CYTOKINE SYNTHESIS ANALYZED AT THE SINGLE-CELL LEVEL BEFORE AND AFTERREVACCINATION WITH TETANUS TOXOID

Tetanus toroid (TT) is a potent immunogen which evokes strong antibody responses after immunization. Here, TT was used as a model antigen to study the production of cytokines at the single-cell level during the in vitro immune response to a specific recall antigen. Peripheral blo od mononuclear cells were obtained from healthy volunteers before and 9 weeks after TT vaccination and were cultured with antigen in vitro. The kinetics of cytokine synthesis as well as frequencies of cytokine- producing cells were determined at the single-cell level by immunofluo rescent intracellular staining of the cytokine protein. The phenotype of the producer cells was revealed by concomitant staining of surface markers. Two patterns of cytokine synthesis were induced by TT: (i) T lymphocytes expressed a number of lymphokines (interleukin (IL)-2, IL- 3, IL-4, IL-10, interferon (IFN)-gamma and tumor necrosis factor (TNF) -beta), each with distinct kinetics of synthesis. This cytokine expres sion was strictly dependent on the previous exposure of the donor to T T and positively correlated with the level of tetanus immunity, as jud ged by TT-specific Ab levels in plasma as well as lymphoproliferation. Cells producing IL-2, IFN-gamma and particularly TNF-beta dominated t his in vitro response. After 96-120 h in culture, 1.0-1.3 % of the cel ls produced TNF-beta, i.e. frequencies at least tenfold higher than fo r any of the other lymphokines assayed. The addition of IL-2 to the cu ltures caused a fourfold increase and a kinetics shift in the producti on of TNF-beta, which peaked already at 24 h. Exogenously added IL-2 a lso caused a five- to tenfold increase in the number of IL-2 and IFN-g amma producers but no apparent change in the kinetics of intracellular lymphokine appearance. (ii) The cytokines IL-1 alpha, IL-1 beta, IL-6 and TNF-alpha were produced by monocytes. This inflammatory monokine response was independent of the TT-specific immune status of the donor s, characterized by a rapid onset and was transient.