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

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.