Detection of individual interleukin 2-producing cells after anti-CD3 antibody activation

A new intracytoplasmic immunofluorescence staining technique to detect and quantify human interleukin-2 (IL-2)-producing lymphocytes is described. Blood mononuclear cells (MNC) were cultured and stimulated by mitogenic anti-CD3 antibody to produce IL-2. The cells were then fixed and subsequently permeabilized in suspension by the detergent saponin. Cytoplasmic IL-2 could then be demonstrated using polyclonal IL-2 specific antibodies and indirect immunofluorescence staining. A characteristic morphology of the IL-2 staining was noted with a local circle-shaped accumulation in the cytoplasm in a perinuclear position, probably reflecting the presence of the lymphokine in the Golgi stacks. A rapid, but transient IL-2 production peak 6 h after initiation of the cultures was observed. Only 1% of the cells produced IL-2, but each IL-2 stained cell was very bright, indicating a low capacity of anti-CD3 antibody to induce IL-2 production rather than an insensitivity of our detection system. Of the IL-2-producing cells 90% were CD4-positive T cells and 10% were CD8-positive T cells as revealed by two-colour staining of cell surface antigens and cytoplasmic IL-2.