Induction and Measurement of Cytotoxic T Lymphocyte Activity

John Wonderlich1, Gene Shearer1, Alexandra Livingstone2, Andrew Brooks3, Mark J. Soloski4, Matthew M. Presby4

1 National Cancer Institute, Bethesda, Maryland, 2 Imperial College, London, United Kingdom (JAM test), 3 University of Melbourne, Victoria, Australia (CTL activity in vivo), 4 Johns Hopkins School of Medicine, Baltimore, Maryland (non‐radioactive assay for in vitro CTL activity)
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.11
DOI:  10.1002/cpim.38
Online Posting Date:  February, 2018
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Cytotoxic T cells (CTLs) are important immune effector cells in the adaptive immune response. It has been well documented that CTLs are important in host immune responses to viral and bacterial intracellular pathogens, tumors, and transplanted tissues. The properties of CTLs have been studied extensively in murine models, and their roles validated in the human setting. Frequently, the presence of these cells correlates well with protective immunity, so the ability to readily measure the activity of these cells is an important immunological measurement. In this unit, several assays are described that are commonly utilized to induce CTLs and to measure CTL activity both in vitro and in vivo. These assays are adaptable to many experimental and/or disease models, and in the case of the in vitro assays can be applied to measure CTL activity in human samples. © 2018 by John Wiley & Sons, Inc.

Keywords: cytotoxic T cells; CTLs; T cell clones; target cells; Cr‐release; in vitro assays; in vivo assays

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • 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
  • Basic Protocol 3: A Non‐Radioactive Assay for Measuring In Vitro 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 4: Measurement of CTL Activity In Vivo
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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 2A)
  • 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; Kruisbeek, ) and cell viability test using trypan blue exclusion ( appendix 3B; Strober, )
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 2A; 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; Kruisbeek, ) and cell viability test using trypan blue exclusion ( appendix 3B; Strober, )
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 [unit 1.9 ((Reeves and Reeves, ) and unit 3.1 (Kruisbeek, )] 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 2A)
  • 1 mCi/ml [3H]thymidine (5 Ci/mmol; Amersham)
  • Experimental and control effector cell populations (see protocol 1)
  • Cell washing medium: HBSS ( appendix 2A) 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; Strober, )
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.

Basic Protocol 3: A Non‐Radioactive Assay for Measuring In Vitro CTL Activity

  Materials
  • Antigen‐expressing target cells
  • Control bystander cells (antigen‐negative)
  • DMEM medium with 10% FBS and antibiotics ( appendix 2A)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Cellstripper (Corning, cat. no. 25‐056‐CI)
  • Carboxyfluorescein succinimidyl ester (CFSE; Biolegend, cat. no. 422701)
  • RPMI 1640 with 10% FBS and antibiotics ( appendix 2A)
  • Effector T cell populations (CTL clone/lines or in vivo generated)
  • Lympholyte M (murine lymphocyte isolation; Cedarlane, cat. no. CL5030) or Ficoll‐Paque Plus (human lymphocyte isolation, GE HeathCare, cat. no. 17‐1440‐03)
  • FACS wash buffer: PBS with 2% FBS and 5 mM sodium azide
  • Anti‐CD8 PE antibody
  • TO‐PRO‐3 iodide (ThermoFisher, cat. no. T3605)
  • 15‐ml centrifuge tubes
  • Low‐speed tabletop centrifuge (e.g., Fisher Centrific) with 96‐well plate adapter
  • 96‐well round‐bottom microtiter tissue culture plates
  • Additional reagents and equipment include counting viable cells by trypan blue exclusion ( appendix 3B; Strober, ), and flow cytometry (Chapter 5)

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

  Materials
  • Hanks balanced salt solution (HBSS; appendix 2A), protein‐free
  • TNBS solution (see recipe)
  • 10% heat‐inactivated (1 hr at 56°C) FBS in PBS ( appendix 2A), 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 2A)
  • 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; Cooper and Patterson, )
  • 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; Donovan and Brown, ), euthanasia of mice (unit 1.8; Donovan and Brown, ), dissection of mice to obtain spleen or lymph nodes (unit 1.9; Reeves and Reeves, ), preparation of single‐cell suspensions from spleen or lymph nodes (unit 3.1; Kruisbeek, ), counting viable cells ( appendix 3B; Strober, ), and flow cytometry (Chapter 5)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

  Aichele, P., Brduscha–Riem, K., Oehen, S., Odermatt, B., Zinkernagel, R. M., Hengartner, H., & 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., & 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., & 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.
  Cooper, H. M. and Patterson, Y. (2008). Production of polyclonal antisera. Curr. Protoc. Immunol. 82, 2.4.1–2.4.10. doi: 10.1002/0471142735.im0204s82.
  Davis, L. S., Lipsky, P. E., & Bottomly, K. 2001. Measurement of human and murine interleukin 2 and interleukin 4. Curr. Protoc. Immunol. 37, 6.3.1–6.3.13. doi: 10.1002/0471142735.im0603s37.
  Donovan, J. and Brown, P. 2006a. Parenteral injections. Curr. Protoc. Immunol. 73, 1.6.1–1.6.10. doi: 10.1002/0471142735.im0106s73.
  Donovan, J. and Brown, P. (2006b). Euthanasia. Curr. Protoc. Immunol. 73, 1.8.1–1.8.4. doi: 10.1002/0471142735.im0108s73.
  Gately, M. K., & 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.
  Godoy‐Ramirez, K., Mäkitalo, B., Thorstensson, R., Sandström, E., Biberfeld, G., & Gaines, H. 2005. A novel assay for assessment of HIV‐specific cytotoxicity by multiparameter flow cytometry. Cytometry A 68, 71–80.
  Green, W. R., Ballas, Z. K., & 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., & Martz, E. (eds.). 1985. Mechanisms of Cell–Mediated Cytotoxicity, Vol. II. Plenum, New York.
  Henkart, P., & Yue, C. C. 1988. The role of cytoplasmic granules in lymphocyte cytotoxicity. Prog. Allergy 40, 82–110.
  Kourilsky, P., & Claverie, J. M. 1989. MHC–antigen interaction: What does the T cell receptor see. Adv. Immunol. 45, 107–193.
  Kruisbeek, A. M. 2001. Isolation of mouse mononuclear cells. Curr. Protoc. Immunol. 39, 3.1.1–3.1.5. doi: 10.1002/0471142735.im0301s39.
  Kruisbeek, A. M., Shevach, E. and Thornton, A. M. 2004. Proliferative assays for T cell function. Curr. Protoc. Immunol. 60, 3.12.1–3.12.20. doi: 1002/0471142735.im0312s60.
  Laky, K. and Kruisbeek, A. M. 2016. In vivo depletion of T lymphocytes. Curr. Protoc. Immunol. 113, 4.1.1–4.1.9. doi: 10.1002/0471142735.im0401s113.
  Lovchik, J. C., & 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.
  McFarland, H. I. and Rosenberg, A. S. 2009. Skin allograft rejection. Curr. Protoc. Immunol. 84, 4.4:4.4.1–4.4.13. doi: 10.1002/0471142735.im0404s84.
  Möller, G. (ed.) 1983. Mechanism of action of cytotoxic T cells. Immunol. Rev. 72 (entire volume).
  Muchmore, A. V., Nelson, D. L., Kirschner, H., & Blaese, R. M. 1975. A reappraisal of the effector cells mediating mitogen induced cellular cytotoxicity. Cell. Immunol. 19, 78–90.
  Nakagawa, Y., Watari, E., Shimizu, M., & Takahashi, H. 2011 One‐step simple assay to determine antigen‐specific cytotoxic activities by single‐color flow cytometry. Biomed. Res. 32, 159–66.
  Oehen, S., & 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., & 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., & Radsak, M. P. 2005. Cutting edge: Priming of CTL by transcutaneous peptide immunization with imiquimod. J. Immunol. 174, 2476–2480.
  Reeves, J. and Reeves, P. 2001. Removal of lymphoid organs. Curr. Protoc. Immunol. 1, 1.9.1–1.9.3. doi: 10.1002/0471142735.im0109s01.
  Segal, D. M., & Snider, D. P. 1989. Targeting and activation of cytotoxic lymphocytes. Chem. Immun. 47, 179–213.
  Shearer, G. M., & Schmitt–Verhulst, A.‐M. 1977. Major histocompatibility complex–restricted cell–mediated immunity. Adv. Immunol. 25, 55–91.
  Simpson, E., & 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., & 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., & Bevan, M. J. 1985. Hybrid antibodies can target sites for attack by T cells. Nature (Lond.) 314, 628–631.
  Strober, W. 2015. Trypan blue exclusion test of cell viability. Curr. Protoc. Immunol. 111, A3.B.1–A3.B.3. doi: 10.1002/0471142735.ima03bs111.
  Townsend, A., & 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.
  Trowsdale, J. and Campbell, R. D. 2001. Human MHC genes and products. Curr. Protoc. Immunol. 27, A.1K.1–A.1K.14. doi: 10.1002/0471142735.ima01ks27.
  Waterfield, J. D., Waterfield, E. M., Anaclerio, A., & 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., & Cohn, Z. A. 1988. Perforin–dependent and –independent pathways of cytotoxicity. Immunol. Rev. 103, 161–202.
  Zhang, L., Manirarora J. N., & Wei, C. H. 2014. Evaluation of immunosuppressive function of regulatory T cells using a novel in vitro cytotoxicity assay. Cell Biosci. 4, 51. doi: 10.1186/2045‐3701‐4‐51.
  Zinkernagel, R. M., & 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., & Chen, Y. U. 1980. Cell mediated cytolytic responses. In Selected Methods in Cellular Immunology (B.B. Mishell, S.M. Shiigi, C. Henry, & R.I. Mishell, eds.) pp. 124–137. W. H. Freeman, New York.
  Two reviews of the chromium–release assay and its applications.
  Simpson, E., & Chandler, P. 1986. Analysis of cytotoxic T cell responses. In Cellular Immunology, Vol. 2, 4th ed. (D. M. Weir, L. A. Herzenberg, C. Blackwell, & L. A. Herzenberg, eds.) pp. 68.1–68.16. Blackwell Scientific, Oxford.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library