Simultaneous production of interleukin 2, interleukin 4 and interferon‐γ by activated human blood lymphocytes

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Abstract
The production of interleukin 2 (IL 2), IL 4 and interferon‐γ (IFN‐γ) by in vitro activated unselected human blood mononuclear cells was studied at a single‐cell level. Individual lymphokine‐synthesizing cells were identified by intracellular immunofluorescent staining using cytokine‐specific monoclonal or polyclonal antibodies. Cultures from adult blood donors revealed a biphasic kinetic production pattern for IL 2 and IFN‐γ with peaks occurring 4–6 and 24–30 h after initiation of the cultures. Approximately 20%–40% of the lymphocytes produced IL 2 and IFN‐γ. In contrast, only 1%–3% of the lymphocytes synthesized IL 4 with maximal frequency after 6 h of culture. CD4+ as well as CD8+ T cells contributed to the synthesis of all three lymphokines studied. CD4+CD45R− T cells were the major producers of IL 2 and IL 4, while CD8+CD45R− T cells were the most common phenotype of IFN‐γ‐synthesizing cells. By performing two‐color immunofluorescence studies we observed that among IL 4‐producing cells every second one made simultaneously IL 2 and every fourth one made IFN‐γ. Mononuclear cells from umbilical cord blood could be stimulated to make IL 2 to the same extent as cells from adult blood donors. No IL 4 production and a strikingly reduced frequency of IFN‐γ producers were noted in cell cultures from neonates. IL 2, IL 4 and IFN‐γ accumulated in the Golgi system, which resulted in a characteristic morphology of the staining, eliminating problems with evaluation of background signals.