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

  • 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

  • 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

  • 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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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
  Website of the NIH NIAID Tetramer Facility that supplies MHC‐tetramers to investigators.
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