title: Intracellular staining for TNF and IFN-gamma detects different frequencies of antigen-specific CD8(+) T cells.
abstract: 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.
authors.affiliation: Department of Microbiology and Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: V P
authors.initials: VP
authors.lastname: Badovinac
authors.forename: J T
authors.initials: JT
authors.lastname: Harty
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D020126:Brefeldin A / Q000494:pharmacology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000382:microbiology / Q000821:virology; D005260:Female; D005453:Fluorescence; D007371:Interferon-gamma / Q000032:analysis* / Q000276:immunology; D007424:Intracellular Fluid; D007476:Ionophores / Q000494:pharmacology; D008089:Listeria monocytogenes / Q000276:immunology; D018655:Lymphocyte Count; D008217:Lymphocytic choriomeningitis virus / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008985:Monensin / Q000494:pharmacology; D013194:Staining and Labeling / Q000379:methods; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D007476:Ionophores; D014409:Tumor Necrosis Factor-alpha; D020126:Brefeldin A; D007371:Interferon-gamma; D008985:Monensin