Measuring the Activity of Cytolytic Lymphocytes

B. Paige Lawrence1

1 Washington State University, Pullman, Washington
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 18.6
DOI:  10.1002/0471140856.tx1806s22
Online Posting Date:  January, 2005
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Abstract

The protocols in this unit describe how to measure the activity of the two most common types of lymphocytes that have cytolytic activity: cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Measurement of CTL activity requires priming of cells, which can be performed either in vivo or in vitro. Priming leads to a population of differentiated, antigen‐specific CTL. Protocols in which CTL are primed in vivo and in vitro are included in this unit. In contrast to CTL, the cytolytic activity of NK cells can be measured in the absence of antigen priming. In all of these protocols, single‐cell suspensions of effector and target cells are prepared separately. The target cells are labeled with 51Cr, and then the effector and target cells are co‐cultured for several hours. The amount of target cell lysis is determined by measuring the amount of 51Cr released into the culture medium. For all of the protocols, the effector cells are derived from mouse spleen. The target cells are either spleen cells from mice of a different MHC haplotype, virus‐infected cells, tumor cells, or cultured cell lines. These protocols provide a methodological framework that can be adapted for measuring the activity of cytolytic lymphocytes in a variety of experimental paradigms.

Keywords: cytolytic activity; CTL; NK cells; chromium release assay

     
 
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Table of Contents

  • Basic Protocol 1: Chromium Release Assay for Measuring CTL Activity Following in Vitro Sensitization of Mouse Spleen Cells
  • Alternate Protocol 1: Chromium Release Assay for Measuring CTL Activity Following in Vivo Sensitization with Allogeneic Cells
  • Alternate Protocol 2: Chromium Release Assay for Measuring CTL Activity Following in Vivo Sensitization with Influenza a Virus
  • Basic Protocol 2: Chromium Release Assay for Measuring NK Cell Activity
  • Support Protocol 1: Preparation of a Single ‐Cell Suspension of Murine Spleen Cells
  • Support Protocol 2: Growth and Maintenance of P815 Cells
  • Support Protocol 3: Growth and Maintenance of Yac‐1 Tumor Cells
  • Support Protocol 4: Growth and Maintenance of MC57G Fibroblasts
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Chromium Release Assay for Measuring CTL Activity Following in Vitro Sensitization of Mouse Spleen Cells

  Materials
  • Stimulator/target cells (e.g., spleen cells), single‐cell suspension
  • Control target cells (to test specificity of cytolytic activity; e.g., syngeneic spleen cells), optional
  • Effector cells (e.g., alloantigen‐sensitized spleen cells, also called responder cells)
  • Control effector cells (non‐sensitized spleen cells or cells that were sensitized using an irrelevant antigen)
  • Complete RPMI (cRMPI, see recipe)
  • 0.5 mg/ml mitomycin C in cRPMI
  • Lipopolysaccharide (LPS) or concanavalin A (Con A) (both available from Sigma)
  • Phosphate buffered saline (PBS), sterile and low‐endotoxin (e.g., GIBCO, Invitrogen)
  • Serum‐free RPMI (sfRPMI, see recipe)
  • ≥5 mCi/ml Na 251CrO 4, sterile, aqueous solution (10 to 35 mCi/ml and 200 to 500 mCi/mg Cr; e.g., Amersham Biosciences)
  • 0.5% SDS (see recipe)
  • Coulter counter or hemacytometer
  • 24‐ or 96‐well flat‐bottom cell culture plates
  • 25‐cm2 tissue culture flasks or 100‐mm tissue culture dishes
  • 15‐ml and 50‐ml conical centrifuge tubes with screw caps (sterile, polystyrene or polypropylene; e.g., Corning or Falcon)
  • 96‐well round‐bottom microtiter plates
  • Multi‐channel micropipettor (50‐ to 200‐µl) with disposable tips
  • Reagent reservoirs, disposable
  • 12 × 75–mm glass test tubes
  • Gamma counter
  • Additional reagents and equipment for counting cells and determining viability ( appendix 3B)

Alternate Protocol 1: Chromium Release Assay for Measuring CTL Activity Following in Vivo Sensitization with Allogeneic Cells

  Materials
  • DBA mouse (see protocol 6)
  • Target cells (e.g., a single‐cell suspension of P815 mastocytoma cells; ATCC no. TIB‐64)
  • Hank's balanced salt solution (HBSS, e.g., GIBCO, Invitrogen; see recipe)
  • C57Bl/6 mice
  • Phosphate buffered saline (PBS), sterile and low‐endotoxin (e.g., GIBCO, Invitrogen)
  • Serum‐free RPMI (sfRPMI, see recipe)
  • ≥5 mCi/ml Na 251CrO 4 sterile, aqueous solution (10 to 35 mCi/ml and 200 to 500 mCi/mg Cr; e.g., Amersham Biosciences)
  • Control target cells (e.g., syngeneic spleen cells), optional
  • Complete RPMI (cRMPI, see recipe)
  • Effector cells (e.g., P815‐sensitized spleen cells; see protocol 5)
  • Control effector cells (non‐sensitized spleen cells or cells that were sensitized using an irrelevant antigen)
  • 15‐ and 50‐ml conical centrifuge tubes with screw caps (sterile, polystyrene or polypropylene; e.g., Corning, Falcon)
  • 26‐G × 1/2‐in. needle and 1‐ml syringe
  • Additional reagents and equipment for counting cells ( appendix 3B), and chromium release assay (see protocol 1)

Alternate Protocol 2: Chromium Release Assay for Measuring CTL Activity Following in Vivo Sensitization with Influenza a Virus

  Materials
  • Influenza A virus stock (mouse‐adapted, inactivated, e.g., SPAFAS/Charles River)
  • Phosphate buffered saline (PBS), sterile and low‐endotoxin (e.g., GIBCO)
  • Mice
  • Effector cells (i.e., spleen cells from an infected mouse; see protocol 5)
  • Control effector cells (non‐sensitized spleen cells or cells from a mouse that was sensitized using an irrelevant antigen)
  • Complete RPMI (cRMPI, see recipe)
  • Serum‐free RPMI (sfRPMI, see recipe)
  • 0.5 mg/ml mitomycin C in cRPMI
  • Target cells (i.e., virus‐infected or viral peptide–pulsed cells, e.g., MC57G cells, ATCC# CRL‐2295; see protocol 8)
  • Control target cells (e.g., 51Cr‐labeled MC57G cells that were not exposed to virus or viral peptide), optional
  • ≥5 mCi/ml Na 251CrO 4 sterile, aqueous solution (10 to 35 mCi/ml and 200 to 500 mCi/mg Cr; e.g., Amersham Biosciences)
  • 0.5% SDS (see recipe)
  • 15‐ and 50‐ml conical centrifuge tubes with screw caps (sterile, polystyrene or polypropylene; e.g., Corning or Falcon)
  • 24‐well tissue culture plates
  • Beckman Allegra GS‐6 (or equivalent) refrigerated centrifuge with swinging bucket rotor
  • 20‐, 200‐, and 1000‐µl micropipettors with disposable tips
  • 96‐well U‐bottom plates
  • 12 × 75–mm glass test tubes
  • Gamma counter
  • Additional reagents and equipment for counting cells ( appendix 3B)

Basic Protocol 2: Chromium Release Assay for Measuring NK Cell Activity

  Materials
  • Target cells (e.g., a single cell suspension of Yac‐1 tumor cells, ATCC no. TIB‐160; see protocol 7)
  • Yac‐1 growth medium (see recipe)
  • Control target cells (to test specificity of cytolytic activity; e.g., syngeneic spleen cells), optional
  • Phosphate buffered saline (PBS), sterile and low‐endotoxin (e.g., GIBCO, Invitrogen)
  • Complete RPMI (cRMPI, see recipe)
  • Serum‐free RPMI (sfRPMI, see recipe)
  • ≥5 mCi/ml Na 251CrO 4 aqueous solution (10 to 35 mCi/ml and 200 to 500 mCi/mg Cr; Amersham Biosciences)
  • Effector cells (spleen cells; see protocol 5)
  • Control effector cells (e.g., a single cell suspension of thymocytes from a syngenic mouse)
  • 0.5% SDS (see recipe)
  • 500 µg/ml polyinosinic acid‐polycytidylic acid (poly I:C) in HBSS or PBS, optional
  • 25‐cm2 culture flasks
  • 15‐ and 50‐ml conical centrifuge tubes with screw caps (sterile, polystyrene or polypropylene; e.g., Corning, Falcon)
  • 96‐well round‐bottom microtiter plates
  • Beckman Allegra GS‐6 (or equivalent) refrigerated centrifuge with swinging bucket rotor
  • 12 × 75–mm glass test tubes
  • Gamma counter
  • Additional reagents and equipment for counting cells ( appendix 3B), and chromium release assay (see protocol 1)

Support Protocol 1: Preparation of a Single ‐Cell Suspension of Murine Spleen Cells

  Materials
  • Mouse of appropriate strain
  • 70% ethanol
  • sfRPMI (see recipe), ice cold
  • cRPMI (see recipe), ice cold
  • Sterile distilled water, endotoxin tested for cell culture (e.g., GIBCO, Invitrogen), ice cold
  • 10× HBSS (e.g., GIBCO, Invitrogen; see recipe)
  • 15‐ml centrifuge tubes
  • 4‐in. dressing forceps
  • 4 3/4‐in. surgical scissors
  • 60 × 15–mm cell culture dishes
  • Frosted microscope slides (25 × 75 × 1–mm)
  • Plastic transfer pipets (optional)
  • 5‐ and 10‐ml sterile serological pipets
  • Refrigerated centrifuge (e.g., Beckman GS‐6 or equivalent)
  • Additional reagents and equipment for counting cells ( appendix 3B)

Support Protocol 2: Growth and Maintenance of P815 Cells

  • P815 cells (ATCC no. TIB‐64)
  • DBA mice
  • Biological safety cabinet (level 2)
  • 10‐ml syringe
  • 20‐ to 22‐G × 1‐in. needles
  • Small scissors
  • 4‐in. fine‐tipped forceps
  • 50‐ml centrifuge tubes
  • Vortexer

Support Protocol 3: Growth and Maintenance of Yac‐1 Tumor Cells

  • Yac‐1 cells (ATCC no. TIB‐160)
  • Yac‐1 cell growth medium (see recipe)
  • Biological safety cabinet (level 2)
  • 25‐ and 75‐cm2 tissue culture flasks
  • Additional reagents and equipment for counting cells and determining viability ( appendix 3B)

Support Protocol 4: Growth and Maintenance of MC57G Fibroblasts

  Materials
  • MC57G (ATCC #CRL‐2295)
  • MC57G growth medium (see recipe)
  • Cell culture–grade sterile PBS (e.g., from GIBCO, Invitrogen)
  • 0.25% trypsin solution in PBS
  • 15‐ and 50‐ml conical centrifuge tubes
  • 37°C water bath
  • 75‐cm2 tissue culture flasks
  • 37°C, 5% CO 2 humidified incubator
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Figures

Videos

Literature Cited

   Barrett, T. and Inglis, S.C. 1985. Growth, purification and titration of influenza viruses. In Virology: A Practical Approach (B.W. J. Mahy, ed.), pp. 119‐150. IRL Press, Washington, D.C.
   Cho, Y., Basta, S., Chen, W., Bennink, J.R., and Yewdell, W. 2003. Heat‐aggregated noninfectious influenza virus induces a more balanced CD8+T lymphocyte immunodominance hierarchy than infectious virus. J. Virol. 77:4679‐4684.
   Kagi, D., Ledermann, B., Burki, K., Zinkernagel, R.M., and Hengartner, H. 1996 Molecular mechanisms of lymphocyte‐mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo. Annu. Rev. Immunol. 14:207‐232.
   Kos, F. and Engleman, E. 1996. Role of natural killer cells in the generation of influenza virus–specific cytotoxic T cells. Cell Immunol. 173:1‐6.
   Mitchell, K.A. and Lawrence, B.P. 2003a. Exposure to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) renders influenza virus–specific CD8+ T cells hyporesponsive to antigen. Toxicol. Sci. 74:74‐84.
   Mitchell, K.A. and Lawrence, B.P. 2003b. T cell receptor transgenic mice provide novel insights into understanding cellular targets of TCDD: Suppression of antibody production, but not the response of CD8+ T cells, during infection with influenza virus. Toxicol. Appl. Pharmacol. 192:275‐286.
   Nonacs, R., Humborg, C., Tam, F., and Steinman, R. 1992. Mechanisms of mouse spleen dendritic cell function in the generation of influenza‐specific cytolytic T lymphocytes. J. Exp. Med. 176:519‐529.
   Russell, J.H. and Ley, T.J. 2002. Lymphocyte‐mediated cytotoxicity Annu. Rev. Immunol. 20:323‐370.
   Warren, T.K., Mitchell, K.A., and Lawrence, B.P. 2000. Exposure to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) suppresses the humoral and cell‐mediated immune responses to influenza A virus without affecting cytolytic activity in the lung. Toxicol. Sci. 56:114‐123.
   Yokoyama, W.M. and Plougastel, B.F.M. 2003. Immune functions encoded by the natural killer gene complex. Nat. Rev. Immunol. 3:304‐316.
Internet Resources
   http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm
  A pdf manual of Biosafety in Microbiological and Biomedical Laboratories, 4th Edition (1999), U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, and National Institutes of Health. US Government Printing Office Washington, D.C.
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