Detection of intracellular antigen-specific cytokines in human T cell populations

Determination of antigen-specific cytokine responses of T lymphocytes after vaccination is made difficult by the low frequency of responder cells. In order to detect these responses, the profile of intracellular cytokines was analyzed using flow cytometry after antigenic expansion. Peripheral blood mononuclear cells were stimulated with antigens for 5 days, further expande d with interleukin (IL)-2, and then restimulated on day 10. Cytokine produc tion was detected by intracellular staining with monoclonal antibodies afte r saponin-based permeabilization. Influenza expansion resulted in specific interferon-gamma (IFN-gamma) production of 6%-20%, with less IL-4 productio n (0%-2%), Tetanus toroid resulted in even greater production. IL-4 and IFN -gamma were produced mainly by memory cells of the CD45RO(+) phenotype. IFN -gamma production was contributed by both CD4 and CD8 populations. These me thods were then applied to a clinical trial of a candidate human immunodefi ciency virus type 1 vaccine. Antigen-specific increases in IFN-gamma were m easured, which corresponded to antibody production, lymphoproliferation, an d skin testing.