Intracellular staining for TNF and IFN-gamma detects different frequencies of antigen-specific CD8(+) T cells

J Immunol Methods. 2000 Apr 21;238(1-2):107-17. doi: 10.1016/s0022-1759(00)00153-8.


CD8(+) T lymphocytes are important mediators of adaptive immunity against certain viral, protozoan and bacterial pathogens. Activated CD8(+) T cells are able to induce cytolysis of infected cells (perforin and CD95-CD95L mediated pathways) and also elaborate cytokines, including IFN-gamma and TNF after appropriate MHC class I-peptide recognition. New technologies for the detection of antigen-specific CD8(+) T cells, including tetrameric MHC class I-peptide complexes, intracellular IFN-gamma staining and IFN-gamma ELISPOT analysis have revised our understanding of the magnitude of the CD8(+) T cell response to infection. Here, using intracellular cytokine staining, we compare detection of IFN-gamma and TNF in the analysis of pathogen-specific CD8(+) T cell lines and CD8(+) T cells after primary viral infection (LCMV) or secondary bacterial infection (Listeria monocytogenes). Under multiple conditions and with multiple epitopes, we find that staining for intracellular IFN-gamma consistently detects a higher frequency of antigen-specific CD8(+) T cells than detection of intracellular TNF. However, (a) intracellular staining for TNF can be used to detect antigen-specific CD8(+) T cell responses and (b) intracellular staining for cytokines is a useful approach for in vitro characterization of antigen-specific CD8(+) T cell lines.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology
  • Brefeldin A / pharmacology
  • CD8-Positive T-Lymphocytes* / immunology
  • CD8-Positive T-Lymphocytes* / microbiology
  • CD8-Positive T-Lymphocytes* / virology
  • Female
  • Fluorescence
  • Interferon-gamma / analysis*
  • Interferon-gamma / immunology
  • Intracellular Fluid
  • Ionophores / pharmacology
  • Listeria monocytogenes / immunology
  • Lymphocyte Count
  • Lymphocytic choriomeningitis virus / immunology
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Monensin / pharmacology
  • Staining and Labeling / methods
  • Tumor Cells, Cultured
  • Tumor Necrosis Factor-alpha / analysis*
  • Tumor Necrosis Factor-alpha / immunology


  • Antibodies, Monoclonal
  • Ionophores
  • Tumor Necrosis Factor-alpha
  • Brefeldin A
  • Interferon-gamma
  • Monensin