Phenotypic characterization of individual interferon‐γ‐producing cells after OKT3 antibody activation

This is the first study using a direct approach to determine the phenotype of interferon gamma (IFN‐γ)‐producing cells after polyclonal T cell activation. Blood mononuclear cells (MNC) were cultured and stimulated by OKT3 antibody to produce IFN‐γ. They were then stained with a panel of monoclonal antibodies with specificity for cell surface antigens, fixed and permeabilized in suspension and stained for cytoplasmic IFN‐γ using monoclonal antibodies and indirect immunofluorescence technique. IFN‐γ appeared mainly as a local, polar accumulation in the cytoplasm in a juxtanuclear position, probably identical to the Golgi apparatus. After a culture period of 20 h, 6–11% and 0.2–0.6% of the OKT3‐activated MNC from adults or newborns, respectively, produced IFN‐γ in cultures. Unstimulated cultures contained maximally 0.1% IFN‐γ‐positive MNC. The majority of the IFN‐γ‐producing MNC were T cells and expressed the T11 antigen and receptors for interleukin 2. To our surprise most of these T cells also carried receptors for complement 3bi (OKM1) but did not express class II molecules. Less than 50% displayed T4 or T8 antigens in three out of the four experiments performed. Natural killer cells, as recognized by Leu7 or B73.1 antibodies, contributed only marginally to the IFN‐γ synthesis whereas B cells (OKB7) and monocytes (Leu M3) showed no production of IFN‐γ.