Comparing Individual NK Cell Activity In Vitro

Heidi Sierich1, Thomas Eiermann1

1 University Medical Center Hamburg‐Eppendorf (UKE), Hamburg, Germany
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.32
DOI:  10.1002/0471142735.im1432s100
Online Posting Date:  February, 2013
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Abstract

Natural killer (NK) cells play an important role in the innate immune system by eliminating infected and mutated cells. Their cytotoxic capacities vary markedly among individuals. The cytotoxic activity can be measured in peripheral blood mononuclear cells (PBMCs) using the NK cell–specific target cell line K562. In this chapter, we present a protocol for the standardization and normalization of cell preparation and NK cell cytotoxicity measurement in a 51Cr‐release assay. By following these protocols, it is possible to compare the NK cell activity of numerous—if necessary selected—individuals in vitro. Curr. Protoc. Immunol. 100:14.32.1‐14.32.11. © 2013 by John Wiley & Sons, Inc.

Keywords: 51Cr release assay; K562; cytotoxicity; responsiveness; licensing

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Preparation of Effector PBMCs
  • Basic Protocol 2: 51Cr‐Release Assay for the Measurement of NK Cell Cytotoxicity Against K562 Cells
  • Basic Protocol 3: Determination of Percentage of NK Cells by Facs Analysis
  • Support Protocol 1: Culture of K562 Target Cell Line
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Effector PBMCs

  Materials
  • Heparinized blood ( appendix 3F)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Ficoll‐Paque PLUS (GE Healthcare) or comparable reagent for density centrifugation (unit 7.1)
  • Freezing solution 1: RPMI 1640 medium with 50% fetal bovine serum (FBS, heat inactivated); always prepare fresh, keep at 4°C
  • Freezing solution 2: RPMI 1640 medium with 20% DMSO (> 99.5%); always prepare fresh, keep at 4°C
  • 100% isopropanol
  • Centrifuge
  • 50‐ml conical polypropylene centrifuge tubes (e.g., BD Falcon)
  • 2‐ml cryotubes
  • Cryo 1°C freezing container (“Mr. Frosty”; Nalgene)
  • Additional reagents and equipment for Ficoll‐Hypaque gradient centrifugation (optional, unit 7.1) and determining cell number using trypan blue dye exclusion ( appendix 3B)

Basic Protocol 2: 51Cr‐Release Assay for the Measurement of NK Cell Cytotoxicity Against K562 Cells

  Materials
  • K562 cells, maintained in culture for maximum 2 month (see protocol 4)
  • Cell culture medium (CM): RPMI 1640 medium with 10% FBS (store up to 4 weeks at 4°C)
  • 5 µCi/µl 51Cr stock solution
  • PBMCs, frozen ( protocol 1)
  • 2% (v/v) Triton X‐100
  • 25‐cm2 tissue culture dish
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Centrifuge
  • 96‐well round or V‐bottom culture plates
  • Multichannel (12‐channel) pipettor with 100‐µl disposable tips
  • Gamma counter: “Wallac 1470 Wizard” or equivalent, with racks and tubes
  • Additional reagents and equipment for thawing cryopreserved cells ( appendix 3G) and determining viable cell number using trypan blue exclusion ( appendix 3B)

Basic Protocol 3: Determination of Percentage of NK Cells by Facs Analysis

  Materials
  • PBMC suspension ( protocol 1) used in protocol 2
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • FACS buffer (see recipe)
  • Human anti‐CD3/FITC mAb, clone OKT3 (eBioscience, cat. no. 11‐0037‐41)
  • Human antiCD56/PE mAb, clone B159 (eBioscience, cat. no. 56 1903)
  • Isotype control mAb(s)
  • Additional reagents and equipment for determining cell number using trypan blue dye exclusion ( appendix 3B) and flow cytometric analysis of cells (unit 5.1)

Support Protocol 1: Culture of K562 Target Cell Line

  Materials
  • K562 cells (ATCC no. CCL‐243)
  • Cell culture medium (CM): RPMI 1640 medium with 10% FBS (store up to 4 weeks at 4°C)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 25‐cm2 tissue culture flasks
  • 50‐ml conical polypropylene centrifuge tubes (e.g., BD Falcon)
  • Centrifuge
  • Additional reagents and equipment for thawing cryopreserved cells ( appendix 3G) and determining cell number using trypan blue dye exclusion ( appendix 3A)
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Figures

Videos

Literature Cited

   Biassoni, R., Falco, M., Cambiaggi, A., Costa, P., Verdiani, S., Pende, D., Conte, R., Di Donato, C., Parham, P., and Moretta, L. 1995. Amino acid substitutions can influence the natural killer (NK)‐mediated recognition of HLA‐C molecules: Role of serine‐77 and lysine‐80 in the target cell protection from lysis mediated by “group 2” or “group 1” NK clones. J. Exp. Med. 182:605‐609.
   Brodin, P., Karre, K., and Hoglund, P. 2009. NK cell education: Not an on‐off switch but a tunable rheostat. Trends Immunol. 30:143‐149.
   Cooley, S., Trachtenberg, E., Bergemann, T.L., Saeteurn, K., Klein, J., Le, C.T., Marsh, S.G., Guethlein, L.A., Parham, P., Miller, J.S., and Weisdorf, D.J. 2009. Donors with group B KIR haplotypes improve relapse‐free survival after unrelated hematopoietic cell transplantation for acute myelogenous leukemia. Blood 113:726‐732.
   Duske, H., Sputtek, A., Binder, T., Kröger, N., Schrepfer, S., and Eiermann, T. 2011. Assessment of physiologic natural killer cell cytotoxicity in vitro. Hum. Immunol. 72:1007‐1012.
   Fauriat, C., Ivarsson, M.A., Ljunggren, H.G., Malmberg, K.J., and Michaëlsson, J. 2010. Education of human natural killer cells by activating killer cell immunoglobulin‐like receptors. Blood 115:1166‐1174.
   Joncker, N.T., Shifrin, N., Delebecque, F., and Raulet, D.H. 2010. Mature natural killer cells reset their responsiveness when exposed to an altered MHC environment. J. Exp. Med. 207:2065‐2072.
   Kroger, N., Binder, T., Zabelina, T., Wolschke, C., Schieder, H., Renges, H., Ayuk, F., Dahlke, J., Eiermann, T., and Zander, A. 2006. Low number of donor activating killer immunoglobulin‐like receptors (KIR) genes but not KIR‐ligand mismatch prevents relapse and improves disease‐free survival in leukemia patients after in vivo T‐cell depleted unrelated stem cell transplantation. Transplantation 82:1024‐1030.
   Long, E.O. 1999. Regulation of immune responses through inhibitory receptors. Annu. Rev. Immunol. 17:875‐904.
   McQueen, K.L., Dorighi, K.M., Guethlein, L.A., Wong, R., Sanjanwala, B., and Parham, P. 2007. Donor‐recipient combinations of group A and B KIR haplotypes and HLA class I ligand affect the outcome of HLA‐matched, sibling donor hematopoietic cell transplantation. Hum. Immunol. 68:309‐323.
   Moretta, L., Bottino, C., Pende, D., Castriconi, R., Mingari, M.C., and Moretta, A. 2006. Surface NK receptors and their ligands on tumor cells. Semin. Immunol. 18:151‐158.
   Uhrberg, M., Parham, P., and Wernet, P. 2002. Definition of gene content for nine common group B haplotypes of the Caucasoid population: KIR haplotypes contain between seven and eleven KIR genes. Immunogenetics 54:221‐229.
   Vivier, E., Tomasello, E., Baratin, M., Walzer, T., and Ugolini, S. 2008. Functions of natural killer cells. Nat. Immunol. 9:503‐510.
   Wagtmann, N., Rajagopalan, S., Winter, C.C., Peruzzi, M., and Long, E.O. 1995. Killer cell inhibitory receptors specific for HLA‐C and HLA‐B identified by direct binding and by functional transfer. Immunity 3:801‐809.
Key Reference
   Duske et al. 2011. See above.
  Contains a comprehensive discussion of optimal conditions and quality control for the sample preparation and standardization/normalization of the CRA.
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