In Situ Staining Using MHC Class I Tetramers

Pamela J. Skinner1, Ashley T. Haase1

1 University of Minnesota, Minneapolis, Minnesota
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 17.4
DOI:  10.1002/0471142735.im1704s64
Online Posting Date:  January, 2005
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Abstract

The invention of MHC‐tetramer technology to label antigen‐specific T cells has lead to a greatly enhanced understanding of T lymphocyte biology. This protocol describes the use of MHC class I tetramers to stain antigen‐specific T cells in tissue sections. In situ tetramer staining (IST) can be used to determine the localization, abundance, and phenotype of antigen‐specific T cells in native environments and in three‐dimensional space. IST is broadly applicable because it can be used to stain essentially any antigen‐specific T cell in any tissue for which MHC tetramers are available. IST can be combined with histochemistry and/or immunohistochemistry to permit visualization and characterization of antigen‐specific T cells relative to other cell types in stained tissue sections. Thus, IST is a useful and valuable component of MHC‐tetramer technology.

Keywords: In situ tetramer staining (IST); MHC class I tetramers; CD8+ T cells; vibratome; confocal microscopy

     
 
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Table of Contents

  • Basic Protocol 1: In Situ MHC Class I Tetramer Staining using Fresh Tissue Sections
  • Alternate Protocol 1: Direct MHC‐Tetramer Staining
  • Alternate Protocol 2: In Situ MHC Class I Tetramer Staining using Frozen Tissue Sections
  • Support Protocol 1: Use of Tissue Chambers for Staining Free‐Floating Sections
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: In Situ MHC Class I Tetramer Staining using Fresh Tissue Sections

  Materials
  • Sterile PBS ( appendix 2A), 4°C and room temperature
  • Tissue (e.g., mouse spleen)
  • PBS‐buffered, 4% low‐melt agarose, ∼40°C (see recipe)
  • Loctite Quick Set Instant Adhesive
  • Blocking solution with and without Triton X‐100 (see recipe)
  • 0.5 µg/ml FITC‐conjugated MHC class I tetramers
  • Mouse or nonrabbit antibodies directed at extracellular epitopes (e.g., anti‐CD8 antibodies)
  • PBS‐buffered 4% paraformaldehyde (see recipe)
  • Rabbit anti‐FITC antibodies (e.g., BioDesign)
  • 0.01 M urea: dissolve 0.3 g urea in 500 ml H 2O; store up to 12 months at room temperature
  • Nonrabbit antibodies directed at intracellular epitopes (optional)
  • PBS containing Triton X‐100: add 1.5 ml (0.3% final) Triton X‐100 to 500 ml PBS ( appendix 2A); store up to 12 months at room temperature
  • Fluorescently labeled antibodies (e.g., goat‐anti‐rabbit‐Cy3, goat‐anti‐rabbit‐Alexa 488) that have been highly cross‐absorbed to other species IgG for use with multiple labeling
  • Glycerol/gelatin/n‐propyl gallate mounting media (see recipe)
  • Vibratome with block
  • Surgical scissors or scalpel
  • No. 2 camel‐hair paintbrushes: trim to desired thickness with razor blade if needed
  • Tissue chambers (see protocol 4)
  • 24‐well flat‐bottom tissue culture plates with lids
  • Aluminum foil
  • Cardboard slide folder (e.g., Fisher)
  • Confocal microscope

Alternate Protocol 1: Direct MHC‐Tetramer Staining

  Materials
  • OCT freezing medium (e.g., Tissue‐Tek, Sakura Finetek)
  • Tissue
  • Small cryomolds
  • Cryostat, −20°C
  • Silane‐ or lysine‐coated microscope slides
  • Humid chamber

Alternate Protocol 2: In Situ MHC Class I Tetramer Staining using Frozen Tissue Sections

  Materials
  • 14‐ml polypropylene round‐bottom snap‐cap tube (e.g., Falcon)
  • Wire mesh (e.g., Screen 40 Mesh for CD‐1, Sigma)
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Figures

Videos

Literature Cited

   Altman, J.D., Moss, P.A., Goulder, P.J., Barouch, D.H., McHeyzer‐Williams, M.G., Bell, J.I., McMichael, A.J., and Davis, M.M. 1996. Phenotypic analysis of antigen‐specific T lymphocytes. Science 274:94‐96.
   Andersen, M.H., Pedersen, L.O., Capeller, B., Brocker, E.B., Becker, J.C., and thor Straten, P. 2001. Spontaneous cytotoxic T‐cell responses against survivin‐derived MHC class I‐restricted T‐cell epitopes in situ as well as ex vivo in cancer patients. Cancer Res. 61:5964‐5968.
   Daniels, M.A. and Jameson, S.C. 2000. Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers. J. Exp. Med. 191:335‐346.
   Dickinson, A.M., Wang, X.N., Sviland, L., Vyth‐Dreese, F.A., Jackson, G.H., Schumacher, T.N., Haanen, J.B., Mutis, T., and Goulmy, E. 2002. In situ dissection of the graft‐versus‐host activities of cytotoxic T cells specific for minor histocompatibility antigens. Nat. Med. 8:410‐414.
   Haanen, J.B., van Oijen, M.G., Tirion, F., Oomen, L.C., Kruisbeek, A.M., Vyth‐Dreese, F.A., and Schumacher, T.N. 2000. In situ detection of virus‐ and tumor‐specific T‐cell immunity. Nature Medicine 6:1056‐1060.
   McGavern, D.B., Christen, U., and Oldstone, M.B. 2002. Molecular anatomy of antigen‐specific CD8(+) T cell engagement and synapse formation in vivo. Nat. Immunol. 3:918‐925.
   Mothe, B.R., Horton, H., Carter, D.K., Allen, T.M., Liebl, M.E., Skinner, P., Vogel, T.U., Fuenger, S., Vielhuber, K., Rehrauer, W., Wilson, N., Franchini, G., Altman, J.D., Haase, A., Picker, L.J., Allison, D.B., and Watkins, D.I. 2002. Dominance of CD8 responses specific for epitopes bound by a single major histocompatibility complex class I molecule during the acute phase of viral infection. J. Virol. 76:875‐884.
   Schmitz, J.E., Veazey, R.S., Kuroda, M.J., Levy, D.B., Seth, A., Mansfield, K.G., Nickerson, C.E., Lifton, M.A., Alvarez, X., Lackner, A.A., and Letvin, N.L. 2001. Simian immunodeficiency virus (SIV)‐specific cytotoxic T lymphocytes in gastrointestinal tissues of chronically SIV‐infected rhesus monkeys. Blood 98:3757‐3761.
   Schrama, D., Pedersen, L.O., Keikavoussi, P., Andersen, M.H., Straten, Pt.P., Brocker, E.B., Kampgen, E., and Becker, J.C. 2002. Aggregation of antigen‐specific T cells at the inoculation site of mature dendritic cells. J. Invest. Dermatol. 119:1443‐1448.
   Skinner, P.J. and Haase, A.T. 2002. In situ tetramer staining. J. Immunol. Methods 268:29‐34.
   Skinner, P.J., Daniels, M.A., Schmidt, C.S., Jameson, S.C., and Haase, A.T. 2000. Cutting edge: In situ tetramer staining of antigen‐specific T cells in tissues. J. Immunol. 165:613‐617.
   Stevceva, L., Alvarez, X., Lackner, A.A., Tryniszewska, E., Kelsall, B., Nacsa, J., Tartaglia, J., Strober, W., and Franchini, G. 2002. Both mucosal and systemic routes of immunization with the live, attenuated NYVAC/simian immunodeficiency virus SIV(gpe) recombinant vaccine result in gag‐specific CD8(+) T‐cell responses in mucosal tissues of macaques. J. Virol. 76:11659‐11676.
   Stevceva, L., Moniuszko, M., Alvarez, X., Lackner, A.A., and Franchini, G. 2004. Functional simian immunodeficiency virus Gag‐specific CD8+ intraepithelial lymphocytes in the mucosae of SIVmac251‐ or simian‐human immunodeficiency virus KU2‐infected macaques. Virology 319:190‐200.
   Stratmann, T., Martin‐Orozco, N., Mallet‐Designe, V., Poirot, L., McGavern, D., Losyev, G., Dobbs, C.M., Oldstone, M.B., Yoshida, K., Kikutani, H., Mathis, D., Benoist, C., Haskins, K., and Teyton, L. 2003. Susceptible MHC alleles, not background genes, select an autoimmune T cell reactivity. J. Clin. Invest. 112:902‐914.
   Yang, J., Jaramillo, A., Liu, W., Olack, B., Yoshimura, Y., Joyce, S., Kaleem, Z., and Mohanakumar, T. 2003. Chronic rejection of murine cardiac allografts discordant at the H13 minor histocompatibility antigen correlates with the generation of the H13‐specific CD8+ cytotoxic T cells. Transplantation 76:84‐91.
Key References
   Altman et al., 1996. See above.
  Original paper describing the invention of MHC tetramers to label antigen‐specific T cells.
   Haanen et al., 2000. See above.
  Original paper describing direct IST
   Skinner et al., 2000. See above.
  Original paper describing indirect IST
Internet Resources
   http://www.niaid.nih.gov/reposit/tetramer/overview.html
  Website of the NIH NIAID Tetramer Facility that supplies MHC‐tetramers to investigators.
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