In Vitro Models of Human T Cell Anergy

Leonard J. Appleman1, Vassiliki Boussiotis1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.36
DOI:  10.1002/0471142735.im0736s65
Online Posting Date:  March, 2005
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Abstract

T cell anergy (acquired unresponsiveness) is an important mechanism for tolerance of self antigens. Human helper T cell clones rendered anergic in vitro are valuable tools for studying the molecular mechanisms of T cell tolerance. This unit outlines the induction and assessment of anergy in human CD4+ helper T cell clones that are reactive against MHC class II alloantigens.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Induction and Evaluation of Anergy in Alloantigen‐Specific T Cell Clones
  • Support Protocol 1: Generation and Maintenance of Alloreactive Human T Cell Clones
  • Support Protocol 2: Maintenance of Alloreactive CD4+ T Cell Clone Cultures
  • Support Protocol 3: Construction of Artificial Antigen‐Presenting Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Induction and Evaluation of Anergy in Alloantigen‐Specific T Cell Clones

  Materials
  • Stimulator cells: NIH‐3T3 cells transfected with the human HLA‐DR7 cDNA, NIH‐3T3 cells transfected with the human HLA‐DR7 and B7‐1 cDNAs, and NIH‐3T3 cells transfected with the neomycin resistance plasmid (t‐DR7, t‐DR7/B7, and t‐neoR cells, respectively; see protocol 4)
  • Mitomycin C (Sigma)
  • Complete DMEM/F12 culture medium (see recipe)
  • Complete RPMI culture medium (see recipe)
  • CD4+ T cell clones in co‐culture with t‐DR7/B7 or Epstein‐Barr virus (EBV)–immortalized B cells (see protocol 2)
  • Recombinant human interleukin 2
  • Phorbol myristate acetate (PMA)
  • Ionomycin
  • 24‐ and 96‐well flat‐bottom culture plates (tissue culture–treated; Corning or Nalge Nunc)
  • Gamma irradiator: 67Cs
  • Additional reagents and equipment for Ficoll gradient centrifugation (unit 7.1), ELISA (unit 6.4), and assessment of [3H]thymidine incorporation (unit 7.10; appendix 3D)
NOTE: The responder PBMCs must be negative for the HLA allele of interest to mount an alloantigen T cell response.

Support Protocol 1: Generation and Maintenance of Alloreactive Human T Cell Clones

  Materials
  • NIH‐3T3 cells transfected with the human HLA‐DR7 and B7‐1 cDNAs (t‐DR7/B7 cells; see protocol 4)
  • Complete DMEM/F12 culture medium (see recipe) with and without 50 µg/ml mitomycin C (Sigma)
  • PBS ( appendix 2A)
  • Trypsin‐EDTA solution (Invitrogen)
  • RPMI medium containing 2.5% FBS (see recipe)
  • Complete RPMI culture medium (see recipe)
  • Blood or discarded blood product (buffy coat or leukopak) from a DR7 subject
  • PBMCs from a DR7+ or DR7 subject
  • Recombinant human IL‐2
  • Phytohemagglutinin (PHA)
  • Fluorescent anti‐CD4 and anti‐CD8 monoclonal antibodies
  • 60‐cm2 tissue culture–coated dishes
  • Inverted‐phase microscope
  • 15‐ml conical tubes
  • Sorvall centrifuge equipped with an RC‐3B rotor
  • 24‐ and 96‐well flat‐bottom culture plates (tissue culture–treated; Corning or Nalge Nunc)
  • Gamma irradiator: 67Cs
  • Additional reagents and equipment for the collection of blood from human subjects ( appendix 3F), Ficoll gradient centrifugation (unit 7.1), trypan blue staining ( appendix 3B), staining of cells with immunofluorescent antibodies (unit 21.4), flow cytometric analysis (unit 5.4), preparation of EBV‐immortalized B cells (unit 7.22), and assessment of [3H]thymidine incorporation (unit 7.10; appendix 3D)
CAUTION: EBV may cause disease in nonimmune individuals. In addition, it has been associated with a number of lymphoproliferative disorders. Thus, only individuals known to have serum antibodies to EBV should handle cell lines infected with the virus, and all applicable biosafety guidelines must be followed (see Chapter 7).CAUTION: Gamma irradiation should be performed only by personnel trained in the proper use of this technique. Standard precautions for preventing exposure and contamination of personnel and equipment should be followed at all times.NOTE: Radiation sensitivity varies among EBV‐immortalized B cell lines. Thus, optimum radiation doses may need to be determined empirically for individual lines.

Support Protocol 2: Maintenance of Alloreactive CD4+ T Cell Clone Cultures

  Materials
  • Mitomycin‐treated NIH‐3T3 cells transfected with the human HLA‐DR7 and B7‐1 cDNAs (mitomycin‐treated t‐DR7/B7 cells; see Support Protocols protocol 21 and protocol 43)
  • Complete DMEM/F12 culture medium (see recipe)
  • Complete RPMI culture medium (see recipe) with and without 50 U/ml recombinant human IL‐2
  • DR7‐reactive CD4+ T cell clones (see protocol 2)
  • PBMCs from a DR7+ or DR7 subject
  • 24‐well flat‐bottom culture plates (tissue culture–treated; Corning or Nalge Nunc)
  • Gamma irradiator: 67Cs
  • Additional reagents and equipment for Ficoll gradient centrifugation (unit 7.1) and preparation of EBV‐immortalized B cells (unit 7.22)
CAUTION: EBV may cause disease in nonimmune individuals. In addition, it has been associated with a number of lymphoproliferative disorders. Thus, only individuals known to have serum antibodies to EBV should handle cell lines infected with the virus, and all applicable biosafety guidelines must be followed (see Chapter 7).CAUTION: Gamma irradiation should be performed only by personnel trained in the proper use of this technique. Standard precautions for preventing exposure and contamination of personnel and equipment should be followed at all times.NOTE: Radiation sensitivity varies among EBV‐immortalized B cell lines. Thus, optimum radiation doses may need to be determined empirically for individual lines.

Support Protocol 3: Construction of Artificial Antigen‐Presenting Cells

  Materials
  • NIH‐3T3 cells
  • Complete DMEM/F12 culture medium (see recipe)
  • Expression constructs encoding the α and β subunits of an HLA class II allele (separate construct for each subunit), a selectable marker, and B7‐1
  • G418 sulfate (Genetecin; Invitrogen)
  • Fluorescent anti‐DR7 and anti‐B7‐1 antibodies
  • Freezing medium ( appendix 3G)
  • Additional reagents and equipment for transfection of DNA constructs into mammalian cells and selection of transfectants (unit 10.15, unit 10.16, and unit 10.1710.17), staining of cells with immunofluorescent antibodies (unit 21.4), flow cytometric analysis (unit 5.3) and cell sorting (unit 5.4), cloning by limiting dilution (unit 2.5), and cryopreservation of T cell clones (unit 7.19)
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Figures

Videos

Literature Cited

   Billingham, R.E., Brent, L., and Medawar, P.B. 1953. Actively acquired tolerance of foreign cells. Nature 172:603‐606.
   Boussiotis, V.A., Freeman, G.J., Gray, G., Gribben, J., and Nadler, L.M. 1993. B7 but not intercellular adhesion molecule‐1 costimulation prevents the induction of human alloantigen‐specific tolerance. J. Exp. Med. 178:1753‐1763.
   Brent, L. 2003. The 50th anniversary of the discovery of immunologic tolerance. N. Engl. J. Med. 349:1381‐1383.
   Gimmi, C.D., Freeman, G.J., Gribben, J.G., Gray, G., and Nadler, L.M. 1993. Human T‐cell clonal anergy is induced by antigen presentation in the absence of B7 costimulation. Proc. Natl. Acad. Sci. U.S.A. 90:6586‐6590.
   Groux, H., Bigler, M., de Vries, J.E., and Roncarolo, M.G. 1996. Interleukin‐10 induces a long‐term antigen‐specific anergic state in human CD4+ T cells. J. Exp. Med. 184:19‐29.
   Jenkins, M.K. and Schwartz, R.H. 1987. Antigen presentation by chemically modified splenocytes induces antigen‐specific T cell unresponsiveness in vitro and in vivo. J. Exp. Med. 165:302‐319.
   Jenkins, M.K., Pardoll, D.M., Mizuguchi, J., Chused, T.M., and Schwartz, R.H. 1987. Molecular events in the induction of a nonresponsive state in interleukin 2‐producing helper T‐lymphocyte clones. Proc. Natl. Acad. Sci. U.S.A. 84:5409‐5413.
   Jenkins, M.K., Ashwell, J.D., and Schwartz, R.H. 1988. Allogeneic non‐T spleen cells restore the responsiveness of normal T cell clones stimulated with antigen and chemically modified antigen‐presenting cells. J. Immunol. 140:3324‐3330.
   Jenkins, M.K., Chen, C.A., Jung, G., Mueller, D.L., and Schwartz, R.H. 1990. Inhibition of antigen‐specific proliferation of type 1 murine T cell clones after stimulation with immobilized anti‐CD3 monoclonal antibody. J. Immunol. 144:16‐22.
   Kearney, E.R., Pape, K.A., Loh, D.Y., and Jenkins, M.K. 1994. Visualization of peptide‐specific T cell immunity and peripheral tolerance induction in vivo. Immunity 1:327‐339.
   Lamb, J.R., Skidmore, B.J., Green, N., Chiller, J.M., and Feldmann, M. 1983. Induction of tolerance in influenza virus‐immune T lymphocyte clones with synthetic peptides of influenza hemagglutinin. J. Exp. Med. 157:1434‐1447.
   LaSalle, J.M., Tolentino, P.J., Freeman, G.J., Nadler, L.M., and Hafler, D.A. 1992. Early signaling defects in human T cells anergized by T cell presentation of autoantigen. J. Exp. Med. 176:177‐186.
   Phelan, M.C. 1998. Basic techniques for mammalian cell tissue culture. In Current Protocols in Cell Biology (J.S. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott‐Schwartz, and K.M. Yamada, eds.) pp. 1.1.1‐1.1.10. John Wiley & Sons, Hoboken, N.J.
   Quill, H. and Schwartz, R.H. 1987. Stimulation of normal inducer T cell clones with antigen presented by purified Ia molecules in planar lipid membranes: Specific induction of a long‐lived state of proliferative nonresponsiveness. J. Immunol. 138:3704‐3712.
   Rellahan, B.L., Jones, L.A., Kruisbeek, A.M., Fry, A.M., and Matis, L.A. 1990. In vivo induction of anergy in peripheral V beta 8+ T cells by staphylococcal enterotoxin B. J. Exp. Med. 172:1091‐1100.
   Sloan‐Lancaster, J., Evavold, B.D., and Allen, P.M. 1993. Induction of T‐cell anergy by altered T‐cell‐receptor ligand on live antigen‐presenting cells. Nature 363:156‐159.
   Staveley‐O'Carroll, K., Sotomayor, E., Montgomery, J., Borrello, I., Hwang, L., Fein, S., Pardoll, D., and Levitsky, H. 1998. Induction of antigen‐specific T cell anergy: An early event in the course of tumor progression. Proc. Natl. Acad. Sci. U.S.A. 95:1178‐1183.
Key References
   Boussiotis et al., 1993. See above.
   Gimmi et al., 1993. See above.
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