for PBS)
  • 90% (v/v) methanol
  • FACS buffer (see recipe)
  • Anti‐phospho‐Stat3 (Y705), PE‐conjugated (e.g., BD PharMingen, pretitrated as 10 µl for 5 × 105 cells)
  • Anti‐CD8a (Ly‐2), APC‐conjugated (e.g., BD PharMingen)
  • 24‐well flat‐bottom tissue culture plates
  • Centrifuge with plate carrier
  • 96‐well U‐bottom tissue culture plates
  • 5‐ml flow cytometer tubes
  • Flow cytometry software: e.g., FlowJo (http://www.flowjo.com) or CellQuest (BD Biosciences)
  • Additional reagents and equipment for preparation of splenocytes ( protocol 2, step 1) and flow cytometric analysis (units 5.3& 5.4).
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All incubations should be carried out in a humidified 37°C, 6% CO 2 incubator.
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Figures

Videos

Literature Cited

Literature Cited
   Alves, N.L., Arosa, F.A., and Van Lier, R.A.W. 2005. IL‐21 sustains CD28 expression on IL‐15‐activated human naive CD8+ T cells. J. Immunol. 175:755.
   Asao, H., Okuyama, C., Kumaki, S., Ishii, N., Tsuchiya, S., Foster, D., and Sugamura, K. 2001. Cutting edge: The common gamma‐chain is an indispensable subunit of the IL‐21 receptor complex. J. Immunol. 167:1‐5.
   Gagnon, J., Ramanathan, S., Leblanc, C., and Ilangumaran, S. 2006. Regulation of IL‐21 signaling by suppressor of cytokine signaling‐1 (SOCS1) in CD8(+) T lymphocytes. Cell. Signal. 19:806‐816.
   Habib, T., Senadheera, S., Weinberg, K., and Kaushansky, K. 2002. The common gamma chain (gamma c) is a required signaling component of the IL‐21 receptor and supports IL‐21‐induced cell proliferation via JAK3. Biochemistry 41:8725‐8731.
   Jin, H., Carrio, R., Yu, A., and Malek, T.R. 2004. Distinct activation signals determine whether IL‐21 induces B cell costimulation, growth arrest, or Bim‐dependent apoptosis. J. Immunol. 173:657‐665.
   Leonard, W.J. 2001. Cytokines and immunodeficiency diseases. Nat. Rev. Immunol. 1:200‐208.
   Leonard, W.J. and Spolski, R. 2005. Interleukin‐21: A modulator of lymphoid proliferation, apoptosis and differentiation. Nat. Rev. Immunol. 5:688‐698.
   Mehta, D.S., Wurster, A.L., Whitters, M.J., Young, D.A., Collins, M., and Grusby, M.J. 2003. IL‐21 induces the apoptosis of resting and activated primary B cells. J. Immunol. 170:4111‐4118.
   Ozaki, K., Kikly, K., Michalovich, D., Young, P.R., and Leonard, W.J. 2000. Cloning of a type I cytokine receptor most related to the IL‐2 receptor beta chain. Proc. Natl. Acad. Sci. U.S.A. 97:11439‐11444.
   Ozaki, K., Spolski, R., Feng, C.G., Qi, C.F., Cheng, J., Sher, A., Morse, H.C. 3rd, Liu, C., Schwartzberg, P.L., and Leonard, W.J. 2002. A critical role for IL‐21 in regulating immunoglobulin production. Science 298:1630‐1634.
   Ozaki, K., Spolski, R., Ettinger, R., Kim, H.P., Wang, G., Qi, C.F., Hwu, P., Shaffer, D.J., Akilesh, S., Roopenian, D.C., Morse, H.C. 3rd, Lipsky, P.E., and Leonard, W.J. 2004. Regulation of B cell differentiation and plasma cell generation by IL‐21, a novel inducer of Blimp‐1 and Bcl‐6. J. Immunol. 173:5361‐5371.
   Parrish‐Novak, J., Dillon, S.R., Nelson, A., Hammond, A., Sprecher, C., Gross, J.A., Johnston, J., Madden, K., Xu, W., West, J., Schrader, S., Burkhead, S., Heipel, M., Brandt, C., Kuijper, J.L., Kramer, J., Conklin, D., Presnell, S.R., Berry, J., Shiota, F., Bort, S., Hambly, K., Mudri, S., Clegg, C., Moore, M., Grant, F.J., Lofton‐Day, C., Gilbert, T., Rayond, F., Ching, A., Yao, L., Smith, D., Webster, P., Whitmore, T., Maurer, M., Kaushansky, K., Holly, R.D., and Foster, D. 2000. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function. Nature 408:57‐63.
   Strengell, M., Sareneva, T., Foster, D., Julkunen, I., and Matikainen, S. 2002. IL‐21 up‐regulates the expression of genes associated with innate immunity and Th1 response. J. Immunol. 169:3600‐3605.
   Suto, A., Nakajima, H., Hirose, K., Suzuki, K., Kagami, S., Seto, Y., Hoshimoto, A., Saito, Y., Foster, D.C., and Iwamoto, I. 2002. Interleukin 21 prevents antigen‐induced IgE production by inhibiting germ line C(epsilon) transcription of IL‐4‐stimulated B cells. Blood 100:4565‐4573.
   Wang, G., Tschoi, M., Spolski, R., Lou, Y., Ozaki, K., Feng, C., Kim, G., Leonard, W.J., and Hwu, P. 2003. In vivo antitumor activity of interleukin 21 mediated by natural killer cells. Cancer Res. 63:9016‐9022.
   Zeng, R., Spolski, R., Finkelstein, S.E., Oh, S., Kovanen, P.E., Hinrichs, C.S., Pise‐Masison, C.A., Radonovich, M.F., Brady, J.N., Restifo, N.P., Berzofsky, J.A., and Leonard, W.J. 2005. Synergy of IL‐21 and IL‐15 in regulating CD8+ T cell expansion and function. J. Exp. Med. 201:139‐148.
   Zeng, R., Spolski, R., Casas, E., Zhu, W., Levy, D.E., and Leonard, W.J. 2007. The molecular basis of IL‐21‐mediated proliferation. Blood 109:4135‐4142.
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