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Induction and Measurement of Cytotoxic T Lymphocyte Activity

John Wonderlich¹, Gene Shearer¹, Alexandra Livingstone², Andrew Brooks³

¹National Cancer Institute, Bethesda, Maryland
²Imperial College, London, United Kingdom, UK
³University of Melbourne, Victoria, Australia, 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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Unit Introduction
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 1 and 2)
Sensitization medium (see recipe)
Source of stimulator cells: unmodified or hapten-modified (see Support Protocol 3) mouse spleen cells
Complete RPMI-10 medium (appendix 2)
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 2; 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 PBS or 1 mg/ml LPS in H₂O (to stimulate splenic T and B cells, respectively)
~1 mCi/ml Na₂⁵¹CrO₄ 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 Basic Protocol 1)
Control effector cells (nonsensitized spleen cells or spleen cells sensitized against irrelevant antigen)
2% (v/v) Triton X-100 in H₂O
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 H₂O (for B cell blasts)
Cell culture medium: e.g. IMDM-7 medium (appendix 2)
1 mCi/ml [³H]thymidine (5 Ci/mmol; Amersham)
Experimental and control effector cell populations (see Basic Protocol 1)
Cell washing medium: HBSS (appendix 2) 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% CO₂ incubator unless otherwise specified.

Support Protocol 3: TNP Modification of Target/Stimulator Cells

Materials

Hanks balanced salt solution (HBSS; appendix 2), protein-free
TNBS solution (see recipe)
10% heat-inactivated (1 hr at 56°C) FBS in PBS (appendix 2), ice-cold

Additional reagents and equipment for CTL generation (see Basic Protocol 1) or ⁵¹Cr labeling (see Basic Protocol 2)

Support Protocol 4: Viral Infection of Target/Stimulator Cells

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 2)

Additional reagents and equipment for CTL generation (see Basic Protocol 1) and ⁵¹Cr labeling (see Basic Protocol 2)

CAUTION: Although the virus is “egg-adapted,” standard precautions for working with a live virus should be taken.

Alternate Protocol 2: Polyclonal Induction of CTL Activity

Materials

Sensitization medium with 2 µg/ml Con A
1.0 M -methyl-D-mannoside (MM) in complete RPMI-10 medium

Additional reagents and equipment for CTL generation (see Basic Protocol 1) and ⁵¹Cr labeling (see Basic Protocol 2)

Alternate Protocol 3: Redirection of CTL Lysis

Materials

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

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

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

Figure 3.11.1

Induction and detection of cytotoxic T lymphocyte (CTL) activity using chromium- release assay. Relationships of protocols presented in this unit are indicated.

View Image
Figure 3.11.2

Lytic units (LU). Population A has >20 LU/10⁶ cells, and C has <20 LU/10⁶ cells.

View Image

Literature Cited

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.
	Berke, G. 1989. Functions and mechanisms of lysis induced by cytotoxic T lymphocytes and natural killer cells. In Fundamental Immunology (W.E. Paul, ed.) pp. 735-764. Raven Press, New York.
	Bevan, M.J. 1975. The major histocompatibility complex determines susceptibility to cytotoxic T cells directed against minor histocompatibility. J. Exp. Med. 142:1349-1364.
	Bevan, M.J. and Cohn, M. 1975. Cytotoxic effects of antigen- and mitogen-induced T cells on various targets. J. Immunol. 114:559-565.
	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.
	Möller, G. (ed.) 1983. Mechanism of action of cytotoxic T cells. Immunol. Rev. 72 (entire volume).
	Muchmore, A.V., Nelson, D.L., Kirschner, H., and Blaese, R.M. 1975. A reappraisal of the effector cells mediating mitogen induced cellular cytotoxicity. Cell. Immunol. 19:78-90.
	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.
	Townsend, A. and Bodmer, H. 1989. Antigen recognition by class I-restricted T lymphocytes. Annu. Rev. Immunol. 7:601-624.
	Trinchieri, G. 1989. Biology of natural killer cells. Adv. Immunol. 47:187-376.
	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.
	Young, Y.D., Liu, C.C., Perschini, P.M., and Cohn, Z.A. 1988. Perforin-dependent and -independent pathways of cytotoxicity. Immunol. Rev. 103:161-202.
	Zinkernagel, R.M. and Doherty, P.C. 1979. MHC- restricted cytotoxic T cells: Studies on the biological role of polymorphic major transplantation antigens determining T cell restriction-specificity, function, and responsiveness. Adv. Immunol. 27:51-177.
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.
	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.
Two reviews of the chromium-release assay and its applications.