Induction and Measurement of Cytotoxic T Lymphocyte Activity

John Wonderlich1, Gene Shearer1, Alexandra Livingstone2, Andrew Brooks3

1 National Cancer Institute, Bethesda, Maryland, 2 Imperial College, London, United Kingdom, 3 University of Melbourne, Victoria, Australia
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.11
DOI:  10.1002/0471142735.im0311s72
Online Posting Date:  May, 2006
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Abstract

Cytotoxic T lymphocytes (CTL) kill target cells on the basis of cell‐surface antigen recognition and are important in the host response to tumors, transplants, and viruses. This unit presents several protocols for generating and measuring CTL activity. The first describes generating CTL against some of the most commonly used target antigens. Two methods for the quantitation of CTL activity are described based on the two pathways used bt CTL to kill target cells. In one pathway, they release lytic granules containing perforin and granzymes, leading to apoptosis and target cell lysis. In a second pathway, they trigger apoptosis via Fas/Fas ligand interactions. In the chromium‐release assay provided here, labeled antigenic targets are recognized and lysed, releasing radioactivity into the supernatant. In the JAM test protocol, CTL activity is determined by measuring degradation of radioactively labeled DNA in target cells that have undergone apoptotic cell death. Rather than measuring release of radioactivity, the JAM test measures the amount of DNA retained in target cells that are not killed by CTL. Two support protocols detail the generation of CTL precursors (CTLp) against antigens that require priming in vivo. A second set of support protocols describe the preparation of both stimulator and target cells for these responses using two representative antigens, trinitrophenyl and viruses. Finally, two alternate protocols illustrate how to determine total CTLp activity in a population that might express cytolytic activity. These protocols bypass MHC restriction and the original antigen specificity of CTLp by polyclonal stimulation of CTLp with mitogens followed by attachment of CTL to target cells and subsequent cytolysis.

     
 
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Table of Contents

  • Basic Protocol 1: Induction of Cytolytic Activity in CTL Precursors
  • Basic Protocol 2: Chromium‐Release Assay for Measuring CTL Activity
  • Alternate Protocol 1: DNA Fragmentation Assay (JAM Test) for Measuring CTL Activity
  • In Vivo Priming to Generate CTL Precursors
  • Support Protocol 1: In Vivo Priming of Mice with Minor Histocompatibility Antigens
  • Support Protocol 2: In Vivo Priming of Mice with Viral Antigens
  • Modification of Stimulator and Target Cells with Antigens
  • Support Protocol 3: TNP Modification of Target/Stimulator Cells
  • Support Protocol 4: Viral Infection of Target/Stimulator Cells
  • Assessment of all CTL Activity Regardless of Specificity
  • Alternate Protocol 2: Polyclonal Induction of CTL Activity
  • Alternate Protocol 3: Redirection of CTL Lysis
  • Basic Protocol 3: Measurement of CTL Activity In Vivo
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Induction of Cytolytic Activity in CTL Precursors

  Materials
  • Source of responder cells: unprimed or in vivo primed mouse spleen cells (see Support Protocols protocol 41 and protocol 52)
  • Sensitization medium (see recipe)
  • Source of stimulator cells: unmodified or hapten‐modified (see protocol 6) mouse spleen cells
  • Complete RPMI‐10 medium ( appendix 2A2)
  • 0.5 mg/ml mitomycin C prepared in HBSS (stored in dark)
  • Con A or IL‐2 (optional)
  • 15‐ml disposable polystyrene conical tubes with screw caps (e.g., Falcon)
  • Sorvall H‐1000B rotor (or equivalent)
  • 24‐well flat‐bottom microtiter plate, 2‐ml capacity, with lids (e.g., Costar)
  • Additional materials for preparing single‐cell suspensions (unit 3.1) and cell viability test using trypan blue exclusion ( appendix 3B)
NOTE: The mice should be free of infectious agents that can affect results, such as mycoplasma and immunosuppressive viruses (e.g., Sendai).

Basic Protocol 2: Chromium‐Release Assay for Measuring CTL Activity

  Materials
  • Target cells (e.g., single‐cell suspension of splenic lymphoblasts, tissue culture cells, or tumor cells)
  • Control target cells (matched to test target cells except for differences in antigen expression)
  • Complete RPMI‐10 medium ( appendix 2A2; antibiotics not necessary in medium used for this assay)
  • Sensitization medium (see recipe)
  • Mitogen solution (optional; for preparing splenic lymphoblasts): 1 mg/ml Con A in PBSor 1 mg/ml LPS in H 2O (to stimulate splenic T and B cells, respectively)
  • ∼1 mCi/ml Na 251CrO 4 in isotonic medium, sterile and pyrogen‐free (200 to 500 µCi/µg; DuPont NEN or Amersham)
  • Fetal bovine serum (FBS), heat‐inactivated 1 hr at 56°C
  • Effector cells (including CTL; see protocol 1)
  • Control effector cells (nonsensitized spleen cells or spleen cells sensitized against irrelevant antigen)
  • 2% (v/v) Triton X‐100 in H 2O
  • 25‐cm2 tissue culture flasks (e.g., Corning)
  • 24‐well flat‐bottom microtiter plates, 2‐ml capacity, with lids (e.g., Costar)
  • Nylon filtration fabric, 112‐µm mesh (optional; Tetco)
  • 15‐ml disposable polystyrene conical tubes with screw caps (e.g., Falcon)
  • Sorvall H‐1000B rotor (or equivalent) and microtiter plate carrier
  • Multiwick supernatant harvesting system (Skatron)
  • 96‐well round‐bottom microtiter plates with lids to fit supernatant harvesting system (Costar)
  • Multichannel pipettor (50‐ to 200‐µl) with disposable tips
  • 51Cr counting tubes (Skatron)
  • Additional materials for preparing single‐cell suspension (unit 3.1) and cell viability test using trypan blue exclusion ( appendix 3B)
CAUTION: Follow standard radiation‐safety procedures when working with 51Cr solution and 51Cr‐labeled target cells.

Alternate Protocol 1: DNA Fragmentation Assay (JAM Test) for Measuring CTL Activity

  Materials
  • Antigenic and control target cells: splenocytes (units 1.9& 3.1) or tumor cells
  • Mitogen stock solution (for use with splenocytes): 1 mg/ml concanavalin A (Con A; Sigma) in PBS (for T cell blasts) or 5 mg/ml lipopolysaccharide (LPS; Difco) in H 2O (for B cell blasts)
  • Cell culture medium: e.g. IMDM‐7 medium ( appendix 2A2)
  • 1 mCi/ml [3H]thymidine (5 Ci/mmol; Amersham)
  • Experimental and control effector cell populations (see protocol 1)
  • Cell washing medium: HBSS ( appendix 2A2) containing 2% FBS; alternatively use RPMI, IMDM or other cell culture medium appropriate to cell line used, supplemented with 2% FBS
  • 25‐cm2 (e.g., Corning) or 75‐cm2 (e.g., Falcon) tissue culture flasks
  • 6‐ml round‐bottom and 15‐ml conical polystyrene tubes (e.g., Falcon)
  • Sorvall RT‐6000B centrifuge and H‐1000B rotor (or equivalent centrifuge and rotor) and microtiter plate carrier
  • 50‐ml conical polypropylene tubes (e.g., Falcon)
  • 96‐well round‐bottom microtiter plates with lids (e.g., Greiner)
  • Multichannel pipettor (50 to 200 µl capacity) with disposable tips
  • Semiautomated microtiter plate cell harvester and glass microfiber strips (e.g., Cambridge Technology)
  • Additional reagents and equipment for counting viable cells ( appendix 3B)
CAUTION: Follow standard radiation‐safety procedures when working with [3H]thymidine solution and labeled cells.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: In Vivo Priming of Mice with Minor Histocompatibility Antigens

  Materials
  • Hanks balanced salt solution (HBSS; appendix 2A2), protein‐free
  • TNBS solution (see recipe)
  • 10% heat‐inactivated (1 hr at 56°C) FBS in PBS ( appendix 2A2), ice‐cold
  • Additional reagents and equipment for CTL generation (see protocol 1) or 51Cr labeling (see protocol 2)

Support Protocol 2: In Vivo Priming of Mice with Viral Antigens

  Materials
  • Target cells susceptible to viral infection (e.g., mitogen‐activated T lymphoblasts for influenza virus)
  • Allantoic fluid containing −1000 to 3000 hemagglutinin units (HA U) influenza virus per ml (store at −70°C)
  • Complete RPMI‐10 medium ( appendix 2A2)
  • Additional reagents and equipment for CTL generation (see protocol 1) and 51Cr labeling (see protocol 2)
CAUTION: Although the virus is “egg‐adapted,” standard precautions for working with a live virus should be taken.

Support Protocol 3: TNP Modification of Target/Stimulator Cells

  Materials
  • Sensitization medium with 2 µg/ml Con A
  • 1.0 M α‐methyl‐D‐mannoside (αMM) in complete RPMI‐10 medium
  • Additional reagents and equipment for CTL generation (see protocol 1) and 51Cr labeling (see protocol 2)

Support Protocol 4: Viral Infection of Target/Stimulator Cells

  Materials
  • Phytohemagglutinin (PHA), purified (Burroughs Wellcome) or Con A (Pharmacia Biotech or Miles Labs)
  • Additional reagents and equipment for CTL generation (see protocol 1) and 51Cr labeling (see protocol 2)

Alternate Protocol 2: Polyclonal Induction of CTL Activity

  Materials
  • Mice
  • Virus of interest (e.g., HSV‐1) or synthetic peptide antigen
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Complete Freund's adjuvant (also see unit 2.4)
  • Hanks' balanced salt solution (HBSS; appendix 2A)
  • Complete RPMI‐10 ( appendix 2A)
  • PBS ( appendix 2A) containing 0.1% (w/v) BSA
  • 5 mM carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes) in DMSO (store in aliquots at −20°C)
  • PBS ( appendix 2A) containing 1% FBS and 5 mM EDTA
  • Additional reagents and equipment for injection of mice (unit 1.6), euthanasia of mice (unit 1.8), dissection of mice to obtain spleen or lymph nodes (unit 1.9), preparation of single‐cell suspensions from spleen or lymph nodes (unit 3.1), counting viable cells ( appendix 3B), and flow cytometry (Chapter 5)
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Figures

Videos

Literature Cited

   Aichele, P., Brduscha‐Riem, K., Oehen, S., Odermatt, B., Zinkernagel, R.M., Hengartner, H., and Pircher, H. 1997. Peptide antigen treatment of naive and virus‐immune mice: Antigen‐specific tolerance versus immunopathology. Immunity 6:519‐529.
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   Brunner, K.T., Mauel, J., Cerottini, J.‐C., and Chapuis, B. 1968. Quantitative assay of the lytic action of immune lymphoid cells on 51Cr labeled allogenic target cells in vitro: Inhibition by isoantibody and by drugs. Immunology 14:181‐186.
   Gately, M.K. and Martz, E. 1977. Comparative studies on the mechanisms of nonspecific, Con A– dependent cytolysis and specific T cell–mediated cytolysis. J. Immunol. 119:1711‐1722.
   Green, W.R., Ballas, Z.K., and Henney, C.S. 1978. Studies on the mechanism of lymphocyte‐mediated cytolysis. XI. The role of lectin in lectin‐dependent cell‐mediated cytotoxicity. J. Immunol. 121:1566‐1572.
   Henkart, P. and Martz, E. (eds.) 1985. Mechanisms of Cell‐Mediated Cytotoxicity, Vol. II. Plenum, New York.
   Henkart, P. and Yue, C.C. 1988. The role of cytoplasmic granules in lymphocyte cytotoxicity. Prog. Allergy 40:82‐110.
   Kourilsky, P. and Claverie, J.M. 1989. MHC‐antigen interaction: What does the T cell receptor see. Adv. Immunol. 45:107‐193.
   Lovchik, J.C. and Hong, R. 1977. Antibody‐dependent cell‐mediated cytolysis (ADCC): Analysis and projections. Prog. Allergy 22:1‐44.
   Matzinger, P. 1991. A simple assay for DNA fragmentation and cell death. J. Immunol. Methods 145:185‐192.
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   Oehen, S. and Brduscha‐Riem, K. 1998. Differentiation of naive CTL to effector and memory CTL: Correlation of effector function with phenotype and cell division. J. Immunol. 161:5338‐5346.
   Oritz de Landazuri, M. and Herberman, R.B. 1972. Specificity of cellular immune reactivity to virus‐induced tumors. Nature New Biol. 238:18‐19.
   Rechtsteiner, G., Warger, T., Osterloh, P., Schild, H., and Radsak, M.P. 2005. Cutting edge: Priming of CTL by transcutaneous peptide immunization with imiquimod. J. Immunol. 174:2476‐2480.
   Segal, D.M. and Snider, D.P. 1989. Targeting and activation of cytotoxic lymphocytes. Chem. Immun. 47:179‐213.
   Shearer, G.M. and Schmitt‐Verhulst, A.‐M. 1977. Major histocompatibility complex–restricted cell‐mediated immunity. Adv. Immunol. 25:55‐91.
   Simpson, E. and Gordon, R.D. 1977. Responsiveness to H‐Y antigen Ir gene complementation and target cell specificity. Immunol. Rev. 35:59‐75.
   Singer, A., Kruisbeek, A.M., and Andrysiak, P.M. 1984. T cell‐accessory cell interactions that initiate allospecific cytotoxic T lymphocyte responses: Existence of both Ia‐restricted and Ia‐ unrestricted cellular interaction pathways. J. Immunol. 132:2199‐2209.
   Staerz, U.D., Kanagawa, O., and Bevan, M.J. 1985. Hybrid antibodies can target sites for attack by T cells. Nature (Lond.) 314:628‐631.
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   Waterfield, J.D., Waterfield, E.M., Anaclerio, A., and Möller, G. 1976. Lymphocyte‐mediated cytotoxicity against tumor cells: Specificity and characterization of concanavalin A‐activated cytotoxic effector lymphocytes. Transplant. Rev. 29:277‐310.
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Key References
   Grabstein, K. and Chen, Y.U. 1980. Cell mediated cytolytic responses. In Selected Methods in Cellular Immunology (B.B. Mishell, S.M. Shiigi, C. Henry and R.I. Mishell, eds.) pp. 124‐137. W.H. Freeman, New York.
  Two reviews of the chromium‐release assay and its applications.
   Simpson, E. and Chandler, P. 1986. Analysis of cytotoxic T cell responses. In Cellular Immunology, Vol. 2, 4th ed. (D.M. Weir, L.A. Herzenberg, C. Blackwell, and L.A. Herzenberg, eds.) pp. 68.1‐68.16. Blackwell Scientific, Oxford.
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