Isolation and Characterization of Human Natural Killer Cell Subsets

Megan A. Cooper1, Michael A. Caligiuri1

1 The Ohio State University Comprehensive Cancer Center, Columbus, Ohio
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.34
DOI:  10.1002/0471142735.im0734s60
Online Posting Date:  May, 2004
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Natural killer (NK) cells are innate immune lymphocytes that play a critical role in the host defense against pathogens through their production of cytokines and cytotoxic activity. Human NK cells can be divided into two subsets, each with distinct phenotypic and functional properties, based on cell‐surface density expression of CD56 (CD56bright and CD56dim). This unit describes the purification of human NK cell subsets from blood, protocols for the further characterization of NK cell function, and further background information on these cell types.

Keywords: lymphocyte; human natural killer (NK) cell; CD56; innate immunity

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Isolation of Human NK Cells from PBMC
  • Alternate Protocol 1: Enrichment of NK Cells
  • Support Protocol 1: Titration of Fluorescently Conjugated Monoclonal Antibodies
  • Support Protocol 2: Phenotypic and Functional Analysis of Human NK Cell Subsets
  • Support Protocol 3: Labeling P815 Cells for NK Cell ADCC Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Isolation of Human NK Cells from PBMC

  Materials
  • Sodium heparin
  • Human peripheral blood obtained as a leukopac (available from the American Red Cross or local blood banks) or obtained via venipuncture or leukapheresis ( appendix 3F)
  • RosetteSep antibody (human NK cell enrichment) cocktail (StemCell Technologies)
  • PBS ( appendix 2A)
  • RPMI‐1640 medium, 4°C
  • RPMI‐10HAB (see recipe), freshly filtered with 0.2‐µm culture filter (4°C/room temperature)
  • Red blood cell (RBC) lysis buffer (see recipe), room temperature
  • Mouse IgG fluorescently‐conjugated isotype control antibody (Coulter)
  • Anti‐human CD56 fluorescently‐conjugated antibody (Ab) (Coulter, NKH‐1 clone, preferably PE‐ or PC5‐conjugated)
  • RPMI‐30HAB (see recipe), freshly filtered with 0.2‐µm culture filter (4°C)
  • 50‐ml sterile, conical screw‐top polypropylene centrifuge tubes
  • Tabletop rotator (Adams Nutator, Fisher Scientific)
  • 6‐well cell culture dish, optional
  • 37°C, 5% CO 2 humidified incubator
  • Benchtop swinging bucket refrigerated centrifuge (Beckman Allegra 6 with a GH 3.8 rotor or similar)
  • 5‐ml sterile polypropylene or polystyrene flow cytometry tubes with caps (Fisher Scientific)
  • Sterile cell strainer (30‐ to 70‐µm, Fisher Scientific)
  • Flow cytometer capable of cell sorting (Coulter EPICS series, Becton‐Dickinson FACS series, DakoCytomation Moflo, or similar instrument)
  • Additional reagents and equipment for isolation of PBMC by Ficoll‐Hypaque gradient centrifugation (unit 7.1) and trypan blue exclusion test of cell viability ( appendix 3B)

Alternate Protocol 1: Enrichment of NK Cells

  Materials
  • Enriched NK cells (not previously stained with an antibody)
  • Staining buffer (see recipe)
  • Anti‐CD56 fluorescently conjugated Ab (NKH1 clone, Coulter, preferably PE‐ or PC5‐conjugated)
  • Fluorescently conjugated isotype control–Ab (mouse IgG, Coulter)
  • 5‐ml flow cytometry tubes
  • Flow cytometer (Coulter, Becton‐Dickinson, DakoCytomation)

Support Protocol 1: Titration of Fluorescently Conjugated Monoclonal Antibodies

  Materials
  • P815 cell line (ATCC# TIB‐64)
  • Rabbit anti‐mouse lymphocyte Ab (Accurate Chemical)
  • RPMI‐10 ( appendix 2A)
  • 51Chromium51 (Cr)
  • Additional reagents and equipment for analysis of ADCC activity (unit 7.27) and counting cells by trypan blue exclusion ( appendix 3B)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Andre, P., Spertini, O., Guia, S., Rihet, P., Dignat‐George, F., Brailly, H., Sampol, J., Anderson, P.J., and Vivier, E. 2000. Modification of P‐selectin glycoprotein ligand‐1 with a natural killer cell‐restricted sulfated lactosamine creates an alternate ligand for L‐selectin. Proc. Natl. Acad. Sci. U.S.A. 97:3400‐3405.
   Baume, D.M., Robertson, M.J., Levine, H., Manley, T.J., Schow, P.W., and Ritz, J. 1992. Differential responses to interleukin‐2 define functionally distinct subsets of human natural killer cells. Eur. J. Immunol. 22:1‐6.
   Caligiuri, M.A., Murray, C., Robertson, M.J., Wang, E., Cochran, K., Cameron, C., Schow, P., Ross, M.E., Klumpp, T.R., Soiffer, R.J., Smith, K.A., and Ritz, J. 1993. Selective modulation of human natural killer cells in vivo after prolonged infusion of low dose recombinant interleukin‐2. J. Clin. Invest. 91:123‐132.
   Caligiuri, M.A., Zmuidzinas, A., Manley, T.J., Levine, H., Smith, K.A., and Ritz, J. 1990. Functional consequences of interleukin‐2 receptor expression on resting human lymphocytes. Identification of a novel natural killer cell subset with high affinity receptors. J. Exp. Med. 171:1509‐1526.
   Carson, W.E., Fehniger, T.A., Haldar, S., Eckhert, K., Lindemann, M.J., Lai, C.F., Croce, C.M., Baumann, H., and Caligiuri, M.A. 1997. A potential role for interleukin‐15 in the regulation of human natural killer cell survival. J. Clin. Invest. 99:937‐943.
   Cooper, M.A., Fehniger, T.A., Ponnappan, A., Mehta, V., Wewers, M.D., and Caligiuri, M.A. 2001a. Interleukin‐1β costimulates interferon‐γ production by human natural killer cells. Eur. J. Immunol. 31:792‐801.
   Cooper, M.A., Fehniger, T.A., Turner, S.C., Chen, K.S., Ghaheri, B.A., Ghayur, T., Carson, W.E., and Caligiuri, M.A. 2001b. Human natural killer cells: A unique innate immunoregulatory role for the CD56bright subset. Blood 97:3146‐3151.
   Cooper, M.A., Fehniger, T.A., and Caligiuri, M.A. 2001c. The biology of human natural killer‐cell subsets. Trends Immunol. 22:633‐640.
   Fehniger, T.A., Herbein, G., Yu, H., Para, M.I., Bernstein, Z.P., O'Brien, W.A., and Caligiuri, M.A. 1998. Natural killer cells from HIV‐1+ patients produce C‐C chemokines and inhibit HIV‐1 infection. J. Immunol. 161:6433‐6438.
   Fehniger, T.A., Shah, M.H., Turner, M.J., VanDeusen, J.B., Whitman, S.P., Cooper, M.A., Suzuki, K., Wechser, M., Goodsaid, F., and Caligiuri, M.A. 1999. Differential cytokine and chemokine gene expression by human NK cells following activation with IL‐18 or IL‐15 in combination with IL‐12: Implications for the innate immune response. J. Immunol. 162:4511‐4520.
   Fehniger, T.A., Cooper, M.A., Nuovo, G.J., Cella, M., Facchetti, F., Colonna, M., and Caligiuri, M.A. 2003. CD56bright natural killer cells are present in human lymph nodes and are activated by T cell‐derived IL‐2: A potential new link between adaptive and innate immunity. Blood 101:3052‐3057.
   Heid, C.A., Stevens, J., Livak, K.J., and Williams, P.M. 1996. Real time quantitative PCR. Genome Res. 6:986‐994.
   Lanier, L.L., Le, A.M., Civin, C.I., Loken, M.R., and Phillips, J.H. 1986. The relationship of CD16 (Leu‐11) and Leu‐19 (NKH‐1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes. J. Immunol. 136:4480‐4486.
   Leibson, P.J. 1997. Signal transduction during natural killer cell activation: Inside the mind of a killer. Immunity 6:655‐661.
   Long, E.O., Burshtyn, D.N., Clark, W.P., Peruzzi, M., Rajagopalan, S., Rojo, S., Wagtmann, N., and Winter, C.C. 1997. Killer cell inhibitory receptors: Diversity, specificity, and function. Immunol. Rev. 155:135‐144.
   Moretta, A., Bottino, C., Vitale, M., Pende, D., Cantoni, C., Mingari, M.C., Biassoni, R., and Moretta, L. 2001. Activating receptors and coreceptors involved in human natural killer cell‐mediated cytolysis. Annu. Rev. Immunol. 19:197‐223.
   Nagler, A., Lanier, L.L., Cwirla, S., and Phillips, J.H. 1989. Comparative studies of human FcRIII‐positive and negative natural killer cells. J. Immunol. 143:3183‐3191.
   Nagler, A., Lanier, L.L., and Phillips, J.H. 1990. Constitutive expression of high affinity interleukin 2 receptors on human CD16‐natural killer cells in vivo. J. Exp. Med. 171:1527‐1533.
   Nishikawa, K., Saito, S., Morii, T., Hamada, K., Ako, H., Narita, N., Ichijo, M., Kurahayashi, M., and Sugamura, K. 1991. Accumulation of CD16‐CD56+ natural killer cells with high affinity interleukin 2 receptors in human early pregnancy decidua. Int. Immunol. 3:743‐750.
   Robertson, M.J., Soiffer, R.J., Wolf, S.F., Manley, T.J., Donahue, C., Young, D., Herrmann, S.H., and Ritz, J. 1992. Response of human natural killer (NK) cells to NK cell stimulatory factor (NKSF): Cytolytic activity and proliferation of NK cells are differentially regulated by NKSF. J. Exp. Med. 175:779‐788.
   Voss, S.D., Daley, J., Ritz, J., and Robertson, M.J. 1998. Participation of the CD94 receptor complex in costimulation of human natural killer cells. J. Immunol. 160:1618‐1626.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library