Granule Enzyme Exocytosis Assay for Cytotoxic T Lymphocyte Activation

Rolf Taffs1, Michail Sitkovsky2, Hajime Takayama3

1 Food and Drug Administration, Bethesda, Maryland, 2 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, 3 Mitsubishi‐Kasei Institute of Life Sciences, Tokyo, Japan
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.16
DOI:  10.1002/0471142735.im0316s25
Online Posting Date:  May, 2001
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Abstract

Upon activation by specific target cells, cytotoxic T lymphocytes (CTL) release into the culture medium the content of cytoplasmic granules that contain serine esterases. The amount of enzyme released during CTL activation can be easily quantitated by spectrophotometric measurement of the colored product of the enzymatic degradation of a synthetic substrate. In the primary method presented here, CTL are activated with monoclonal antibodies prepared against the T cell receptor (TCR) complex, then activation is quantitated according to the amount of serine esterase released in the supernatant. Alternate protocols describe the activation of CTL by a combination of protein kinase C and calcium ionophores (a TCR‐independent approach) and by the more conventional approach of target‐cell mediation. In a third approach, β‐glucuronidase rather than esterase activity is measured, as this enzyme is also present in granules released upon CTL activation. This unit therefore includes a colorimetric assay for CTL‐induced β‐glucuronidase activity employing the substrate phenolphthalein glucuronic acid as well as a corresponding automated fluorimetric assay employing the substrate 4‐methylumbelliferyl‐D‐glucuronide. Finally, the quantitation of granule exocytosis resulting from cell damage or death induced by the activating agent, rather than CTL activation, is described.

     
 
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Table of Contents

  • Basic Protocol 1: Esterase Assay to Measure Activation of CTL by Anti‐TCR Antibody
  • Alternate Protocol 1: Induction of Granule Exocytosis by Phorbol Esters and Calcium Ionophores
  • Alternate Protocol 2: Induction of Granule Exocytosis by Target Cells
  • Alternate Protocol 3: Assay for CTL‐Induced β‐Glucuronidase Activity
  • Alternate Protocol 4: β‐Glucuronidase Assay Using a 96‐Well‐Plate Fluorescence Scanner
  • Support Protocol 1: Assay for Lactate Dehydrogenase to Measure Granule Release by Damaged Cells
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Esterase Assay to Measure Activation of CTL by Anti‐TCR Antibody

  Materials
  • 5 µg/ml monoclonal antibody against T cell receptor (anti‐TCR MAb; Table 97.80.4711) in PBS
  • Complete RPMI‐10 medium ( appendix 2A) or medium recommended for the particular cell line
  • Cytotoxic T lymphocytes (CTL), prepared as in unit 3.11 and suspended in complete RPMI‐10 medium
  • 1% (v/v) Triton X‐100
  • recipe20 mM BLT (N‐α‐benzyloxycarbonyl‐L‐lysine thiobenzyl ester) stock (see recipe)
  • recipe22 mM DTNB [5,5′‐dithio‐bis(2‐nitrobenzoic acid)] stock (see recipe)
  • Phosphate‐buffered saline (PBS appendix 2A), pH 7.2 to 7.4
  • 0.1 M phenylmethanesulfonyl fluoride (PMSF) in dimethylsulfoxide (DMSO; Sigma, tissue culture grade) or 100% methanol
  • 96‐well round‐bottom microtiter plates (flexible polyvinyl chloride; Dynatech)
  • 12 × 75–mm glass or polypropylene culture tubes
  • Sorvall H‐1000B rotor and plate carrier (or equivalent)
  • 50‐ml conical tubes
  • Additional materials for preparation of CTL (unit 3.11)CAUTION:PMSF is highly toxic and should be handled with care.

Alternate Protocol 1: Induction of Granule Exocytosis by Phorbol Esters and Calcium Ionophores

  • 1 mg/ml phorbol myristate acetate (PMA; Sigma) in DMSO
  • 1 mg/ml calcium ionophore A23187 (Sigma) in DMSO
CAUTION: Preparation and handling of phorbol esters and calcium ionophores must be performed with regard to their hazardous biological activities as toxic agents and tumor promoters.

Alternate Protocol 2: Induction of Granule Exocytosis by Target Cells

  • Specific target cells (bearing antigen recognized by the TCR on CTL), suspended in complete RPMI‐10 medium (unit 3.11)

Alternate Protocol 3: Assay for CTL‐Induced β‐Glucuronidase Activity

  • recipe0.1 M acetate buffer, pH 4.6 (see recipe) containing 0.04% (v/v) Triton X‐100
  • 0.01 M phenolphthalein glucuronic acid in PBS (pH 7.0), prepared just before use
  • recipeβ‐glucuronidase stop solution (see recipe)

Alternate Protocol 4: β‐Glucuronidase Assay Using a 96‐Well‐Plate Fluorescence Scanner

  • recipeFluorogenic substrate solution (see recipe)
  • 96‐well fluorescence scanner: e.g., Titertek Fluoroskan II with Fluoroplate (Labsystems) or spectrofluorometer (e.g., CytoFluor 2350, Millipore)

Support Protocol 1: Assay for Lactate Dehydrogenase to Measure Granule Release by Damaged Cells

  • 2 mM NADH 2, prepared just prior to assay
  • recipe100 mM phosphate buffer, pH 7.4 (see recipe)
  • 4 mM sodium pyruvate, prepared just prior to assay
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Literature Cited

Literature Cited
   Brittinger, G., Hirschhorn, R., Douglas, S.D., and Weissman, G. 1968. Studies on lysosomes. XI. Characterization of a hydrolase‐rich fraction from human lymphocytes. J. Cell Biol. 37:394‐411.
   Coleman, P.L. and Green, G.D.J. 1981. A coupled photometric assay for plasminogen activator. Methods Enzymol. 80:408‐414.
   Pasternack, M.S. and Eisen, H.N. 1985. A novel serine esterase expressed by cytotoxic T lymphocytes. Nature (Lond.) 314:743‐745.
   Pasternack, M.S., Verret, C.R., Liu, M.A., and Eisen, H.N. 1986. Serine esterase in cytolytic T lymphocytes. Nature (Lond.) 322:740‐742.
   Schnyder, J. and Baggiolini, G. 1978. Secretion of lysosomal hydrolases by stimulated and nonstimulated macrophages. J. Exp. Med. 148:435‐450.
   Takayama, H. and Sitkovsky, M.V. 1987. Antigen receptor‐regulated exocytosis in cytotoxic T lymphocytes. J. Exp. Med. 166:725‐743.
   Takayama, H., Trenn, G., and Sitkovsky, M.V. 1987. A novel cytotoxic T lymphocyte activation assay: Optimized conditions for antigen receptor triggered granule enzyme secretion. J. Immunol. Methods. 104:183‐190.
Key Reference
   Takayama et al., 1987. See above.
  Compares various culture and assay conditions relevant to performing reproducible granule exocytosis assays at optimal and suboptimal levels of CTL activation.
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