Measurement of Polyclonal and Antigen‐Specific Cytotoxic T Cell Function

William E. Biddison1, Rudolf Lichtenfels2, Medi Adibzadeh2, Roland Martin2

1 National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, 2 Department of Neurology, Tübingen, Germany
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.17
DOI:  10.1002/0471142735.im0717s17
Online Posting Date:  May, 2001
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Abstract

Measurement of in vitro cytotoxic function of human T cells can be accomplished by polyclonal stimulation of T cell effectors using anti‐CD3 antibody, which stimulates all cytolytic effector cells, or with a specific stimulating antigen. Accordingly, two sets of assays of cytolytic T cell function are described in this unit, one for measuring anti‐CD3‐mediated cytotoxicity and the other for measuring antigen‐specific cytotoxicity. Although the calcein release assay (CARE‐LASS) described here is for use with antigen‐activated cytotoxic T lymphocytes (CTL) as well as natural killer (NK) or lymphokine‐activated killer (LAK) cells, minor changes in the protocols that address polyclonal T cell activation are described that make them suitable for use with calcein‐labeled target cells.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of Anti‐CD3‐Mediated Cytotoxicity
  • Alternate Protocol 1: Measurement of Cytotoxicity Using Solid‐Phase Anti‐CD3 Hybridoma Cells
  • Basic Protocol 2: Measurement of Antigen‐Mediated T Cell Cytotoxicity
  • Basic Protocol 3: Measurement of CTL Activity by the Calcein Release Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Anti‐CD3‐Mediated Cytotoxicity

  Materials
  • Target cells: Epstein‐Barr virus (EBV)–transformed B lymphoblastoid cells (ATCC CRL #1612; unit 7.22)
  • Complete RPMI‐5 medium ( appendix 2A)
  • 1 mCi/ml Na 2[51Cr]O 4 (51Cr; ≥300 mCi/mg; Amersham)
  • T cell effector population (units 7.2 & 7.4)
  • recipeAnti‐CD3 antibody (IgG2a; see recipe)
  • 2% (v/v) Triton X‐100
  • 24‐well flat‐bottom microtiter plate (Costar)
  • Sorvall centrifuge with H‐1000B rotor (or equivalent), tubes, and microplate carrier
  • 96‐well round‐bottom microtiter plates (Costar)
  • Additional reagents and equipment for determining cell viability by trypan blue exclusion ( appendix 3B)
CAUTION: Follow standard radiation safety procedures when working with 51Cr solution and 51Cr‐labeled target cells.NOTE: All tissue culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 1: Measurement of Cytotoxicity Using Solid‐Phase Anti‐CD3 Hybridoma Cells

  Additional Materials
  • OKT3 hybridoma antibody‐producing cells (ATCC #CRL 8001)
  • Anti–influenza nucleoprotein hybridoma (ATCC #HB65)
NOTE: All tissue culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Measurement of Antigen‐Mediated T Cell Cytotoxicity

  Materials
  • Complete RPMI‐5 medium ( appendix 2A) with and without 1× MEM nonessential amino acids (JRH Biosciences)
  • Infectious allantoic fluid containing recipeinfluenza viruses (see recipe)
  • Population of specific cytolytic T cells from humans recently immunized against influenza virus (see units 7.2 & 7.4 for purification methods)
  • 5% FBS in PBS ( appendix 2A)
  • 6‐ml tissue culture tubes (Falcon)
  • Additional reagents and equipment for measurement of T cell cytotoxicity (see protocol 1) and for determining cell viability by trypan blue exclusion ( appendix 3B)
NOTE: All tissue culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise noted.

Basic Protocol 3: Measurement of CTL Activity by the Calcein Release Assay

  Materials
  • HBSSF: recipeHanks balanced salt solution (HBSS; appendix 2A) without phenol red, Ca2+, or Mg2+, supplemented with 5% FBS
  • 1 mg/ml antigen stock solution or infectious agent (e.g., recipeinfluenza viruses; see recipe) to sensitize target cells
  • 2.5 mM Calcein‐AM (Molecular Probes) in DMSO
  • Effector CTL: cytotoxic T lymphocytes specific for antigen of interest and for irrelevant antigen as control (unit 7.19)
  • Lysis buffer: 50 mM sodium borate/0.1% (v/v) Triton X‐100, pH 9.0
  • 15‐ml conical centrifuge tubes
  • Sorvall centrifuge with H‐1000B rotor (or equivalent) equipped with tube and microplate carriers
  • 96‐well round‐bottom microtiter plates
  • Automated fluorescence measurement system accommodating microtiter plates (e.g., CytoFluor 2350, Millipore)
  • Additional reagents and equipment for generating EBV‐transformed B lymphoblastoid cells (unit 7.22) or preparing single‐cell suspensions of tumor cells (as for NK/LAK activity assay; unit 7.18), determining cell viability by trypan blue exclusion ( appendix 3B), and sensitizing target cells with antigens (as for measuring antigen‐mediated T cell cytotoxicity; see protocol 3)
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Figures

Videos

Literature Cited

Literature Cited
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   Blomberg, K.C.G., Hemmilä, I., and Lövgren, T. 1986. Europium‐labeled target cells in an assay of natural killer cell activity. II. A novel non‐radioactive method based on time‐resolved fluorescence. Significance and specificity of the method. J. Immunol. Methods 92:117.
   Brenan, M. and Parish, C.R. 1988. Automated fluorometric assay for T cell cytotoxicity. J. Immunol. Methods 112:121‐131.
   Brunner, K.T., Mauel, J., Ceffotini, M.C., and Chapius, B. 1968. Quantitative assay of the lytic action of immune lymphoid cells on 51Cr‐labeled allogeneic target cells in vitro: Inhibition by isoantibody and by drugs. Immunology 14:181‐196.
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   Tax, W.J.M., Hermes, F.F.M., Willems, R.W., Capel, J.A., Koene, R.A.P. 1984. Fc receptors for mouse IgG1 on human monocytes: Polymorphism and role in antibody‐induced T cell proliferation. J. Immunol. 133:1185‐1191.
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