Measurement of Interleukin 15

Raymond J. Paxton1

1 Immunex Corporation, Seattle, Washington
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.22
DOI:  10.1002/0471142735.im0622s18
Online Posting Date:  May, 2001
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Abstract

Interleukin 15 (IL‐15) is a small helical cytokine that was first characterized by its ability to stimulate proliferation of the murine T cell line CTLL‐2, but is now known to have activity on a variety of cell types. This unit describes two protocols that can be used to measure IL‐15 in culture supernatants and serum samples. The first method uses an enzyme‐linked immunosorbent assay (ELISA) that is specific for human IL‐15, although it can also detect simian IL‐15. The second method relies on the ability of IL‐15 to support proliferation of CTLL‐2 cells. The CTLL‐2 assay is slightly more sensitive than the ELISA, but it is not specific for IL‐15, because CTLL‐2 cells also respond to IL‐2 and IL‐4. The CTLL‐2 assay can be used to measure human, simian, or murine IL‐15.

     
 
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Table of Contents

  • Basic Protocol 1: Detection of IL‐15 Using ELISA
  • Alternate Protocol 1: Detection of IL‐15 Activity Using CTLL‐2 Cells
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Detection of IL‐15 Using ELISA

  Materials
  • Coating antibody: purified anti–human IL‐15 MAb M111 (Genzyme)
  • PBS ( appendix 2A)
  • PBS/Tween: 0.05% (v/v) Tween 20 in PBS
  • ≥20 µg/ml recombinant human IL‐15 standard (Genzyme)
  • Culture supernatant or serum samples to be tested (store at −70°C to maintain stability)
  • Normal mouse serum (Life Technologies)
  • Sample diluent: PBS/Tween containing 0.5 M NaCl and (if testing serum samples) 5% (v/v) normal mouse serum
  • Second antibody: rabbit polyclonal anti–human IL‐15 (e.g., Immunex)
  • Second antibody diluent: PBS/Tween containing (if testing serum samples) 5% (v/v) normal mouse serum
  • Horseradish peroxidase (HRPO)–conjugated donkey anti–rabbit IgG (Jackson Immunoresearch)
  • 1 M H 3PO 4 (store ≤1 year at room temperature)
  • TMB peroxidase substrate solution (Kirkegaard & Perry)
  • 96‐well flat‐bottom microtiter plates (e.g., Nunc MaxiSorp)
  • Microtiter plate washer (e.g., Nunc; optional)
  • Microtiter plate reader (e.g., Molecular Devices)
  • ELISA analysis software (e.g., Delta Soft from BioMetallics)

Alternate Protocol 1: Detection of IL‐15 Activity Using CTLL‐2 Cells

  • recipeCTLL‐2 assay medium (see recipe)
  • Log‐phase culture of CTLL‐2 cells (ATCC #TIB 214; see unit 6.3 for cell maintenance, but use CTLL‐2 assay medium supplemented with 10 ng/ml recombinant human IL‐2, from Chiron or Genzyme)
  • 1 mCi/ml [3H]thymidine, sterile (25 Ci/mmol; e.g., Amersham)
  • Cell harvester (e.g., Inotech Biosystems or Packard)
  • 96‐well flat‐bottom tissue culture plates (e.g., Costar)
  • Sorvall RT6000B centrifuge and H1000B rotor
  • β counter: Packard Matrix 96 direct counter or liquid scintillation counter
  • Additional reagents and equipment for determining cell viability by trypan blue exclusion ( appendix 3B)
NOTE: All incubations should be carried out in a humidified 37°C, 10% CO 2 incubator unless otherwise specified.
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Figures

Videos

Literature Cited

Literature Cited
   Anderson, D.M., Johnson, L., Glaccum, M.B., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Valentine, V., Kirstein, M.N., Shapiro, D.N., Morris, S.W., Grabstein, K., and Cosman, D. 1995a. Chromosomal assignment and genomic structure of IL15. Genomics 25:701‐706.
   Anderson, D.M., Kumaki, S., Ahdieh, M., Bertles, J., Tometsko, M., Loomis, A., Giri, J., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Valentine, V., Shapiro, D.N., Morris, S.W., Park, L.S., and Cosman, D. 1995b. Functional characterization of the human interleukin‐15 receptor α chain and close linkage of IL15RA and IL2RA genes. J. Biol. Chem. 270:29862‐29869.
   Armitage, R.J., Macduff, B.M., Eisenman, J., Paxton, R., and Grabstein, K.H. 1995. IL‐15 has stimulatory activity for the induction of B cell proliferation and differentiation. J. Immunol. 154:483‐490.
   Bamford, R.N., Grant, A.J., Burton, J.D., Peters, C., Kurys, G., Goldman, C.K., Brennan, J., Roessler, E., and Waldmann, T.A. 1994. The interleukin (IL) 2 receptor β chain is shared by IL‐2 and a cytokine, provisionally designated IL‐T, that stimulates T‐cell proliferation and the induction of lymphokine‐activated killer cells. Proc. Natl. Acad. Sci. U.S.A. 91:4940‐4944.
   Bamford, R.N., Battiata, A.P., Burton, J.D., Sharma, H., and Waldmann, T.A. 1996. Interleukin (IL) 15/IL‐T production by the adult T‐cell leukemia cell line HuT‐102 is associated with a human T‐cell lymphotrophic virus type I R region/IL‐15 fusion message that lacks many upstream AUGs that normally attenuate IL‐15 mRNA translation. Proc. Natl. Acad. Sci. U.S.A. 93:2897‐2902.
   Burton, J.D., Bamford, R.N., Peters, C., Grant, A.J., Kurys, G., Goldman, C.K., Brennan, J., Roessler, E., and Waldmann, T.A. 1994. A lymphokine, provisionally designated interleukin T and produced by a human adult T‐cell leukemia line, stimulates T‐cell proliferation and the induction of lymphokine‐activated killer cells. Proc. Natl. Acad. Sci. U.S.A. 91:4935‐4939.
   Carson, W.E., Giri, J.G., Lindemann, M.J., Linett, M.L., Ahdieh, M., Paxton, R., Anderson, D., Eisenman, J., Grabstein, K., and Caligiuri, M.A. 1994. Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL‐2 receptor. J. Exp. Med. 180:1395‐1403.
   Carson, W.E., Ross, M.E., Baiocchi, R.A., Marien, M.J., Boiani, N., Grabstein, K., and Caligiuri, M.A. 1995. Endogenous production of interleukin 15 by activated human monocytes is critical for optimal production of interferon‐γ by natural killer cells in vitro . J. Clin. Invest. 96:2578‐2582.
   Doherty, T.M., Seder, R.A., and Sher, A. 1996. Induction and regulation of IL‐15 expression in murine macrophages. J. Immunol. 156:735‐741.
   Gillis, S., Ferm, M.M., Ward, O.U., and Smith, K.A. 1978. T‐cell growth factor: Parameters of production and a quantitative microassay for activity. J. Immunol. 120:2027‐2031.
   Giri, J.G., Ahdieh, M., Eisenman, J., Shanebeck, K., Grabstein, K., Kumaki, S., Namen, A., Park, L.S., Cosman, D., and Anderson, D. 1994. Utilization of the β and γ chains of the IL‐2 receptor by the novel cytokine IL‐15. EMBO J. 13:2822‐2830.
   Giri, J.G., Kumaki, S., Ahdieh, M., Friend, D.J., Loomis, A., Shanebeck, K., DuBose, R., Cosman, D., Park, L.S., and Anderson, D.M. 1995. Identification and cloning of a novel IL‐15 binding protein that is structurally related to the α chain of the IL‐2 receptor. EMBO J. 14:3654‐3663.
   Grabstein, K.H., Eisenman, J., Shanebeck, K., Rauch, C., Srinivasan, S., Fung, V., Beers, C., Richardson, J., Schoenborn, M.A., Ahdieh, M., Johnson, L., Alderson, M.R., Watson, J.D., Anderson, D.M., and Giri, J.G. 1994. Cloning of a T cell growth factor that interacts with the β chain of the interleukin‐2 receptor. Science 264:965‐968.
   McInnes, I.B., Mughales, J.A., Field, M., Leung, B.P., Huang, F., Dixon, R., Sturrock, R.D., Wilkinson, P.C., and Liew, F.Y. 1996. The role of interleukin‐15 in T cell migration and activation in rheumatoid arthritis. Nature Med. 2:175‐182.
   Munger, W., Quinn DeJoy, S., Jeyaseelan, R. Sr., Torley, L.W., Grabstein, K.H., Eisenman, J., Paxton, R., Cox, T., Wick, M.M., and Kerwar, S.S. 1995. Studies evaluating the antitumor activity and toxicity of interleukin‐15, a new T cell growth factor: Comparison with interleukin‐2. Cell. Immunol. 165:289‐293.
   Quinn, L.S., Haugk, K.L., and Grabstein, K.H. 1995. Interleukin‐15: A novel anabolic cytokine for skeletal muscle. Endocrinology 136:3669‐3672.
   Seder, R.A., Grabstein, K.H., Berzofsky, J.A., and McDyer, J.F. 1995. Cytokine interactions in human immunodeficiency virus‐infected individuals: Roles of interleukin (IL)‐2, IL‐12, and IL‐15. J. Exp. Med. 182:1067‐1078.
   Wilkinson, P.C. and Liew, F.Y., 1995. Chemoattraction of human blood T lymphocytes by interleukin‐15. J. Exp. Med. 181:1255‐1259.
Key Reference
   Grabstein et al. 1994. See above.
  Describes cloning and initial characterization of IL‐15 and shows that IL‐15 interacts with the IL‐2R β chain.
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