Measurement of Cytotoxic Activity of NK/LAK Cells

Theresa L. Whiteside1

1 University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.18
DOI:  10.1002/0471142735.im0718s35
Online Posting Date:  May, 2001
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Abstract

In this unit, methodology is presented for measuring the capacity of human natural killer (NK) or lymphokine‐activated killer (LAK) cells to lyse tumor cell targets. Cytotoxicity of these effector cells is evaluated in a short‐term 51Cr‐release assay using NK‐sensitive tumor cells as the targets for NK cells or NK‐resistant tumor cells as the targets for LAK cells. In the basic protocol, a generic 51Cr‐release assay is described in which PBMC, purified NK cells or interleukin 2 (IL‐2)‐activated lymphocyte populations (LAK cells) are utilized as effector cells. Support protocols describe preparation of nonadherent tumor cells, cells obtained from malignant effusions, trypsinized tumor cells from adherent monolayer culture, or freshly isolated tumor cells from surgical specimens. All of these cell types can serve as the 51Cr‐labeled target cells in the basic protocol. The procedure for labeling target cells from any of these sources with 51Cr is also provided.

     
 
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Table of Contents

  • Basic Protocol 1: 51Cr‐Release Assay for Cytolysis by NK/LAK Cells
  • Support Protocol 1: Preparation of Nonadherent Tumor Target Cells
  • Support Protocol 2: Preparation of Tumor Target Cells from Malignant Effusions
  • Support Protocol 3: Preparation of Tumor Target Cells Starting with Monolayers of Adherent Tumor Cells Maintained in Culture
  • Support Protocol 4: Preparation of Tumor Target Cells Starting with Solid Tumor Surgical Specimens
  • Support Protocol 5: Labeling of Target Cells for Cytotoxicity Assay
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: 51Cr‐Release Assay for Cytolysis by NK/LAK Cells

  Materials
  • Effector cells: NK or LAK cells (unit 7.7) or PBMC separated from heparinized blood by Ficoll‐Hypaque gradient (unit 7.1)
  • RPMI‐10 medium (unit 7.7)
  • 5 × 104 cell/ml suspension of 51Cr‐labeled target cells in RPMI‐10 (see protocol 6)
  • 5% (v/v) Triton X‐100 in PBS
  • 10‐ml round‐bottom polypropylene snap‐cap tubes
  • 96‐well round‐bottom sterile microtiter plates (e.g.,Costar)
  • 8‐channel micropipettor capable of delivering 100 µl (e.g., Titertek, ICN Biomedicals) with disposable tips
  • Sorvall RT‐6000 centrifuge with H‐1000B rotor (or equivalent) and microplate carriers
  • γ scintillation counter and counting tubes
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 3A) and determining number of viable cells by trypan blue exclusion ( appendix 3B)
CAUTION: This procedure should be performed in an area designated for radioisotope use; safety regulations must be followed for use and disposal of 51Cr.

Support Protocol 1: Preparation of Nonadherent Tumor Target Cells

  Materials
  • Malignant effusion fluid
  • recipeRPMI‐5AB medium (see recipe), room temperature
  • recipeDiscontinuous Ficoll‐Hypaque gradient (see recipe)
  • RPMI‐2 medium (unit 7.7)
  • Sorvall RT‐6000 centrifuge with H‐1000B rotor (or equivalent)
  • Programmable rate‐controlled cell freezer (Cryomed from Forma Scientific; optional)
  • Liquid nitrogen storage unit (optional)
  • Additional reagents and equipment for determining number of viable cells by trypan blue exclusion ( appendix 3B) and cryopreserving cells ( appendix 3G)

Support Protocol 2: Preparation of Tumor Target Cells from Malignant Effusions

  Materials
  • 0.25% trypsin‐EDTA (Life Technologies)
  • Monolayer of tumor cells growing in culture flask (e.g., FEMX human melanoma cells)
  • recipeRPMI‐5AB medium (see recipe)
  • Inverted microscope
  • Sorvall RT‐6000 centrifuge with H‐1000B rotor (or equivalent)

Support Protocol 3: Preparation of Tumor Target Cells Starting with Monolayers of Adherent Tumor Cells Maintained in Culture

  Materials
  • Freshly resected tumor from surgery
  • HBSS ( appendix 2A) containing 50 µg/ml gentamycin, 4°C
  • recipeDigestion medium (see recipe)
  • RPMI‐2 medium (unit 7.7)
  • recipeRPMI‐5AB medium (see recipe)
  • Trypsinizing flask (Bellco)
  • Submersible magnetic stirrer (e.g., VWR Scientific, Thomas Scientific) and sterile magnetic stir‐bar
  • 50‐ml conical centrifuge tubes (sterile)
  • Nylon mesh, 50‐ to 90‐µm pore size (Martin)
  • Sorvall RT‐6000 centrifuge with H‐1000B rotor (or equivalent)
  • Additional reagents and equipment for preparing single‐cell suspensions of tumor target cells (as for malignant effusions; see protocol 3)

Support Protocol 4: Preparation of Tumor Target Cells Starting with Solid Tumor Surgical Specimens

  Materials
  • Tumor cells (see Support Protocol protocol 21, protocol 32, protocol 43, or protocol 54)
  • 5 mCi/ml Na51CrO 4 (ICN Biomedicals; specific activity 250 to 900 mCi/mg)
  • RPMI‐2 and RPMI‐10 media (unit 7.7)
  • 15‐ml conical polypropylene tube
  • Sorvall RT‐6000 centrifuge with H‐1000B rotor (or equivalent)
  • 1‐ml tuberculin syringe with 25‐G needle
  • γ scintillation counter and counting tubes
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 3A) and determining number of viable cells by trypan blue exclusion ( appendix 3B)
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Figures

Videos

Literature Cited

Literature Cited
   Bryant, J., Day, R., Whiteside, T.L., and Herberman, R.B. 1992. Calculation of lytic units for the expression of cell‐mediated cytotoxicity. J. Immunol. Methods 146:91‐103.
   Caligiuri, M.A., Zmuidzinas, A., Manley, T., Levine, H., Smith, K.A., and Ritz, J. 1990. Functional consequences of IL2 receptor expression on testing human lymphocytes: Identification of a novel NK cell subset. J. Exp. Med. 171:1509‐152
   Grimm, E.A., Mazumder, A., Zhang, H.Z., and Rosenberg, S.A. 1982. Lymphokine‐activated killer cell phenomenon: Lysis of natural killer–resistant fresh solid tumor cells by interleukin 2–activated autologous human peripheral blood lymphocytes. J. Exp. Med. 155:1823‐184
   Karre, K. 1995. Express yourself or die: Peptides, MHC molecules, and NK cells. Science 267:978‐979
   Malnati, M.S., Peruzzi, M., Parker, K.C., Biddison, W.E., Ciccone, E., Moretta, A., and Long, E.O. 1995. Peptide specificity in the recognition of MHC class I by natural killer cell clones. Science 267:1016‐1018
   Melder, R.J., Walker, E., Herberman, R.B., and Whiteside, T.L. 1991. Adhesion characteristics of human interleukin 2–activated natural killer cells. Cell. Immunol. 132:177‐192
   Ortaldo, J.R., Mason, A., and Overton, R. 1986. Lymphokine‐activated killer cells: Analysis of progenitors and effectors. J. Exp. Med. 164:1193‐119
   Trinchieri, G. 1989. Biology of natural killer cells. Adv. Immunol. 47:187‐376
   Whiteside, T.L. and Herberman, R.B. 1990. Characteristics of natural killer cells and lymphokine‐activated killer cells. Their role in the biology and treatment of human cancer. Immunol. Allergy Clin. N. Amer. 10:663‐704
   Whiteside, T.L. and Herberman, R.B. 1994. Role of human natural killer cells in health and disease. Clin. Diag. Lab. Immunol. 1:125‐133
   Whiteside, T.L., Miescher, S., Hurlimann, J., and Von Fliedner, V. 1986a. Separation, phenotyping and limiting dilution analysis of T‐lymphocytes infiltrating human solid tumors. Int. J.Cancer 37:803‐811
   Whiteside, T.L., Miescher, S., MacDonald, H.R., and Von Fliedner, V., 1986b. Separation of tumor‐infiltrating lymphocytes from tumor cells in human solid tumors. J. Immunol. Methods 90:221‐223.
   Whiteside, T.L., Bryant, J., Day, R., and Herberman, R.B. 1990. Natural killer cytotoxicity in the diagnosis of immune dysfunction: Criteria for a reproducible assay. J. Clin. Lab. Analysis 4:102‐114.
   Whiteside, T.L., Letessier, E., Hirabayashi, H., Vitolo, D., Bryant, J., Barnes, L., Snyderman, C., Johnson, J.T., Myers, E., Herberman, R.B., Rubin, J., Kirkwood, J.M., and Vlock, D.R. 1993. Evidence for local and systemic activation of immune cells by peritumoral injections of interleukin 2 in patients with advanced squamous cell carcinoma of the head and neck. Cancer Res. 53:5654‐566
Key Reference
   Whiteside et al., 1990. See above
  Contains a comprehensive discussion of optimal conditions and quality control for the NK‐cell cytotoxicity assay.
   Whiteside, T.L., Rinaldo, C.R., and Herberman, R.B. 1992. Cytolytic cell functions. In Manual of Clinical Laboratory Immunology, 4th ed. (N.R. Rose, E.C. deMacario, J.L. Fahey, H. Friedman, and G.M. Penn, eds.) pp. 33:220‐230, American Society for Microbiology, Washington, D.C.
  Deals with methods for various cytotoxicity assays, including determenations of LAK activity.
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