Evaluation of Human Natural Killer Cell Activities in Whole Blood

Maren Claus1, Carsten Watzl1

1 Heidelberg University, Heidelberg, Germany
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.39
DOI:  10.1002/0471142735.im0739s91
Online Posting Date:  November, 2010
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Abstract

Natural killer (NK) cells are important effector cells of the innate immune system. Activation of NK cells results in their cytotoxic activity against locally attached target cells and leads to the secretion of cytokines. These activities are usually measured in purified NK cells or isolated peripheral blood mononuclear cells. In this unit, we describe a protocol to measure NK cell cytotoxicity (lysis of 51Cr labeled target cells), degranulation (externalization of CD107a), and cytokine production (intracellular FACS analysis of IFN‐γ) in whole‐blood samples. Using these protocols, it is possible to perform a comprehensive analysis of NK cell function with as little as 3.5 ml of heparinized whole blood. Curr. Protoc. Immunol. 91:7.39.1‐7.39.17. © 2010 by John Wiley & Sons, Inc.

Keywords: natural killer cells; cytotoxicity; cytokine production; degranulation

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Determination of NK Cell Numbers in Whole‐Blood Samples
  • Basic Protocol 2: 51Cr Release Assay Using Whole‐Blood Samples
  • Basic Protocol 3: Whole‐Blood Degranulation (CD107a Externalization) Assay
  • Basic Protocol 4: Whole‐Blood IFN‐γ Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Determination of NK Cell Numbers in Whole‐Blood Samples

  Materials
  • Peripheral venous blood, collected in heparinized tubes, 3 ml
  • NK cell culture medium (see recipe)
  • Recombinant human IL‐2 (NIH cytokine repository; stock concentration, 100,000 U/ml)
  • Recombinant human IL‐12 (Pan‐Biotech, http://www.pan‐biotech.com/; stock concentration, 10 µg/ml)
  • Recombinant human IL‐18 (MBL International, http://www.mblintl.com; stock concentration, 40 µg/ml)
  • PE‐conjugated anti‐CD56 and PE/Cy5‐conjugated anti‐CD3 mAbs (BD Biosciences) and respective isotype controls
  • FITC‐conjugated beads (2000 beads/µl; Right Reference Standard, Bangs Laboratories)
  • FACS Lysing Solution (BD Biosciences)
  • FACS buffer (see recipe)
  • 25‐cm2 cell culture flasks (Nunc)
  • 37°C, 5% CO 2 humidified incubator
  • FACS tubes (BD Falcon, 35 2054)
  • Refrigerated centrifuge
  • Flow cytometer (also see Chapter 5)

Basic Protocol 2: 51Cr Release Assay Using Whole‐Blood Samples

  Materials
  • Peripheral venous blood, collected in heparinized tubes, 3 ml
  • NK cell culture medium (see recipe)
  • Recombinant human IL‐2 (NIH cytokine repository; stock concentration, 100,000 U/ml)
  • Recombinant human IL‐12 (Pan‐Biotech, http://www.pan‐biotech.com/; stock concentration, 10 µg/ml)
  • Recombinant human IL‐18 (MBL International, http://www.mblintl.com; stock concentration, 40 µg/ml)
  • K562 target cells (ATCC no. CCL‐243™)
  • Assay medium (see recipe)
  • [51Cr] sodium chromate; DuPont NEN or Amersham)
  • 2% (v/v) Triton X‐100
  • 25‐cm2 cell culture flasks (Nunc)
  • 15‐ml conical tubes (BD Falcon)
  • End‐over‐end rotator
  • 37°C, 5% CO 2 incubator
  • Centrifuge
  • 96 well plates, V‐bottom (Nunc)
  • Harvesting frames: Supernatant Collection System (Molecular Devices), including harvesting frames (Cat. No. 9000‐0308), harvesting press (Cat. No. 9000‐0305), and transfer fork (Cat. No. 9000‐0307)
  • γ‐counter

Basic Protocol 3: Whole‐Blood Degranulation (CD107a Externalization) Assay

  Materials
  • Peripheral venous blood, collected in heparinized tubes, 3 ml
  • NK cell culture medium (see recipe)
  • Recombinant human IL‐2 (NIH cytokine repository; stock concentration, 100,000 U/ml)
  • Recombinant human IL‐12 (Pan‐Biotech, http://www.pan‐biotech.com/; stock concentration, 10 µg/ml)
  • Recombinant human IL‐18 (MBL International, http://www.mblintl.com; stock concentration, 40 µg/ml)
  • K562 target cells (ATCC no. CCL‐243™)
  • FITC‐conjugated anti‐CD56, PE‐conjugated anti‐CD3 and PE/Cy5‐conjugated anti‐CD107a mAbs (BD Biosciences) and respective isotype controls
  • FACS Lysing Solution (BD Biosciences)
  • FACS buffer (see recipe)
  • 25‐cm2 cell culture flasks (Nunc)
  • 37°C, 5% CO 2 incubator
  • FACS tubes (BD Falcon, 35 2054)
  • Centrifuge
  • Flow cytometer (also see Chapter 5)
  • Additional reagents and equipment for counting cells ( appendix 3A)

Basic Protocol 4: Whole‐Blood IFN‐γ Assay

  Materials
  • Peripheral venous blood, collected in heparinized tubes, 3 ml
  • NK cell culture medium (see recipe)
  • Recombinant human IL‐2 (NIH cytokine repository; stock concentration, 100,000 U/ml)
  • Recombinant human IL‐12 (Pan‐Biotech, http://www.pan‐biotech.com/; stock concentration, 10 µg/ml)
  • Recombinant human IL‐18 (MBL International, http://www.mblintl.com; stock concentration, 40 µg/ml)
  • K562 target cells (ATCC no. CCL‐243™)
  • 10 mg/ml Brefeldin A stock solution (Sigma‐Aldrich)
  • FITC‐conjugated anti‐IFN‐γ, PE‐conjugated anti‐CD56 and PE/Cy5‐conjugated anti‐CD3 mAbs (BD Biosciences) and respective isotype controls
  • FACS Lysing Solution (BD Biosciences)
  • FACS buffer (see recipe)
  • Perm2 (BD Biosciences)
  • 25‐cm2 cell culture flasks (Nunc)
  • 37°C, 5% CO 2 incubator
  • FACS tubes (BD Falcon, 35 2054)
  • Centrifuge
  • Flow cytometer
  • Additional reagents and equipment for counting cells ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
   Alter, G., Malenfant, J.M., and Altfeld, M. 2004. CD107a as a functional marker for the identification of natural killer cell activity. J. Immunol. Methods 294:15‐22.
   Aste‐Amezaga, M., D'Andrea, A., Kubin, M., and Trinchieri, G. 1994. Cooperation of natural killer cell stimulatory factor/interleukin‐12 with other stimuli in the induction of cytokines and cytotoxic cell‐associated molecules in human T and NK cells. Cell Immunol. 156:480‐492.
   Biron, C.A. and Brossay, L. 2001. NK cells and NKT cells in innate defense against viral infections. Curr. Opin. Immunol. 13:458‐464.
   Claus, M., Greil, J., and Watzl, C. 2009. Comprehensive analysis of NK cell function in whole blood samples. J. Immunol. Methods 341:154‐164.
   Hefeneider, S.H., Conlon, P.J., Henney, C.S., and Gillis, S. 1983. In vivo interleukin 2 administration augments the generation of alloreactive cytolytic T lymphocytes and resident natural killer cells. J. Immunol. 130:222‐227.
   Imai, K., Matsuyama, S., Miyake, S., Suga, K., and Nakachi, K. 2000. Natural cytotoxic activity of peripheral‐blood lymphocytes and cancer incidence: An 11‐year follow‐up study of a general population. Lancet 356:1795‐1799.
   Kiessling, R., Klein, E., and Wigzell, H. 1975. “Natural” killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype. Eur. J. Immunol. 5:112‐117.
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   Lauwerys, B.R., Garot, N., Renauld, J.C., and Houssiau, F.A. 2000. Cytokine production and killer activity of NK/T‐NK cells derived with IL‐2, IL‐15, or the combination of IL‐12 and IL‐18. J. Immunol. 165:1847‐1853.
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   Long, E.O., Barber, D.F., Burshtyn, D.N., Faure, M., Peterson, M., Rajagopalan, S., Renard, V., Sandusky, M., Stebbins, C.C., Wagtmann, N., and Watzl, C. 2001. Inhibition of natural killer cell activation signals by killer cell immunoglobulin‐like receptors (CD158). Immunol. Rev. 181:223‐233.
   Moretta, L. and Moretta, A. 2004. Unravelling natural killer cell function: Triggering and inhibitory human NK receptors. EMBO J. 23:255‐259.
   Moretta, L., Ferlazzo, G., Bottino, C., Vitale, M., Pende, D., Mingari, M.C., and Moretta, A. 2006. Effector and regulatory events during natural killer‐dendritic cell interactions. Immunol. Rev. 214:219‐228.
   Ruggeri, L., Mancusi, A., Burchielli, E., Aversa, F., Martelli, M.F., and Velardi, A. 2007. Natural killer cell alloreactivity in allogeneic hematopoietic transplantation. Curr. Opin. Oncol. 19:142‐147.
   Siegel, J.P., Sharon, M., Smith, P.L., and Leonard, W.J. 1987. The IL‐2 receptor beta chain (p70): Role in mediating signals for LAK, NK, and proliferative activities. Science 238:75‐78.
   Wu, J. and Lanier, L.L. 2003. Natural killer cells and cancer. Adv. Cancer Res. 90:127‐156.
   Zamai, L., Ponti, C., Mirandola, P., Gobbi, G., Papa, S., Galeotti, L., Cocco, L., and Vitale, M. 2007. NK cells and cancer. J. Immunol. 178:4011‐4016.
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