Direct functional analysis of epitope-specific CD8+ T cells in peripheral blood

The functional status of virus-specific CD8(+) T cells is important for the outcome and the immunopathogenesis of viral infections. We have developed an assay for the direct functional analysis of antigen-specific CD8(+) T ce lls, which does not require prolonged in vitro cultivation and amplificatio n of T cells. Whole blood samples were incubated with peptide antigens for <5 h, followed by staining with peptide-MHC tetramers to identify epitope-s pecific T cells, The cells were also stained for the activation marker CD69 or for the production of cytokines such as interferon-gamma (IFN<gamma>) o r turner necrosis factor-alpha (TNF alpha). With the combined staining with tetramer and antibodies to CD69 or cytokines the number of antigen-specifi c CD8(+) T cells as well as the functional response of each individual cell to the cognate antigen can be determined in a single experiment. Virus-spe cific CD8(+) T cells that are nonfunctional, as well as those that are func tional under the same stimulating conditions can be simultaneously detected with this assay, which is not possible by using other T-cell functional as says including cytotoxicity assay, intracellular cytokine staining, and enz yme-linked immunospot (ELISPOT) assay.