Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation

J Immunol Methods. 2003 Oct 1;281(1-2):65-78. doi: 10.1016/s0022-1759(03)00265-5.

Abstract

Flow cytometric detection of antigen-specific CD8+ T cells has previously been limited to MHC-class I tetramer staining or intracellular cytokine production, neither of which measure the cytolytic potential of these cells. Here we present a novel technique to enumerate antigen-specific CD8+ T cells using a marker expressed on the cell surface following activation induced degranulation, a necessary precursor of cytolysis. This assay measures the exposure of CD107a and b, present in the membrane of cytotoxic granules, onto the cell surface as a result of degranulation. Acquisition of cell surface CD107a and b is associated with loss of intracellular perforin and is inhibited by colchicine, indicating that exposure of CD107a and b to the cell surface is dependent on degranulation. CD107a and b are expressed on the cell surface of CD8+ T cells following activation with cognate peptide, concordant with production of intracellular IFNgamma. Finally, CD107-expressing CD8+ T cells are shown to mediate cytolytic activity in an antigen-specific manner. Measurement of CD107a and b expression can also be combined with MHC-class I tetramer labeling and intracellular cytokine staining to provide a more complete assessment of the functionality of CD8+T cells expressing cognate T cell receptors (TCR).

MeSH terms

  • Antigens, CD / analysis
  • CD8-Positive T-Lymphocytes / immunology*
  • Cell Degranulation*
  • Cytotoxicity, Immunologic
  • Enterotoxins / pharmacology
  • Flow Cytometry / methods*
  • Histocompatibility Antigens Class I / analysis
  • Humans
  • Interferon-gamma / biosynthesis
  • Lysosomal-Associated Membrane Protein 1
  • Lysosome-Associated Membrane Glycoproteins
  • Membrane Glycoproteins / analysis
  • Perforin
  • Platelet Membrane Glycoproteins / analysis
  • Pore Forming Cytotoxic Proteins
  • Sensitivity and Specificity
  • Tetraspanin 30

Substances

  • Antigens, CD
  • CD63 protein, human
  • Enterotoxins
  • Histocompatibility Antigens Class I
  • Lysosomal-Associated Membrane Protein 1
  • Lysosome-Associated Membrane Glycoproteins
  • Membrane Glycoproteins
  • Platelet Membrane Glycoproteins
  • Pore Forming Cytotoxic Proteins
  • Tetraspanin 30
  • Perforin
  • enterotoxin B, staphylococcal
  • Interferon-gamma