Immunobiological study of interferon-gamma-producing cells after staphylococcal enterotoxin B stimulation

Staphylococcal enterotoxin B (SEB) induced the production of human interferon-gamma (hIFN-gamma) in peripheral blood mononuclear cells (PBMC). Using specific mouse monoclonal antibodies (mAb) to hIFN-gamma, the patterns of cytoplasmic fluorescence in the PBMC from five individuals were studied. Discrete polar bodies in a ring-formation adjacent to the nuclear membrane was the most frequently observed fluorescent pattern throughout the 76-hr observation period. Additional and different fluorescent patterns such as multifocal and diffused cytoplasmic, as well as granular fluorescence over the whole cytoplasm, may appear during the late induction period (50-76 hr). By using an immunogold-silver (IGS) enhancement method to label cell-surface antigens, it was possible to detect the presence of CD3, CD4, CD25 and OKT11 marker in 55%, 54%, 77%, and 71% of the IFN-gamma producer cells, respectively. Monensin and carboxylcyanide m-chlorophenyl-hydrozone (CCCP) are ionophores known to interrupt subcellular transport of a number of secretory proteins. When monensin or CCCP was added to the induced cultures 2-3 hr before harvests, an increase in the intensity of cytoplasmic fluorescence in IFN-gamma-producing cells was observed; a greater than 10-fold enhancement in the sensitivity of immunostaining was demonstrated in these preparations.